C-type natriuretic peptide and method for the treatment of acute lung injury

Abstract

To provide a type C natriuretic peptide and a method for treating acute lung injury. [Solution] This disclosure relates to the treatment of lung, liver, and / or kidney disorders by administering therapeutically effective doses of (ultra)long-acting C-type natriuretic peptide (CNP), CNP derivatives, (ultra)long-acting CNP derivatives, or (ultra)long-acting CNP receptor (NPRB) agonists to subjects in need. This disclosure also relates to the treatment of non-cardiovascular causes of hypoxia, elevated inflammatory cell levels in the lungs, pulmonary edema, sepsis, bacteremia, fibrosis in general, and / or interstitial lung disease using these.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Patent Application No. 63 / 038,595, filed in December June 2020, which is incorporated herein by reference in its entirety.

[0002] Statement regarding sequence listings The sequence listing relating to this application is provided in text format instead of as a hard copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 74043_Sequence.txt. This text file is 16KB in size and was created on June 11, 2021. [Background technology]

[0003] Acute lung injury and acute respiratory distress syndrome Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by a PaO2 / FiO2 ratio of 200 Torr or less in ARDS and less than 300 Torr in ALI, acute onset of severe arterial hypoxemia with bilateral radiographic infiltration, and no evidence of left atrial hypertension (see, for example, Bernard et al., J. Crit. Care, 1994.9(1):p.72-81; Rubenfeld et al., N Engl J Med, 2005.353(16):p.1685-93; Brun-Buisson et al., Intensive Care Med, 2004.30(1):p.51-61; and Phua et al., Am J Respir Crit Care Med, 2009.179(3):p.220-7). As used herein, PaO2 refers to the partial pressure of arterial oxygen, and FiO2 is the percentage of oxygen in the inspired air (the FiO2 of room air is approximately 0.21, and a normal PaO2 / FiO2 is approximately 500 Torr). ARDS is an extremely potent inflammatory pneumonia response to certain primary and secondary noxious stimuli such as pneumonia (aseptic pneumonia, viral pneumonia, bacterial pneumonia, etc.), sepsis, aspiration, inhalation injury, drowning, and lung resection surgery (see, e.g., Alam et al., Ann Thorac Surg, 2007. 84(4): pp. 1085-91). ARDS is characterized by rapidly developing respiratory failure requiring admission to the intensive care unit (ICU) and ventilatory support. When patients survive ALI / ARDS, their long-term quality of life is often negatively affected due to pulmonary scarring (see, for example, Rubenfeld et al., N Engl J Med, 2005.353(16):p.1685-93; Dowdy et al., Intensive Care Med, 2006.32(8):p.1115-24). To date, no effective drugs have been found to treat acute lung injury (ALI) and ARDS, and there is a considerable need for such drugs.

[0004] Supportive care for ALI includes oxygen therapy to maintain arterial oxygen partial pressure (PaO2) above 55 mmHg or oxygen saturation (SaO2) above 88%, as well as fluid management. However, care must be taken not to supply excessive oxygen to avoid oxygen toxicity (i.e., oxygen should be administered at less than 60%). Furthermore, this measure does not address the underlying alveolar inflammatory edema.

[0005] All drugs previously tested in human clinical trials for the treatment of ALI, including glucocorticoids, surfactants, N-acetylcysteine, inhaled nitric oxide, liposomal PGE1, ketoconazole, lysophyllin, salbutamol, procysteine, activated protein C, and inhaled albuterol, have failed (see, e.g., Johnson ER and Matthay MA, J Aerosol Med Pulm Drug Deliv. 2010, 23(4):243-52). The treatment of ALI remains difficult for those skilled in the art to understand.

[0006] Pulmonary fibrosis (PF) refers to the progressive scarring of lung tissue resulting from a number of conditions, including chronic inflammatory processes (e.g., sarcoidosis, Wegener's granulomatosis), infections, environmental factors (e.g., exposure to asbestos, silica, certain gases), exposure to ionizing radiation (e.g., radiotherapy to treat thoracic tumors), chronic diseases (e.g., lupus, rheumatoid arthritis), or certain medications. Interstitial lung disease (ILD) is another comprehensive term used for PF and is synonymous for the purposes of this specification. Idiopathic pulmonary fibrosis (IPF) is PF of unknown cause. PF or IPF is an incurable type of chronic scarring lung disease affecting 5 million people worldwide, characterized by a progressive and irreversible decline in lung function accompanied by a gradual onset of shortness of breath and dry cough (see, for example, Raghu et al., (2011) American Journal of Respiratory and Critical Care Medicine. 183(6):788-824), and is associated with risk factors including inhalation of chemicals such as smoking, viral infections, or a family history of the disease. Other symptoms may include fatigue, abnormally large, domed fingernails and toenails (watch-dish nails). See, for example, nhlbi.nih.gov / health-topics / idiopathic-pulmonary-fibrosis; en.wikipedia.org / wiki / Idiopathic_pulmonary_fibrosis. Complications may include pulmonary hypertension, heart failure, pneumonia, or pulmonary embolism.

[0007] C-type natriuretic peptide (CNP), when administered continuously before or during a disorder ultimately leading to ALI or sepsis, can alleviate ALI, sepsis, and IPF, but its effectiveness when used after the disorder (e.g., as post-disorder treatment) is unclear. In general, CNP must be administered continuously at low doses and cannot be given as a bolus dose, as its half-life is very short and a bolus dose can cause a rapid drop in blood pressure. Administering it as a high bolus dose to compensate for the short half-life and extend the duration of its presence in the blood results in very high peak plasma concentrations (C). maxThis can cause a dangerous drop in blood pressure. To mitigate these adverse effects, CNP is usually delivered by slow infusion. See, for example, Kimura et al., J Surg Res. 2015, 194(2); 631-637.

[0008] CNP and NPRB receptors CNP was first isolated from pig brain in 1990 by Sudoh et al. and is a peptide consisting of 22 amino acid residues. See, for example, Sudoh et al., Biochem. Biophys. Res. Commun. 1989;159:1427-1434. CNP has a cyclic structure and is structurally similar to related natriuretic peptides, atrial natriuretic peptide (ANP) and type B natriuretic peptide (BNP), but lacks the elongation of the carboxyl terminus. See, for example, Hunt et al., J. Clin. Endocrinol. Metab. 1994;78:1428-1435. CNP is a highly conserved natriuretic peptide across various species. See, for example, Imura et al., Front. Neuroendocrinol. 1992;13:217-249. For example, in humans, the CNP gene (NPPC) is located on chromosome 2, while in mice, the CNP gene is located on chromosome 1. The CNP gene consists of two exons and one intron. See, for example, Ogawa et al., The Journal of Clinical Investigation. 1994;93:1911-192110; and Ogawa et al., Genomics. 1994;15(24):383-387. It is produced as a preprohormone or a 126-amino acid parent CNP peptide, which is converted to a 103-amino acid proCNP after the removal of 23 amino acid residues at the carboxyl terminus, and further processed by the enzyme furin into CNP-53 containing 53 amino acid residues and CNP containing 22 amino acid residues. For example, see Lumsden et al., Curr. Pharm. Des. 2010; 16: 4080-4088; Wu et al., J. Biol. Chem. 2003; 278: 25847-25852; and Chopra et al., Indian J. Endocrinol. Metab. 2013; 17: 83-90.In tissues, the relatively high molecular weight CNP-53 (CNP 51-103) is dominant, while CNP-22 (CNP 82-103) is mainly found in plasma and cerebrospinal fluid. Both contain a 17-amino acid residue ring structure common to all natriuretic peptides. Compared to ANP and BNP, the plasma half-life of CNP is relatively short, approximately 2-3 minutes in humans. See, for example, Potter LR. FEBS J. 2011;278:1808-1817. Normal plasma CNP concentrations (both forms) are in the low femtomole range per milliliter. See, for example, Das BB and Solinger R., Cardiovasc Hematol Agents Med Chem. 2009,7,29-42. CNP is mainly produced and secreted from the endothelium of the vascular system and male gonads and acts as a relaxant peptide. For example, see Suga et al., Endocrinology. 1998;139:1920-1926.

[0009] CNP peptides have two known membrane receptors (namely, natriuretic peptide receptor B (NPRB) and natriuretic peptide receptor C (NPRC)). NPRB receives messages from CNP and activates downstream signaling pathways, while NPRC is primarily a clearance receptor involved in the clearance or degradation of CNP. See, for example, Itoh H and Nakao K, Nihon Rinsho. 1997;55:1923-1936; Koller et al., Science. 1991;252:120-123; Suga et al., Endocrinology. 1992;130:229-239; and Potter LR and Hunter TJBiol. Chem. 2001;276:6057-6060. NPRB is also known by other names such as guanylate cyclase B (GC-B) or natriuretic peptide receptor 2 (NPR2).

[0010] The remaining natriuretic peptide receptor, NPRA, is activated by atrial natriuretic peptide (ANP) and type B natriuretic peptide (BNP), but not by CNP. ANP and BNP activate both NPRA and NPRB, while CNP selectively activates NPRB. All three natriuretic peptides bind to NPRC (which lacks guanylyl cyclase activity) for clearance and degradation. See, for example, Koller et al., Science. 1991;252:120-123; Suga et al., Endocrinology. 1992;130:229-239; and Potter LR and Hunter TJBiol. Chem. 2001;276:6057-6060. The difference in physiological effects between activation of one receptor and activation of both the NPRA and NPRB receptors remains unclear. In addition, the difficulty of administering a single bolus of CNP due to its short half-life (2-13 minutes), and the fact that this bolus administration is associated with a rapid drop in blood pressure, hinders the study of the in vivo effects of CNP. For example, Kimura et al. See al., J Surg Res. 2015, 194(2); 631-637. In fact, it was unclear until the disclosure of this invention whether any NPRB agonist, CNP, or CNP derivative could be administered as a bolus to treat ALI or ARDS without a significant reduction in blood pressure (e.g., a reduction of 20%, 15%, 10%, or 5%) while increasing cyclic GMP by a significant amount (e.g., more than 1.5 times, 2 times, 3 times, 4 times, or 5 times) over a certain duration (i.e., 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 84 hours, or 168 hours) to treat ALI or ARDS.

[0011] CNP expression and secretion are also regulated by various cytokines and growth factors involved in vascular remodeling and inflammation, including tumor necrosis factor (TNF), lipopolysaccharide (LPS), basic fibroblast growth factor (bFGF), interleukin-1 (IL-1), transforming growth factor beta (TGFβ), and thrombin. For example, see: Suga et al., Endocrinology. 1993; 133: 3038-3041; Suga et al., J. Clin. Invest. 1992; 90: 1145-1149; Woodard et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 2002; 282: R156-R165; Hama et al., Biochem. Biophys. Res. Commun. 1994; 198: 1177-1182; and Okahara et al., FEBS Lett. 1995; 373: 108-110. CNP levels are elevated in the blood during endothelial injury, sepsis, hypoxia, and chronic renal failure. See, for example, Hama et al., Biochem. Biophys. Res. Commun. 1994;198:1177-1182. Shear stress also induces CNP gene expression in human endothelial cells. See, for example, Okahara et al., FEBS Lett. 1995;373:108-110. The promoter region of the CNP gene has a binding site for the transcription factor TSC-22 (see, for example, Sellitti et al., Peptides. 2011;32:1964-1971), which is thought to be involved in regulating the function of hematopoietic progenitor cells and is a putative tumor suppressor gene that is hypermethylated and silenced in T or NK LGL leukemia. For example, Yu See et al., Blood. 2009;113(22):5558-67. The CNP gene promoter also has binding sites for transcription factors such as NF-κB, STAT1, ATF6, and E2F1. See, for example, Santhekaduret al., Biomed Pharmacother. 2017;92:826-835. However, it is unclear whether NPRB agonists such as CNP or its derivatives are usable for the treatment of ALI or ARDS. In fact, some inflammations are associated with increased CNP expression and secretion. Indeed, the results of bolus administration of larger doses of CNP, its derivatives, or other NPRB agonists for the treatment of ALI or ARDS are unknown. This uncertainty is further complicated by the complexity and unpredictability of the biological system. Previous studies in healthy human volunteers have demonstrated that CNP bolus injection transiently but significantly reduces both systolic and diastolic blood pressure, significantly increases heart rate, and presents only a limited and transient increase in plasma cyclic GMP for less than 90 minutes. Igaki et al., Hypertens Res 1998;21:7-13. Generally, all CNPs produce hemodynamic effects or similar hypotensive activity in mice, non-human primates, rats, dogs, and humans. See, for example, Wendt et al., J Pharmacol Exp Ther 353:132-149, April 2015. Another CNP variant (BMN-111; sequence) exhibits increased neutral endopeptidase (NEP) resistance. [ka] A new drug is currently under development. Studies of BMN-111 in animals and humans have demonstrated that as the dose increases to the desired therapeutic level, arterial blood pressure (BP) decreases and heart rate (HR) increases. In addition to investigating various variants of CNP, various CNP conjugates have been obtained by conjugating the CNP moiety with either PEG or a proteinaceous compound. These PEGylated CNPs and chimeric CNPs showed hemodynamic responses similar to those observed with non-PEGylated CNP variants. All variants studied to date have shown similar BP-lowering activity. See, for example, Wendt, J., Pharmacol Exp Ther 353:132-149, April 2015. Therefore, although we do not wish to be bound by theory, it appears that increasing drug exposure by increasing the bolus dose of drugs with CNP activity may be associated with unacceptable cardiovascular side effects such as hypotension. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Bernard et al., J.Crit.Care, 1994.9(1):p.72-81 [Non-Patent Document 2] Rubenfeld et al.,N Engl J Med,2005.353(16):p.1685-93 [Non-Patent Document 3] Brun-Buisson et al., Intensive Care Med, 2004.30(1):p.51-61 [Non-Patent Document 4] Phua et al., Am J Respir Crit Care Med, 2009.179(3):p.220-7 [Non-Patent Document 5] Alam et al.,Ann Thorac Surg,2007.84(4):p.1085-91 [Non-Patent Document 6] Dowdy et al.,Intensive Care Med,2006.32(8):p.1115-24

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[0013] Thus, there is a need for more effective and safer treatments for ALI and / or ARDS that maintain or enhance plasma levels of CNP therapeutic agents while avoiding cardiovascular side effects such as hypotension. There is also a need for CNP derivatives or CNP receptor (NPRB) agonists with a long half-life that allows for prolonged presence in the bloodstream, which can be used to treat ALI and / or ARDS. This disclosure aims to meet these needs and also provides related advantages. [Means for solving the problem]

[0014] This summary is provided to give a simplified overview of concepts that will be explained in more detail below in the detailed description. This summary is not intended to identify the main features of the claimed subject matter, nor is it intended to be used as an aid in defining the scope of the claimed subject matter.

[0015] In one embodiment, the present disclosure is characterized by a method for treating a subject having lung, liver, and / or kidney disorders; or symptoms associated with lung, liver, and / or kidney disorders, wherein the subject is treated with a composition comprising a long-acting CNP, a long-acting CNP derivative, a long-acting NPRB agonist, an ultra-long-acting CNP, an ultra-long-acting CNP derivative, an ultra-long-acting NPRB agonist, a long-acting CNP agonist, an ultra-long-acting CNP agonist, or any combination thereof, in a therapeutically effective bolus dose. The composition is administered to a person, wherein the composition does not lower blood pressure by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of a baseline blood pressure measurement, where the baseline blood pressure measurement is the mean blood pressure before administration of the composition, and the composition is administered 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 84 hours). The plasma cyclic GMP level after 168 hours is increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level of the subject before administration of the composition), where lung, liver, and / or kidney damage, or symptoms associated with lung, liver, and / or kidney damage, are as follows: acute lung injury Disorders of lung function include: arterial fibrosis (ALI), acute respiratory distress syndrome (ARDS), pulmonary edema, elevated levels of inflammatory cells in the lungs, increased levels or expression of inflammatory cytokines in the lungs compared to healthy lungs, increased levels of proteins in the alveolar space compared to healthy lungs, hypoarterial oxygenation (hypoarterial oxygenation is defined as blood PaO2 less than 60 mmHg and / or blood hemoglobin oxygen saturation (SpO2) less than 90%), pneumonia, fibrosis (e.g., pulmonary fibrosis, hepatic fibrosis, renal fibrosis), renal impairment, and any combination thereof.

[0016] In another embodiment, the present disclosure features long-acting or ultra-long-acting CNP derivatives comprising U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 4], U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 11], or any combination thereof, where each capital letter except U is an amino acid residue represented by single-letter amino acid nomenclature, U is part of formula (I) or (II), and formula (I) is, (aliphatic) a -(X)- (I) The formula is such that a is 0 or 1 (preferably a is 1); and the aliphatic is optionally substituted C 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 X is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is a linker (γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, and each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. Formula (II) is (Polymer) a -(Y)-(II) where a is 0 or 1 (preferably, a is 1); the polymer is cellulose, poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) (MPEG), poly(lactic-co-glycolic acid), poly(N-vinylpyrrolidone), or derivatives thereof; Y is a 1-10 amino acid residue or peptide sequence, and each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D), a 1-10 amino acid residue or peptide sequence; a non-amino acid linker, a non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; an amino acid residue-containing linker, where the amino acid residue is covalently attached to (Polymer) a an amino acid residue-containing linker; or a peptide linker different from a 1-10 amino acid residue or peptide sequence. In some embodiments, Y is the linker (γE) m -(B) n where B is a 1-8 amino acid residue or peptide sequence, and each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0017] In yet another embodiment, the Disclosure features a method for treating a subject having ALI and / or ARDS, or being at risk of developing ALI and / or ARDS, comprising administering to the subject a therapeutically effective bolus dose of a composition containing a long-acting CNP derivative or an ultra-long-acting CNP derivative, or any combination thereof, including: U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], or GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 4], U-CFGLKLDRIGSxSGLGC (where x is a natural or non-natural amino acid residue) [SEQ ID NO: 11], where U is as defined in formula (I) or (II) above. U can be covalently bonded to the epsilon amino group of an N-terminal G or C residue and / or K residue. The composition does not lower blood pressure by more than 15% (e.g., more than 10% or more than 5%) of the baseline blood pressure measurement, where the baseline blood pressure measurement is the mean blood pressure before administration of the composition; the composition increases the plasma cyclic GMP level 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where the baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level for the subject before administration of the composition).

[0018] In one embodiment, the present disclosure relates to a composition comprising a long-acting CNP derivative comprising the peptide of formula U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 30], wherein x is a natural or non-natural amino acid residue, except that x is a methionine residue; and U is a compound of formula (I): (aliphatic) a -(X)- (I) This is part of it; During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is the linker (γE) m -(B) n The present invention provides a composition in which, in the formula, B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0019] The aforementioned aspects of this disclosure and many of its associated advantages will be more readily understood by referring to the detailed description below in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0020] [Figure 1A]This plot shows plasma CNP levels [mean (SD); n=5] in CD-1 mice after subcutaneous administration of 2.0 mg / kg of native CNP, CNP derivative (dCNP), and ultra-long-acting CNP derivative (VLA-dCNP). The inset is an enlarged scale in the lower left corner showing the low plasma CNP levels (diamond) when native CNP is administered. Error bars represent the standard deviation of the n=5 plasma samples. The baseline CNP level before administration was 1.74 (0.6) ng / mL [mean (SD); n=15]. Figure 1A shows the continued presence of dCNP and VLA-dCNP in plasma after bolus administration in mice. [Figure 1B] Figure 1B is a plot showing plasma cyclic GMP levels in male C57BL / 6J mice measured using the CisBio (Codolet, France) cyclic GMP kit after subcutaneous administration of 1.0 mg / kg of native CNP, a CNP derivative (dCNP), and an ultra-long-acting CNP derivative (VLA-dCNP). Baseline plasma cyclic GMP levels were 20 ((3.7) mean (SEM); n=8) pmol / L or 7 ((1.3) mean (SEM); n=8) ng / mL; [n=8]. At time points of 2 hours or more, subcutaneous administration of native CNP did not show a significant increase in plasma cyclic GMP compared to baseline, while similar administration of long-acting CNP (dCNP and VLA-dCNP) showed a significant increase in cyclic GMP for at least 24 hours. Figure 1B shows the sustained presence of cyclic GMP after bolus administration of dCNP and VLA-dCNP compared to native CNP in mice. [Figure 2A]Figure 2A is a plot showing the corresponding increase in plasma cyclic GMP [mean (SEM); n=12] monitored after bolus administration of 25 μg / Kg ultra-long-acting CNP derivative (VLA-dCNP), ultra-long-acting BNP derivative (VLA-dBNP), and ultra-long-acting ANP derivative (VLA-dANP). Baseline plasma cyclic GMP levels were 8(2) ng / mL [mean (SD; n=12)], similar to those in healthy individuals. See, for example, Igaki, et al., Hypertens Res 1998;21:7-13. All ultra-long-acting formulations of natriuretic peptides increased cyclic GMP above the baseline of 8 ng / mL. Cyclic GMP AUC values ​​were 3,483 ng*h / mL for VLA-dANP, 2,585 ng*h / mL for VLA-dBNP, and 2,627 ng*h / mL for VLA-dCNP. The ultra-long-acting CNP derivative (VLA-dCNP) increased plasma cyclic GMP for 3 days without associated blood pressure reduction. Figure 2A shows the sustained presence of cyclic GMP after bolus administration of VLA-dCNP compared to two other ultra-long-acting natriuretic peptides from the same family. [Figure 2B]Figure 2B is a plot showing the mean arterial pressure [mean (SEM; n=12)] of dogs monitored after bolus administration of 25 μg / Kg ultra-long-acting CNP derivative (VLA-dCNP), ultra-long-acting BNP derivative (VLA-dBNP), and ultra-long-acting ANP derivative (VLA-dANP). VLA-dCNP did not cause a significant decrease in blood pressure from baseline (0 hours) after administration at ultra-high doses. In comparison, other ultra-long-acting natriuretic peptides such as VLA-dBNP and VLA-dANP derivatives caused a decrease in blood pressure of 15% or more. This was particularly true for VLA-dANP, where a decrease in blood pressure could reach as much as 50% in the case of a similar increase in cyclic GMP. In stark contrast, the ultra-long-acting CNP derivative (VLA-dCNP) increased plasma cyclic GMP over 3 days without any associated decrease in blood pressure. Figure 2B demonstrates that there was no decrease in blood pressure after a bolus administration of high-dose VLA-dCNP to dogs, while two other ultra-long-acting natriuretic peptides from the same family showed a significant decrease in blood pressure despite an increase in plasma cyclic GMP (Figure 2A). This indicates that plasma cyclic GMP is not the cause of the decrease in blood pressure. [Figure 3A]Figure 3A shows the timeline of a protocol for evaluating dCNP-suppressed LPS-induced acute lung injury. The protocol included treating mice with LPS (0.05 mg / kg intratracheal administration) and VLA-dCNP (L: 0.1 mg / kg sc; M: 0.3 mg / kg sc; H: 1.0 mg / kg sc), dCNP (H 1.0 mg / kg sc), CNP (1.0 mg / kg sc), atrial natriuretic peptide (ANP) (H 1.0 mg / kg sc), brain natriuretic peptide (BNP) (H 1.0 mg / kg sc), anti-mouse TNFα (TNFα ab) (clone XT3.11; BioXcell West Lebanon, NH) 1.0 mg / kg sc, or vardenafil (VDN) (Cayman Chemicals Ann Arbor, MI) 1.0 mg / kg sc. The test substance was administered immediately after LPS administration. Twenty-four hours after the procedure, mice were sacrificed under isoflurane anesthesia, and bronchoalveolar lavage fluid (BALF) was collected. [Figure 3B] Figure 3B is a bar graph showing the increase in cells, particularly neutrophils, in BALF in ALI and ARDS according to the protocol shown in Figure 3A. A decrease in cells indicated the resolution of ALI / ARDS. Statistical analysis was performed based on Dunnett's test using GraphPad InStat 3 (n=15, 23, 7, 7, 7, 7, 7, 7, 7, and 9; NC, control, CNP(H), dCNP(H), ANP(H), BNP(H), TNFα ab, VDN, VLA-dCNP(H). *P<0.01 (against VLA-dCNP(H))). Figure 3B is a bar graph showing that bolus administration of VLA-dCNP improved LPS-induced cell infiltration in the alveolar space. [Figure 3C]Figure 3C is a bar graph showing the total protein in BALF in ALI and ARDS, according to the protocol shown in Figure 3A. A decrease in total protein indicated the cessation of ALI / ARDS. Statistical analysis was performed based on Dunnett's test using GraphPad InStat 3 (n=15, 23, 7, 7, 7, 7, 7, 7, 7 and 9; NC, control, CNP(H), dCNP(H), ANP(H), BNP(H), TNFα ab, VDN, VLA-dCNP(H). *P<0.01 (against VLA-dCNP(H))). [Figure 4A] Figure 4A is a bar graph showing that VLA-dCNP treatment improved the increase in LPS-induced MPO+ cells, a marker of pro-inflammatory neutrophil granulocytes (i.e., MPO+ cells decreased compared to the control). Statistical analysis was performed based on Dunnett's test using GraphPad InStat 3 Control (n=18, 6, 6, 6, 6, 6, and 6; control, CNP, dCNP, ANP, BNP, TNFα ab, VDN, VLA-dCNP; *P<0.01 (against VLA-dCNP) and **P<0.05 (against VLA-dCNP)). [Figure 4B]Figure 4B is a series of photographs showing that bolus administration or treatment with VLA-dCNP improved LPS-induced inflammatory lung injury. A series of micrographs of paraffin sections of lung tissue stained with hematoxylin-eosin (HE) are shown, showing intensities indicating increased nucleated cell count, extracellular matrix and overall protein, scarring, and / or increased protein permeability into the alveolar space. Inflammatory cell infiltration observed by HE staining indicates inflammation of the lung (the panel shows darker staining as cell count indicating the presence of inflammatory disease and increased protein indicating protein leakage into the alveoli and / or extracellular matrix or scar deposition). For these studies, mice were treated with LPS (Sigma-Aldrich; 0.05 mg / kg intratracheally) and then with ultra-long-acting CNP derivatives or VLA-dCNP (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivatives or dCNP (1.0 mg / kg sc), atrial natriuretic peptide (ANP) (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), antitumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a PDE5 inhibitor called a cyclic GMP degradation inhibitor or vardenafil (VDN) (1.0 mg / kg sc). The test substances were administered immediately after LPS administration. 24 hours after treatment, mice were sacrificial under isoflurane anesthesia, lung tissue was collected, and fixed with 4% paraformaldehyde. Paraffin sections of fixed lung tissue were stained with anti-MPO antibody and hematoxylin-eosin staining. [Figure 5A]Figure 5A is a bar graph showing that bolus administration or treatment of VLA-dCNP and dCNP weakens LPS-induced upregulation of inflammatory cytokines (IL6) in BALF and promotes the resolution of ARDS / ALI. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration) and then with ultra-long-acting CNP derivatives or VLA-dCNP (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivatives or dCNP (1.0 mg / kg sc), atrial natriuretic peptide (ANP) (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), antitumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a PDE5 inhibitor called a cyclic GMP degradation inhibitor or vardenafil (VDN) (1.0 mg / kg sc). 24 hours after treatment, bronchoalveolar lavage fluid (BALF) was collected and IL-6 cytokine levels were measured. Statistical analysis was performed based on Student's t-tests (15, 23, 7, 7, 7, 7, 7, 7 and 9; NC, control, CNP, dCNP, ANP, BNP, TNFα ab, VDN, and VLA-dCNP; *P<0.01 (for VLA-dCNP) and **P<0.05 (for VLA-dCNP)). [Figure 5B] Figure 5B is a bar graph showing that bolus administration or treatment of VLA-dCNP and dCNP weakens LPS-induced upregulation of inflammatory cytokines (TNFα) in BALF and promotes the resolution of ARDS / ALI. The protocol was the same as that described in Figure 5A, except that bronchoalveolar lavage fluid (BALF) was collected and TNFα cytokines were measured. [Figure 5C] Figure 5C is a bar graph showing that bolus administration or treatment of VLA-dCNP and dCNP weakens LPS-induced upregulation of inflammatory cytokines (MCP-1) in BALF and promotes the resolution of ARDS / ALI. The protocol was the same as that described in Figure 5A, except that bronchoalveolar lavage fluid (BALF) was collected and MCP-1 cytokines were measured. [Figures 6A-6D] Figures 6A-6D are bar graphs showing that bolus administration or treatment with VLA-dCNP weakened LPS-induced upregulation of inflammatory cytokines in lung tissue and promoted the resolution of ARDS / ALI. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration) and then with VLA-dCNP (1.0 mg / kg sc). Lung tissue was collected 24 hours after treatment. The concentrations of each cytokine in the extracted lung proteins were measured using an ELISA kit. These cytokines were interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and macrophage chemoattractant protein-1 (MCP-1). Statistical analysis was performed based on Student's t-test (n=10, 10, 9; NC, control, VLA-dCNP; *P<0.05 (compared to control)). Figure 6A is a bar graph showing that bolus administration or treatment with VLA-dCNP weakened the LPS-induced upregulation of IL-6 in lung tissue and promoted the resolution of ARDS / ALI. Figure 6B is a bar graph showing that bolus administration or treatment with VLA-dCNP weakened the LPS-induced upregulation of TNF-α in lung tissue and promoted the resolution of ARDS / ALI. Figure 6C is a bar graph showing that bolus administration or treatment with VLA-dCNP weakened the LPS-induced upregulation of MCP-1 in lung tissue and promoted the resolution of ARDS / ALI. Figure 6D is a bar graph showing that bolus administration or treatment with VLA-dCNP weakened the LPS-induced upregulation of IL-1b in lung tissue and promoted the resolution of ARDS / ALI. [Figure 7A]Figure 7A is a bar graph showing that bolus administration of VLA-dCNP attenuated the expression of LPS-induced inflammatory cytokines, including IL-6, which are generally regulated by the NFκb system, the master regulator of the inflammatory system. This suggests that VLA-dCNP broadly suppressed the inflammatory response in the subjects' bodies, thereby promoting the resolution of ARDS / ALI. Measurement of inflammation-related gene expression in ALI lung tissue. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration), and then treated with ultra-long-acting CNP derivatives or VLA-dCNP (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivatives or dCNP (1.0 mg / kg sc), atrial natriuretic peptide or ANP (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), tumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a cyclic GMP degradation inhibitor or a PDE5 inhibitor called vardenafil (VDN) (1.0 mg / kg sc). Lung tissue was collected 24 hours after treatment. Total RNA was extracted from the collected lung tissue. Statistical analysis was performed based on Student's t-test (n=15, 22, 6, 6, 6, 6, 6, 5, and 9; NC, control, CNP, dCNP, ANP, BNP, TNFα ab, VDN, VLA-dCNP. *P<0.01 (for VLA-dCNP) and **P<0.05 (for VLA-dCNP)). [Figure 7B] Figure 7B is a bar graph showing that bolus administration of VLA-dCNP attenuated the expression of LPS-induced inflammatory cytokines, including iNOS. This suggests that VLA-dCNP broadly suppressed the target inflammatory response and promoted the resolution of ARDS / ALI. The protocol was as described in Figure 7A. [Figure 7C]Figure 7C is a bar graph showing that bolus administration of VLA-dCNP reduced the expression of LPS-induced inflammatory cytokines, including MCP-1, suggesting that VLA-dCNP broadly suppresses the inflammatory response in the subjects' bodies and promotes the resolution of ARDS / ALI. The protocol was as described in Figure 7A. [Figure 7D] Figure 7D is a bar graph showing that bolus administration of VLA-dCNP reduced the expression of LPS-induced inflammatory cytokines, including IL-1b. This suggests that VLA-dCNP broadly suppressed the inflammatory response in the subjects' bodies, thereby promoting the resolution of ARDS / ALI. The protocol was as described in Figure 7A. [Figure 7E] Figure 7E is a bar graph showing that bolus administration of VLA-dCNP reduced the expression of LPS-induced inflammatory cytokines, including IFNg. This suggests that VLA-dCNP broadly suppressed the inflammatory response in the subjects' bodies, thereby promoting the resolution of ARDS / ALI. The protocol was as described in Figure 7A. [Figure 8] Figure 8 is a series of bar graphs showing that bolus administration of VLA-dCNP suppressed inflammation levels in lung tissue and promoted the resolution of ARDS / ALI. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration) and then with VLA-dCNP (1.0 mg / kg sc). Lung tissue was collected 24 hours after treatment. Western blot analysis was performed using antibodies: Elf-1, Tollip, IRAK-1, P-P38, P-P65 and β-actin (internal standard). Statistical analysis was performed based on Student's t-test (n=5, *P<0.05 (against control)). [Figure 9]Figure 9 is a series of bar graphs showing that bolus administration of VLA-dCNP suppressed STAT levels in lung tissue and promoted the resolution of ARDS / ALI. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration) and then with VLA-dCNP (1.0 mg / kg sc). Lung tissue was collected 24 hours after treatment. Western blot analysis was performed using antibodies: anti-STAT-1, P-STAT-1, STAT-2, STAT-3, STAT-6 and β-actin (internal standard). Statistical analysis was performed based on Student's t-test (n=5, *P<0.05 (compared to control)). [Figure 10] Figure 10 is a bar graph showing that bolus administration of VLA-dCNP suppressed Elf-1 expression in human umbilical vein endothelial cells. Human umbilical vein endothelial cells (HUVECs) were inoculated into 12-well plates (1 × 10⁵ cells / well in 2 mL of HuMedia-EG2) while maintained in HuMedia-EG2. After 24 hours, the cells were treated with each concentration of VLA-dCNP (0.07 μM (0.21 μg / mL) or 0.7 μM (2.1 μg / mL)) (in M199 1% BSA) for 6 hours. Protein levels were evaluated by Western blot analysis using anti-Elf-1 and β-actin (internal standard). Statistical analysis was performed based on Student's t-test (n=4, *P<0.05 (compared to control)). [Figure 11]Figure 11 is a bar graph showing that bolus administration of VLA-dCNP suppressed Elf-1 levels in the nuclei of human umbilical vein endothelial cells. Human umbilical vein endothelial cells (HUVECs) were maintained in HuMedia-EG2. Cells were seeded in glass-bottom dishes at a density of 1 × 10⁵ cells / well in 2 mL of HuMedia-EG2. After 24 hours, cells were treated for 6 hours with each concentration of VLA-dCNP (0.07 μM (0.21 μg / mL)) or CNP 0.1 μM (0.21 μg / mL) in M199 (Thermo Fisher Scientific, Waltham MA) supplemented with 1% BSA (Sigma-Aldrich, St. Louis MO). Cells were fixed with 4% paraformaldehyde, treated with anti-Elf-1 Ab (Santa Cruz Biotechnology, Dallas TX), and then incubated with Alexa Fluor 488-labeled secondary antibody (Thermo Fisher Scientific, Waltham MA) and Hoechst 33342. [Figure 12] Figure 12 is a bar graph showing that bolus administration of VLA-dCNP induces Tollip expression in the human lung fibroblast cell line HFL1. Human lung fibroblast cells HFL1 (1.0 × 10⁵ cells / well) were cultured in DMEM medium for 16 hours, and then incubated with 1% BSA-M199 medium containing 0.21 μM (0.66 ug / mL) VLA-dCNP and (NC) without VLA-dCNP. After 12 hours of incubation, the cells were stimulated with LPS (final concentration 1.0 μg / mL). After a further 2 hours of incubation, the cells were harvested and lysed. Intracellular protein expression levels were evaluated by Western blotting with anti-Tollip and β-actin (internal standard). Statistical analysis was performed based on Student's t-test (n = 4, *P < 0.05 (compared to control)). [Figure 13A]Figure 13A is a graph showing that bolus administration of VLA-dCNP had a protective effect against LPS-induced sepsis lethality. Balb / c (11-week-old male) mice were treated with LPS (10 mg / kg ip) and then with each dose of VLA-dCNP (low 0.1 mg / kg sc; medium 0.3 mg / kg sc; high 1.0 mg / kg sc). Survival was observed every 2 hours. Statistical analysis was performed using the log-rank test based on GraphPad Prism 6.0 (n=10, 10, 10, 11). [Figure 13B] Figure 13B is a graph showing C57BL / 6J (6-week-old male) mice treated with LPS (15 mg / kg ip) followed by predetermined bolus doses of VLA-dCNP (low 0.1 mg / kg sc; medium 0.3 mg / kg sc; high 1.0 mg / kg sc). Survival was observed every 2 hours. Statistical analysis was performed using the log-rank test (n=11, 10, 11, 11). VLA-dCNP showed a protective effect against LPS-induced sepsis. [Figure 14A] Figure 14A is a bar graph showing that bolus administration of VLA-dCNP reduced the fibrotic area of ​​the lungs in this animal model of interstitial lung disease (ILD) or idiopathic pulmonary fibrosis (IPF). Male C57BL / 6J mice (6 weeks old) were treated with bleomycin (1.0 mg / kg intratracheally) and then with VLA-dCNP at doses of 0.1 mg / kg sc and 0.3 mg / kg sc. VLA-dCNP was administered 7 days after bleomycin administration (5 times / week). On day 21, mice were sacrificed, lung tissue was collected, and Masson's trichrome staining was performed. Fibrotic area was measured using Image J (NIH, Bethesda, Maryland, USA). Statistical analysis was performed based on Dunnett's test using GraphPad Prism 6. (n=5, 8, 9, 7; negative control, control, VLA-dCNP 0.1, and VLA-dCNP 0.3. *P<0.05 (compared to control)). [Figure 14B]Figure 14B is a series of micrographs showing the Masson trichrome-stained lung tissue sample from Figure 14A. The blue and light blue areas in the lung tissue indicate advanced collagen / fibrosis. [Figure 15A] Figure 15A is a bar graph showing that bolus administration of VLA-dCNP reduced the number of cells in the BALF from acute exacerbations in an idiopathic pulmonary fibrosis (IPF-AE) model. Male C57BL / 6J mice (6 weeks old) were treated with bleomycin (1.0 mg / kg intratracheal administration), and 3 weeks later, the mice were treated with LPS (0.05 mg / kg intratracheal administration) and then with VLA-dCNP at doses of 0.3 mg / kg sc and 1.0 mg / kg sc. VLA-dCNP was administered immediately after LPS administration. Mice were sacrificed 24 hours after treatment. Statistical analysis was performed based on Student's t-test using GraphPad Prism 6 (n=6, 6, 9, 9, 9; negative control, bleomycin, control, VLA-dCNP 0.3, and VLA-dCNP 1.0. *P<0.05 (against control)). [Figure 15B] Figure 15B is a bar graph showing that bolus administration of VLA-dCNP reduced protein levels in BALF from acute exacerbations in an idiopathic pulmonary fibrosis (IPF-AE) model. The protocol is as described in Figure 15A. [Figure 15C] Figure 15C is a bar graph showing that VLA-dCNP attenuated IL-6 in BALF from acute exacerbations in an idiopathic pulmonary fibrosis (IPF-AE) model. The protocol is as described in Figure 15A. [Figure 15D] Figure 15D is a bar graph showing that bolus administration of VLA-dCNP reduced cell count and protein levels in BALF from acute exacerbations in an idiopathic pulmonary fibrosis (IPF-AE) model, and attenuated TNFα. The protocol is as described in Figure 15A. [Figure 16A] Figure 16A shows a series of micrographs of kidney tissue. [Figure 16B]Figure 16B is a graph showing tubular damage as a function of bolus administration of VLA-dCNP in a model of acute kidney injury. [Figure 17A] Figure 17A is a bar graph showing a significant decrease in the liver enzyme aspartate aminotransferase (AST) in a diet-induced model of liver fibrosis when administered to VLA-dCNP or long-acting CNP. [Figure 17B] Figure 17B is a bar graph showing a significant decrease in the liver enzyme alanine aminotransferase (ALT) in a diet-induced model of hepatic fibrosis when administered to VLA-dCNP or long-acting CNP. [Figure 17C] Figure 17C is a bar graph showing a significant decrease in alpha-smooth muscle actin (a-SMA) in a diet-induced model of liver fibrosis when administered with VLA-dCNP or long-acting CNP. [Figure 17D] Figure 17D is a bar graph showing a significant decrease in tumor necrosis growth factor α (TNF-α), a marker of inflammation that induces fibrosis, in a diet-induced model of hepatic fibrosis when administered to VLA-dCNP or long-acting CNP. [Figure 17E] Figure 17E is a bar graph showing a significant decrease in monocyte chemotactic protein 1 (MCP-1), a mediator of macrophage-induced inflammation in liver tissue, in a diet-induced model of hepatic fibrosis when administered with VLA-dCNP or long-acting CNP. [Figure 18A] Figure 18A is a bar graph showing significant improvement in renal function based on a decrease in serum creatinine when VLA-dCNP or long-acting CNP are administered. [Figure 18B] Figure 18B is a bar graph showing a significant improvement in renal function based on a decrease in urinary albumin levels, calculated by the albumin / creatinine ratio, when VLA-dCNP or long-acting CNP were administered. [Figure 18C]Figure 18C is a bar graph showing a significant reduction in renal fibrosis area (%) when VLA-dCNP or long-acting CNP were administered. Fibrosis area was measured using Image J (NIH, Bethesda, Maryland, USA). [Figure 18D] Figure 18D shows a series of representative images of kidney staining with Masson's trichrome (MT). The magnification is ×20. With this Masson's trichrome stain, the nucleus is stained with iron hematoxylin (brown / black in the image), the cytoplasm with acid fuchsin (pink / red in the image), and the collagen fibrillation region with aniline blue (blue in the image). [Figure 19A] Figure 19A is a bar graph showing a significant reduction in fibrosis based on a decrease in hydroxyproline, a major component of collagen in lung tissue, when VLA-dCNP or long-acting CNP are administered. [Figure 19B] Figure 19B is a bar graph showing a significant reduction in fibrotic area (%) in the lung, based on the quantitative evaluation of Masson's trichrome histological staining of lung tissue sections, when VLA-dCNP or long-acting CNP were administered. Fibrotic area was measured using Image J (NIH, Bethesda, Maryland, USA). [Figure 19C] Figure 19C shows a series of representative images of a kidney stained with Masson's trichrome (MT) at a magnification of 20x. [Figure 20] This graph shows plasma CNP levels [mean (SEM); n=5] in CD-1 mice after subcutaneous administration of 2.0 mg / kg of CNP derivative s1 (dCNP-s1) and CNP derivative s2 (dCNP-s2). The inset shows the low plasma CNP levels (diamonds) when native CNP is administered. Error bars represent the standard error of the mean of the n=5 plasma samples. The baseline CNP level before administration was 0.391 (0.02) ng / mL [mean (SEM); n=10]. The long-acting dCNP-s1 and dCNP-s2 provide 10 times higher blood CNP levels compared to native CNP when administered at similar dose weight / kg doses, for a continuous period (at least 8 hours). [Modes for carrying out the invention]

[0021] This disclosure relates to the treatment of lung, hepatic, and / or renal disorders, or symptoms associated with lung, hepatic, and / or renal disorders, such as acute lung injury (ALI), and prevention of more severe forms, namely acute respiratory distress syndrome (ARDS) and its progression to death or pulmonary / hepatic / renal fibrosis, by administering a therapeutically effective dose of a long-acting C-type natriuretic peptide (CNP), CNP derivative, long-acting CNP derivative, or long-acting CNP receptor (NPRB) agonist to a subject in need. This disclosure also relates to the treatment of hypoxemia, elevated inflammatory cell levels in the lungs, pulmonary edema, sepsis, bacteremia, and / or fibrosis (e.g., non-cardiovascular causes of hypoxemia, elevated inflammatory cell levels in the lungs, pulmonary edema, and / or fibrosis).

[0022] This disclosure also relates to the treatment of fibrosis in general, including pulmonary fibrosis, hepatic fibrosis, cirrhosis, and renal glomerulosclerosis, as well as the treatment / protection of renal impairment, which involves administering a therapeutically effective dose of the disclosure as a bolus without lowering blood pressure by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of a baseline blood pressure measurement, where the baseline blood pressure measurement is the mean blood pressure before administration of the composition, and 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 hour to After 84 hours, 2-84 hours, 4-84 hours, 12-84 hours, 1-168 hours, 2-168 hours, 4-168 hours, or 12-168 hours, the plasma cyclic GMP level increases to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times the baseline plasma cyclic GMP level), in which case the baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0023] Unlike conventional methods for treating ALI and sepsis, a therapeutically effective amount of the composition of the present disclosure can be administered as a bolus before, during, and / or after any injury that may lead to acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pulmonary edema, elevated levels of inflammatory cells in the lungs, increased levels or expression of inflammatory cytokines in the lungs (compared to healthy lungs), increased levels of proteins in the alveolar space (compared to healthy lungs), hypoarterial oxygenation (where hypoarterial oxygenation is a blood PaO2 of less than 60 mmHg and / or blood hemoglobin oxygen saturation (SpO2) of less than 90%), sepsis, bacteremia pneumonia, pulmonary / pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), or interstitial lung disease (ILD), without lowering blood pressure by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of a baseline blood pressure measurement, where baseline blood pressure measurement is the mean blood pressure before administration of the composition. A therapeutically effective dose of the composition of this disclosure also increases the plasma cyclic GMP level to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours), where the baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject). In some embodiments, a therapeutically effective dose of the composition of this disclosure may be administered as a bolus after an injury that may lead to the aforementioned disease condition. In some embodiments, a therapeutically effective dose of the composition of this disclosure may be administered as a bolus before an injury that may lead to the aforementioned disease condition. In some embodiments, the therapeutically effective dose of the Disclosure may be administered as a bolus during an impairment that may lead to the aforementioned disease state. Unlike conventional methods of continuous administration, bolus administration of the compositions herein offers advantages such as ease of administration and results in an unexpected reduction in undesirable side effects (such as hypotension).

[0024] definition In various parts of this specification, substituents of the compounds of the disclosure are disclosed in groups or ranges. It is expressly intended that this disclosure includes all individual subcombinations of members of such groups and ranges. For example, "C 1~6 The term "alkyl" is explicitly intended to disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl individually.

[0025] In this specification, single-letter codes for amino acids are used. For example, alanine is A, arginine is R, asparagine is N, aspartic acid is D, cysteine ​​is C, glutamic acid is E, glutamine is Q, glycine is G, histidine is H, isoleucine is I, leucine is L, lysine is K, methionine is M, phenylalanine is F, proline is P, serine is S, threonine is T, tryptophan is W, tyrosine is Y, valine is V, and γE is glutamic acid (where the R group (i.e., side chain) carbonyl (gamma, γ) is not an alpha-carbonyl but a portion used to link to any of the primary amino groups of a peptide or to the N-terminal portion of a peptide). For the purposes of this application, the single-letter code for an amino acid includes the L and / or D amino acid stereoisomers. When amino acids combine to form a peptide, it is understood that an amino acid is referred to as an amino acid residue from which the water element has been removed. Furthermore, when this disclosure refers to an amino acid in a peptide sequence, it is understood that it refers to an amino acid residue.

[0026] As used herein, the term “aliphatic” refers to a compound or group comprising carbon and hydrogen linked to one another by a linear, branched, or non-aromatic ring. Aliphatic compounds or aliphatic groups may be saturated (e.g., alkanes, e.g., hexane and other alkanes, alkyls, e.g., hexyl and other alkyls) or unsaturated (e.g., hexane and other alkenes, as well as alkynes, hexenyl and other alkenyls, as well as alkynyls). Aliphatic compounds and aliphatic groups (e.g., alkyls, alkenyls, or alkynyls) may be substituted with, for example, 1, 2, 3, 4, 5, 6, 7, or 8 substituents (e.g., (=O), hydroxyl, carboxyl, carbonyl, and / or ester groups). For example, an aliphatic group may have a carboxyl group as a substituent as a pendant group and / or at its terminal. When an aliphatic group is part of a compound, it is understood that this aliphatic group may be covalently bonded to the compound via chemical linkages, for example, carbonyl (also represented as C=O, C(O) or C(=O)) (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond, or similar. It is understood that the number of carbons in the aliphatic chain includes the skeletal carbons in the chemical linkages. For example, a saturated C8 aliphatic chain containing a C(=O) linkage may be represented as CH3(CH2)6C(=O) in the straight chain case. As another example, a saturated C8 aliphatic chain having a carboxyl group at the first end and a C(=O) linkage at the second end may be represented as HOC(=O)(CH2)6C(=O) in the straight chain case. For example, a saturated C18 aliphatic chain containing a C(=O) linkage may be represented as CH3(CH2) 16 It can be represented as C(=O). As another example, a saturated C18 aliphatic chain having a carboxyl group at the first end and a C(=O) linkage at the second end can be represented as HOC(=O)(CH2) in the case of a straight chain. 16 It can be represented as C(=O). The aliphatic group may be derived from a fatty acid, and / or the aliphatic group may be derived from a dibasic acid.

[0027] As used herein, the term "alkyl" refers to a linear (e.g., linear) or branched saturated hydrocarbon group. Exemplary alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. Alkyl groups may contain 1 to about 30, 1 to about 24, 2 to about 24, 1 to about 20, 2 to about 20, 1 to about 10, 1 to about 8, 1 to about 6, 1 to about 4, or 1 to about 3 carbon atoms.

[0028] As used herein, the term “fatty acid” refers to an aliphatic chain substituted with a carboxyl group, which may be either saturated or unsaturated. Examples of fatty acids include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, and / or lignoceric acid.

[0029] As used herein, the term “fatty acid ester” refers to a long aliphatic chain (saturated or unsaturated) having a -C(=O)O- moiety at the end of the chain.

[0030] As used herein, the term “fatty acid amide” refers to a long aliphatic chain (saturated or unsaturated) having a -C(=O)NR- moiety at the end of the chain.

[0031] As used herein, the terms “individual,” “subject,” or “patient” are interchangeable and refer to any animal, such as mammals (preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, most preferably humans).

[0032] As used herein, the term “therapeutably effective amount” means an amount of therapeutic agent (i.e., drug or therapeutic composition) that excites a biological or medical response desired by researchers, veterinarians, physicians, or other clinicians in a tissue, system, animal, individual, or human, and such response may include one or more of the following: (1) To prevent disease; for example, to prevent disease, condition or disorder in individuals who may have a predisposition to such disease, condition or disorder but who have not yet experienced or shown any pathology or symptoms of the disease; (2) To suppress a disease; for example, to suppress a disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of a disease, condition, or disorder; and (3) To induce remission of a disease; for example, in an individual experiencing or exhibiting the pathology or symptoms of a disease, condition, or disorder, to induce remission of the disease, condition, or disorder (i.e., to reverse the pathology and / or symptoms), for example, to reduce the severity of the disease, to prolong survival, and / or to prevent death.

[0033] As used herein, the term “bolus dose” refers to a single dose of a drug or other substance administered over a short period of time, for example, less than 10 minutes (e.g., less than 8 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute). In some embodiments, the bolus dose is administered in less than 5 minutes. In some embodiments, the bolus dose is administered in less than 3 minutes. In some embodiments, the bolus dose is administered in less than 1 minute. Administration may include one of the following: injection into any part of the body (e.g., but not limited to, intravascular, subcutaneous, intrathecal, or intradermal); oral (as a dosage form); inhalation (e.g., inhalation by intratracheal inhalation, where the subject is exposed to a high aerosol concentration so that the pharmacoactive ingredient is directly deposited in the lower respiratory tract); or nasal (e.g., as an aerosol, liquid, or powder).

[0034] As used herein, the terms “hypertension reduction,” “blood pressure decrease,” or “low blood pressure” are interchangeable and refer to a statistically significant decrease in a subject’s blood pressure below baseline. Baseline blood pressure is the mean arterial pressure measured before treatment or administration of any drug to a subject, or the mean arterial pressure of a normal, healthy subject. The standard deviation of most blood pressure monitors can be 5–15%, depending on the measurement method and the subject’s posture, mental state, or activity during measurement. For clarity herein, a change in blood pressure is expressed as a statistically significant increase, decrease, or decrease in blood pressure from the mean / average baseline blood pressure before administration of the drug or test substance. Statistically significant means P < 0.05, as is known to those skilled in the art of statistics.

[0035] As used herein, the term "C-type natriuretic peptide" or "CNP" refers to a peptide containing 22 amino acid residues, having a 17-amino acid ring structure formed by disulfide bonds and an additional 5-amino acid residue extension at the N-terminus (GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 10]; where letters follow conventional amino acid nomenclature, amino acid residues C-6 (position 6) and C-22 (position 22) are linked by disulfide bonds). See, for example, Sudoh et al., Biochem. Biophys. Res. Commun. 1989; 159: 1427-1434.

[0036] As used herein, the terms “NPRB receptor,” “Natriuretic peptide receptor B (NPRB),” “NPR2,” “Guanylate cyclase B (GC-B),” or “Natriuretic peptide receptor 2 type B” (NPR2) are interchangeable. In humans, the NPRB receptor is encoded by the NPR2 gene, located on chromosome 9, and on chromosome 4 in mice. See, for example, Nuglozeh et al., Genome. 1997;8:624-625. NPRB expression has been reported in various organs (e.g., heart, brain, uterus, ovaries, kidneys, lungs, liver, and adipocytes), as well as in some cancers. Schulz et al., Cell. 1989; 58: 1155-1162; Nagase et al., J. Hypertens. 1997; 15: 1235-1243; Chrisman, et al., J. Biol. Chem. 1993; 268: 3698-3703. NPRB is selectively activated by CNP but not by ANP or BNP (other known natriuretic peptides). Ubiquitous expression of NPRB plays a role in many physiological functions. Another natriuretic peptide receptor, NPRA, is activated by physiological concentrations of ANP and BNP, but NPRA is not activated by CNP. The difference in the physiological consequences of activation of either the NPRA or NPRB receptor was unclear prior to this disclosure, which makes the method of this disclosure non-obvious and inventive.

[0037] As used herein, the terms “long-acting C-type natriuretic peptide” or “long-acting CNP” refer to CNP formulations in which, when administered as a single bolus dose to mammalian subjects (humans, non-humans, primates, dogs, rats, mice, etc.), the resulting elevation of plasma CNP levels or plasma cyclic GMP levels above baseline persists for a duration of more than 4 hours or more than 6 hours, depending on the species. Long-acting C-type natriuretic peptide or long-acting CNP encompasses ultra-long-acting C-type natriuretic peptide or ultra-long-acting CNP. The elevation of plasma cyclic GMP is either a result of the CNP structural activity itself or originates from a combination of CNP and one or more components of the formulation containing this CNP. Presence (or elevation) in plasma means a detectable presence above the analytical baseline, where the baseline level is measured in the absence of administration of the long-acting CNP formulation. The duration of the sustained elevation of plasma cyclic GMP refers to the duration of the biological activity of the CNP formulation. CNP preparations refer to compositions comprising CNP peptides and one or more excipients or carriers (e.g., polymers, proteins, sugars, surfactants, and / or buffers). The CNP in CNP preparations may or may not be covalently linked to the excipients or carriers. The sustained presence in the blood can be evaluated by pharmacokinetic / pharmacodynamic analysis after administration.

[0038] As used herein, a formulation containing "ultra-long-acting C-type natriuretic peptide" or "ultra-long-acting CNP" refers to a long-acting CNP formulation containing 22 amino acid residues that, when administered to a subject as a single bolus dose, exhibits continuous presence in plasma for 24 hours or more (e.g., up to 2-3 days, or up to 1-4 weeks) or a continuous elevation of plasma cyclic GMP above baseline. Therefore, ultra-long-acting C-type natriuretic peptide or ultra-long-acting CNP is a subset of long-acting C-type natriuretic peptide or long-acting CNP. Presence in plasma means detectable presence exceeding endogenous natural agonists normally produced by the subject or exceeding analytical baseline levels in the absence of administration of a therapeutic CNP formulation. The duration (i.e., length of time) of plasma cyclic GMP elevation or the presence of detectable CNPs above baseline may be 24–192 hours, 24–48 hours, 48–72 hours, 72–96 hours, 96–120 hours, 120–144 hours, 144–168 hours, or 168–192 hours. As described above, CNP formulations are compositions comprising CNP peptides and one or more excipients or carriers (e.g., polymers, proteins, sugars, surfactants, and / or buffers). CNPs in CNP formulations may or may not be covalently linked to the excipients or carriers. Continued presence in the blood can be evaluated by pharmacokinetic / pharmacodynamic analysis after administration.

[0039] As used herein, the term “long-acting CNP derivative” means a CNP derivative that, when administered as a single bolus dose to a mammalian subject or patient, exhibits sustained presence in plasma for more than 4 hours or more than 6 hours, depending on the species, or a sustained elevation of plasma cyclic GMP above baseline. Long-acting CNP derivatives encompass ultralong-acting CNP derivatives. Long-acting properties may derive from the CNP derivative structure itself or from a combination of the CNP derivative and one or more components of a formulation containing this CNP derivative. Presence in plasma or blood means detectable presence exceeding endogenous natural agonists normally produced by mammals or above analytical baseline levels in the absence of administration of a therapeutic compound, peptide, protein, or formulation. Sustained presence in blood may be assessed by post-administration pharmacokinetic / pharmacodynamic analysis. In some embodiments, the CNP derivative is a modified CNP having at least 72% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) sequence homology or sequence identity with respect to the natural CNP. In some embodiments, the CNP derivative is an addition derivative in which the natural CNP is modified by the covalent addition of a chemical moiety (e.g., one or more additional amino acids and / or fatty acids and / or any chemical moiety) at the N-terminus, C-terminus, or R-group of any amino acid residue in the CNP peptide. In some embodiments, the CNP derivative may be a substitution derivative in which 1 to 6 amino acid residues (or 5 to 28% of the amino acid residues) in the natural CNP are replaced with different amino acid residues or non-natural amino acid residues. In certain embodiments, the CNP derivative may be a subtraction derivative in which 1 to 6 amino acid residues (or 5 to 28% of the amino acid residues) in the natural CNP are deleted. In certain embodiments, the CNP derivative may be a subtractive derivative in which 1 to 6 amino acid residues (or 5 to 28% of amino acid residues) in the natural CNP are deleted and / or substituted. A CNP derivative formulation refers to a composition comprising a CNP derivative and one or more excipients or carriers (e.g., polymers, proteins, sugars, surfactants, or buffers).

[0040] As used herein, the term “ultra-long-acting CNP derivative” refers to a long-acting CNP derivative or CNP derivative that, when administered as a single bolus dose to a mammalian subject or patient, exhibits continuous presence in plasma for a duration of 24 hours or more, or a continuous elevation of plasma cyclic GMP above baseline. Therefore, ultra-long-acting CNP derivatives are a subset of long-acting CNP derivatives. Ultra-long-acting CNP derivatives may originate from the CNP derivative structure itself, or from a combination of a CNP derivative and one or more components of a formulation containing this CNP derivative. Presence in plasma refers to detectable presence above baseline plasma levels for analysis in the absence of administration of the ultra-long-acting CNP derivative. The duration of plasma cyclic GMP elevation above baseline or the presence of a detectable CNP derivative may be 24–192 hours, or 24–48 hours, or 48–72 hours, or 72–96 hours, or 96–120 hours, or 120–144 hours, 144–168 hours, or 168–192 hours. Continued presence in the blood can be assessed by post-administration pharmacokinetic / pharmacodynamic analysis. In some embodiments, the CNP derivative may be a modified CNP having 72% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) sequence identity to the native CNP. In some embodiments, the CNP derivative is an addition derivative in which the native CNP is modified by the covalent addition of a chemical moiety (e.g., an additional amino acid, and / or a fatty acid, and / or any chemical moiety) at the N-terminus, C-terminus, or R-group of any amino acid residue in the CNP peptide. In some embodiments, the CNP derivative is a substituted derivative in which 1 to 6 amino acid residues (or 5 to 28% of the amino acid residues) in the natural CNP are replaced with different amino acid residues or non-natural amino acid residues. In certain embodiments, the CNP derivative is a subtractive derivative in which 1 to 6 amino acid residues (or 5 to 28% of the amino acid residues) in the natural CNP are deleted.In certain embodiments, the CNP derivative may be a subtractive derivative in which 1 to 6 amino acid residues (or 5 to 28% of amino acid residues) in the natural CNP are deleted and / or substituted. The CNP derivative formulation is a composition comprising the CNP derivative and one or more excipients or carriers (e.g., polymers, proteins, sugars, surfactants, or buffers).

[0041] As used herein, the terms “CNP formulation” or “CNP derivative formulation” refer to a composition comprising a CNP peptide or derivative thereof, which may or may not be covalently linked to an excipient or carrier (e.g., a polymer, protein, and / or lipid).

[0042] As used herein, the terms “NPRB agonist” or “NPR2 agonist” refer to any compound, peptide, or protein that does not contain a 22-amino acid CNP sequence in its structure and can bind to the cellular catalytic receptor NPRB, stimulating its intracellular guanylyl cyclase activity to increase intracellular cyclic GMP levels or blood cyclic GMP levels, but has limited or no ability to bind to and stimulate the NPRA receptor. Since not all cells express similar levels of NPRB, NPRB agonists are formulated to act primarily on cells expressing NPRB. This selectivity can be readily measured by those skilled in the art by measuring the activity in NPRB-expressing cells compared to the activity in NPRA-expressing cells.

[0043] As used herein, the term “long-acting NPRB agonist” refers to an NPRB agonist as defined above that, when administered as a single bolus dose to a mammalian subject or patient, exhibits a sustained presence in plasma for more than 4 hours or more than 6 hours, depending on the species, or a sustained elevation of plasma cyclic GMP above baseline. Long-acting NPRB agonists encompass ultra-long-acting NPRB agonists. The long-acting nature of an NPRB agonist may be derived from the NPRB agonist structure itself or from a combination of the NPRB agonist and one or more components of a formulation containing this NPRB agonist. Presence in plasma means a detectable presence above baseline levels for analysis in the absence of administration of the long-acting NPRB agonist. A long-acting NPRB agonist formulation, or a long-acting NPRB agonist formulation, is a composition comprising a long-acting NPRB agonist, or a composition comprising a long-acting NPRB agonist and one or more excipients or carriers (e.g., polymers, proteins, sugars, lipids, or buffers). The long-acting NPRB agonist may or may not be covalently linked to the excipient or carrier. The sustained presence in the blood can be evaluated by pharmacokinetic / pharmacodynamic analysis after administration. The sustained plasma elevation of cyclic GMP above baseline can be evaluated by pharmacodynamic analysis after administration.

[0044] As used herein, the term “ultra-long-acting NPRB agonist” refers to a long-acting NPRB agonist that, when administered as a single bolus dose to a mammalian subject or patient, exhibits continuous presence in plasma for 24 hours or more, or a continuous elevation of plasma cyclic GMP above baseline. Ultra-long-acting NPRB agonists are a subset of long-acting NPRB agonists. The ultra-long-acting nature of an NPRB agonist may be derived from the NPRB agonist structure itself, or from a combination of the NPRB agonist and one or more components of a formulation containing this NPRB agonist. Presence in plasma means a detectable presence above baseline levels for analysis in the absence of administration of the ultra-long-acting NPRB agonist. The duration of plasma cyclic GMP elevation above baseline or the presence of a detectable NPRB agonist may be 24–192 hours, 24–48 hours, 48–72 hours, 72–96 hours, 96–120 hours, 120–144 hours, 144–168 hours, or 168–192 hours. A formulation of an ultralong-acting NPRB agonist, or an ultralong-acting NPRB agonist formulation, refers to a composition containing an ultralong-acting NPRB agonist, or a composition containing an ultralong-acting NPRB agonist and one or more excipients or carriers (e.g., polymers, proteins, sugars, lipids, or buffers). The ultralong-acting NPRB agonist may or may not be covalently linked to the excipient or carrier. Continued presence in the blood can be evaluated by post-administration pharmacokinetic / pharmacodynamic analysis. The continuous presence of cyclic GMP in the blood can be evaluated by pharmacokinetic / pharmacodynamic analysis after administration. A continuous increase in plasma cyclic GMP above baseline can also be evaluated by pharmacodynamic analysis after administration.

[0045] As used herein, the phrase "NPRB agonist having limited or no agonist activity to NPRA" refers to an NPRB agonist having more than five times the binding affinity (or lower EC50) to NPRB compared to NPRA.

[0046] As used herein, the term “polymer” refers to a macromolecule that is primarily or entirely formed of many similar repeating units covalently bonded to one another. The term polymer includes cellulose derivatives, poly(ethylene glycol)(PEG), methoxypoly(ethylene glycol)(MPEG), poly(lactic acid-coglycolic acid), and poly(N-vinylpyrrolidone), as well as their derivatives. These polymers may be branched or linear. As used herein, polymers may be attached to peptides, proteins, or linker groups by amide, ester, ether, thioether, thioester, or carbamate bonds, or by linkers containing one of these bonds. Polymers may also be grafted onto each other to create protected graft copolymer excipients, which, when mixed with a pharmaceutically active ingredient, may enhance the pharmacokinetic and pharmacodynamic properties of the pharmaceutically active ingredient by increasing its presence in blood or plasma after in vivo administration.

[0047] As used herein, the term "amino acid" refers to an organic compound with a molecular weight of less than 500 Da, containing an amino functional group (-NH2) and a carboxyl functional group (-COOH), along with a side chain (R group) unique to each amino acid. The main components of an amino acid are carbon (C), hydrogen (H), oxygen (O), and nitrogen (N), although other components may be found in the side chains of certain amino acids. As of 1983, approximately 500 naturally occurring amino acids were known (however, only 20 are found in the mammalian genetic code, and these 20 amino acids are also referred to herein as "natural amino acids"). Amino acids can be alpha-amino acids, in which the amino group is directly bonded to the alpha carbon. Amino acids can be non-alpha-amino acids, in which the primary amino group is bonded to a carbon other than the alpha position. The alpha carbon is the carbon directly adjacent to the carboxyl group.

[0048] The terms “derivative” or “analog,” as used herein, include compounds whose core structure is identical to or similar to that of the parent compound, but which have chemical or physical modifications such as different or additional groups, and include copolymers of the parent compound that can be linked to other atoms or molecules. The terms also include peptides or proteins that have at least 72% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) sequence identity with the parent peptide or protein. The terms also include peptides that have additional groups attached to them, such as additional labels or tags, compared to the parent peptide. The terms also include polymers that have additional groups attached to them, such as alkoxy or methoxy groups, compared to the parent polymer.

[0049] As used herein, “additional derivative” or “expanded derivative” refers to a peptide derivative in which the main skeletal amino acid sequence of the peptide remains the same, but an additional functional group and / or amino acid residue is added to the main skeletal amino acid sequence using one or more reactive moieties in the main skeletal amino acid sequence, thereby producing an additional or expanded derivative. An additional or expanded derivative is different from a truncated and / or substituted peptide derivative in which one or more amino acid residues in the main skeletal amino acid sequence of the peptide are removed and / or replaced with different functional groups and / or amino acids.

[0050] As used herein, the terms “linker group,” “linking group,” or “linker” refer to an atom or chemical part that covalently links or bonds two entities (e.g., parts of two molecules) to each other. For example, a linker precursor (e.g., an amino acid, peptide, or non-amino acid molecule obtained from a commercially available crosslinking agent) is reacted with two entities so that these two entities are linked to each other via a linker group. Once these two entities are linked to each other, the linker group is the part remaining from the linker precursor in the final linked entity. For example, when molecule A is linked to molecule B, the linker group may have two chemical functional groups, one of which reacts with A and the other with B to obtain “A-linker group-B.” In this case, the linker group is the part of the linker precursor remaining after the covalent linking of A and B.

[0051] As used herein, the term "polypeptide" refers to a polymer of amino acids.

[0052] As used herein, the term "peptide" refers to a polypeptide in which three or more amino acids are covalently linked to each other by amide bonds via alpha-amino and alpha-carboxyl groups. The number of amino acid residues in a peptide can range from 3 to approximately 100.

[0053] As used herein, the term "protein" refers to a polypeptide of sufficient size having a three-dimensional structure such as a β-barrel or α-helix.

[0054] As used herein, the term “antibody” refers to a protein produced by immune cells that recognize a specific antigen. Antibodies are proteins produced in response to a specific antigen in the blood and that counteract this antigen. Antibodies chemically bind to substances that the body recognizes as foreign, such as bacteria, viruses, and foreign bodies in the blood.

[0055] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species in which the protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans.

[0056] As used herein, the terms “subcutaneous administration,” “sc,” “sc administration,” “SC,” or “SC administration” refer to the direct delivery of a drug (usually in liquid form) into the adipose tissue just beneath the skin. This delivery is usually performed by direct injection. This injection is shallower than injections into muscle tissue. Healthcare providers often use subcutaneous injection for drugs that are suitable for slow and steady absorption into the bloodstream.

[0057] As used herein, the terms “intravenous administration,” “IV administration,” or “IV injection” refer to the direct delivery of a drug (typically in liquid form) into a vein in an animal or human. This method of delivery is usually by direct injection. Intravenous administration routes can be used for both injections using a syringe at relatively high pressure and infusions using pressure provided by, for example, gravity.

[0058] As used herein, the terms “intramuscular administration,” “IM ​​administration,” or “IM injection” refer to the direct intramuscular delivery of a drug (usually in liquid form) into the muscle of an animal or human. This delivery is usually by direct injection, which allows for rapid absorption of the drug into the bloodstream. In some cases, IM injections may be self-administered. In some embodiments, for example, if a particular therapeutic agent irritates a vein, or if a suitable vein cannot be found, IM injection may be used instead of intravenous injection.

[0059] As used herein, the term “intranasal administration” refers to the delivery of a therapeutic agent (e.g., in the form of a gel, liquid, aerosol, gas, or powder) to the nose of an animal or human by topical application, dripping as a liquid, or gas injection (or blowing or spraying). Depending on the formulation, this mode of administration may deliver the therapeutic agent, for example, to the nasal cavity or lungs (depending on the equipment used) and / or not be absorbed systemically (pure topical administration), and / or be completely absorbed systemically (pure systemic), and / or be more frequently partially absorbed (both topical and systemic). Examples of topical agents for nasal sprays include decongestants for the treatment of colds and allergies, the systemic effects of which are typically minimized. Examples of systemic agents available as nasal sprays include, for example, migraine medications, nicotine replacements, and hormonal agents.

[0060] As used herein, the terms “parenteral” or “non-gastrointestinal” administration refer to routes of administration that do not involve the intestinal or gastrointestinal pathway. Examples of parenteral administration include: subcutaneous (under the skin), intravenous (in the intestinal veins), intraarterial (in the arteries), intramuscular (in the muscles), intraperitoneal (injection or administration into the peritoneal cavity), inhalation (e.g., inhalation via intratracheal inhalation, where the subject is exposed to a high aerosol concentration of the active ingredient so that the active ingredient is directly deposited in the lower respiratory tract), nasal administration (administration through the nose), sublingual and buccal drug application, intrathecal (in the spinal canal), intracerebral (in the cerebrum), intraventricular (in the ventricles), intradermal (in the skin itself), or any other route of administration that does not involve the gastrointestinal tract. As used herein, the term “enteral” means administration to any region of the gastrointestinal tract, including the mouth (oral cavity), pharynx (throat), esophagus, stomach, small intestine, large intestine, rectum, and anus, and administration through an artificial opening in any of these regions.

[0061] As used herein, the terms “therapeutic agent,” “drug,” or “pharmaceutical active ingredient” refer to a substance or molecule that can produce a therapeutic effect on a disease condition.

[0062] As used herein, the term “excipient” refers to a substance formulated or mixed with a pharmaceutically active ingredient to increase the volume of a formulation containing a small amount of potent active ingredient for the purpose of long-term stabilization (and therefore often referred to as “volume extender,” “filler,” or “diluent”), and / or to impart therapeutic enhancement to the pharmaceutically active ingredient in the final dosage form, for example, to promote drug absorption and / or potency / dose, to reduce viscosity, to increase solubility, and / or to prolong the action or presence of the pharmaceutically active ingredient in the blood. The selection of an appropriate excipient depends on the route of administration and dosage form, the pharmaceutically active ingredient, and other factors. Examples of excipients may include: sugars, amino acids, buffers, antioxidants, chelating agents, solvents or vehicles, and / or composite polymers that bind to and stabilize the pharmaceutically active ingredient in vitro and / or in vivo. Excipients were once considered “inactive” components, but it is now understood that they can be “important determinants of dosage form performance.” In other words, the influence of excipients on pharmacokinetics and efficacy may be significant and may require extensive investigation and research. How excipients affect the delivery of active pharmaceutical ingredients is often unpredictable.

[0063] As used herein, the term “treatment” refers to the procedures performed after a diagnosis of a condition.

[0064] As used herein, the term “mitigation” refers to procedures taken to prevent or reduce the likelihood of an anticipated injury or illness.

[0065] As used herein, the term “healthy subject” refers to an individual (human and / or mammal) participating in a research study who has no significant health problems. For the purposes of this disclosure, these are individuals without lung, liver, and / or kidney disease, but within the same age range as individuals with lung, liver, and / or kidney disease, as assessed by a person skilled in the art (physician and / or clinician). As an example, a healthy human adult subject with healthy lungs may have an average vital capacity of approximately 4.8–7.2 L as measured by spirometry, an arterial hemoglobin saturation of 95–100% and / or a blood oxygen level of 80–100 mmHg, and an arterial carbon dioxide level of approximately 35–45 mmHg. Healthy human subjects with a healthy liver may have: approximately 60–83 g / L of total plasma protein, approximately 34–54 g / L of albumin, approximately 0–12 mg / L of total bilirubin in adults (0–10 mg / L for those under 18), approximately 0–3 mg / L of conjugated direct bilirubin, approximately 44–147 international units (IU / L) or 0.73–2.45 microcatars (μkat / L) of serum alkaline phosphatase (ALP) per liter in adults, but approximately twice that amount in children under 18, approximately 5–40 U / L of aspartate aminotransferase (AST), and approximately 7–56 U / L of serum alanine aminotransferase (ALT). Healthy human subjects with healthy kidneys may have renal panel results that do not deviate from the following parameters: glomerular filtration rate greater than 60 mL / min / 1.73 m², serum creatinine of approximately 5.0–15 mg / L (variing by approximately 20% depending on the assay used), serum urea nitrogen (BUN) of 70–240 mg / L, BUN:creatinine of approximately 6–25, serum sodium of approximately 135–145 mM, serum potassium of approximately 3.6–5.2 mM, chloride of approximately 98–112 mM, bicarbonate of approximately 17–29 mM, anion gap of 7–15, and phosphorus of 43–45 mg / L. Furthermore, generally healthy human subjects have a resting heart rate range of 50–90 beats / min in humans, but a wider range is considered acceptable unless there is a sign of thyroid dysfunction or other known serious health problems.

[0066] As used herein, the term "increase in total protein in the lung" or "increase in total protein in BALF" refers to an increase in the protein concentration in bronchoalveolar lavage fluid (BALF) of at least 1.5-fold compared to BALF from healthy control subjects measured in the same manner. This level can be up to 4-fold (e.g., up to 2-fold, up to 3-fold, or up to 4-fold) compared to healthy control subjects.

[0067] As used herein, the term "increase in the level of inflammatory cytokines in BALF" refers to an increase in the concentration of inflammatory cytokines (e.g., IL-6, TNFα (TNF-a), MCP-1, IL-1b) in BALF of at least 4-fold compared to BALF from normal healthy control subjects measured in the same manner. This level can be up to 10-fold to 100-fold (e.g., up to 20-fold, up to 30-fold, up to 40-fold, up to 50-fold, up to 60-fold, up to 70-fold, up to 80-fold, up to 90-fold, or up to 100-fold) compared to the BALF of healthy control subjects.

[0068] As used herein, "liquid" refers to a substance that freely flows at room temperature and as a result, changes shape like, for example, water or oil, but retains a constant volume.

[0069] As used herein, "room temperature" refers to a typical indoor ambient temperature of about 25°C.

[0070] Unless otherwise defined, any feature within any aspect or embodiment of this disclosure may be combined with any feature within any other aspect or embodiment of the invention, and such combinations are incorporated herein. This also applies to, but is not limited to, the endpoints of the scope disclosed herein. For example, where it is disclosed that a given substance is present in a composition in a concentration range of X to Y% or A to B%, this disclosure is understood to expressly disclose not only the ranges X to Y% and A to B%, but also the ranges X to B%, A to Y%, and, where numerically, Y to A% and B to X%. Each of these ranges, and any combination of ranges, is intended and intended to be understood as being directly and uniquely disclosed herein.

[0071] Unless otherwise specified, any range designation in this application using two parenthetical values ​​X and Y or a hyphen ("-") separating two parenthetical ratios shall be understood to mean and disclose a range that includes both endpoint values ​​X and Y. The same applies to ranges expressed as "X~Y". Thus, "X~Y (XY)" and "X~Y of (of The range expressions "X to Y)", "X~Y (from X to Y)", "X~Y of XY", and "X~Y (from XY)" shall be understood as equivalent to meaning and disclosing a range that includes the endpoint X, all values ​​between X and Y (including decimals), and the endpoint Y.

[0072] As used herein, the term “approximately” when referring to a particular value (e.g., the endpoint of a range) includes and discloses a certain variation around that particular enumerated value, in addition to the value itself. Such variation may arise, for example, from normal measurement variations (e.g., measurement variations in weighing or distributing various substances by methods known to those skilled in the art). The term “approximately” is understood to include and disclose a range of variation above and below the indicated particular value, such as “approximately” may include and disclose a variation of ±5.0%, where the percentage value is relative to the particular enumerated value itself. The term “approximately” may include and disclose a variation of ±4.5%. The term “approximately” may include and disclose a variation of ±4.0%. The term “approximately” may include and disclose a variation of ±3.5%. The term “approximately” may include and disclose a variation of ±3.0%. The term “approximately” may include and disclose a variation of ±2.5%. The term "approximately" may encompass and disclose a variation of ±2.0%. The term "approximately" may encompass and disclose a variation of ±1.5%. The term "approximately" may encompass and disclose a variation of ±1.0%. The term "approximately" may encompass and disclose a variation of ±0.5%. The term "approximately" with respect to a specific mentioned value may encompass and disclose the exact specific value itself, regardless of any explicit indication that the exact specific value is included. Even without explicit indication that the term "approximately" includes a specific exact mentioned value, the exact specific value is still included within the range of variation resulting from the term "approximately," and is therefore disclosed in this disclosure. Unless otherwise specified, if the term "approximately" is listed before the first endpoint of a numerical range but not before the second endpoint of that range, the term and the variation implied by the term in the range and disclosure refer to both the first endpoint and the second endpoint of that range. For example, a range listed as "approximately X to Y" should be interpreted as "approximately X to approximately Y". The same applies to ranges listed as ratios.For example, the listed range of weight ratios, "approximately X:Y to A:B," is to be read as the weight ratio "(approximately X):(approximately Y) to (approximately A):(approximately B)."

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification (e.g., definitions) shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to limit the scope. It will be readily apparent that the aspects of this disclosure described and illustrated herein may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly intended herein.

[0074] Furthermore, the specific arrangement shown in the figure should not be considered limiting. It should be understood that other embodiments may include more or fewer of each element shown in the given figure. Moreover, some of the shown elements may be combined or omitted. Furthermore, exemplary embodiments may include elements not shown.

[0075] Treatment method This disclosure features methods of treatment, such as methods for treating subjects (e.g., mammalian subjects, patients) having lung, liver, and / or kidney disorders, or conditions or symptoms associated with lung, liver, and / or kidney disorders. Examples of lung, liver, and / or kidney disorders, or conditions or symptoms associated with lung, liver, and / or kidney disorders, include: acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pulmonary edema, elevated levels of inflammatory cells in the lungs, increased levels or expression of inflammatory cytokines in the lungs (compared to healthy lungs), elevated levels of proteins in the alveolar space (compared to healthy lungs), hypoarterial oxygenation (hypoarterial oxygenation is defined as blood PaO2 less than 60 mmHg and / or blood hemoglobin oxygen saturation (SpO2) less than 90%), sepsis, bacteremia, pneumonia, fibrosis (e.g., pulmonary, hepatic, or renal fibrosis), and / or kidney disorders. In some embodiments, lung, liver, and / or kidney damage, or symptoms associated with lung, liver, and / or kidney injury, may include, for example: acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pulmonary edema, elevated levels of inflammatory cells in the lungs, increased levels or expression of inflammatory cytokines in the lungs (compared to healthy lungs), elevated levels of alveolar proteins (compared to healthy lungs), hypoarterial oxygenation (hypoarterial oxygenation is defined as blood PaO2 less than 60 mmHg and / or blood hemoglobin oxygen saturation (SpO2) less than 90%), pneumonia, fibrosis, and / or kidney damage. Subjects may have hypoarterial oxygenation, defined as blood PaO2 less than 60 mmHg and / or blood hemoglobin oxygen saturation (SpO2) less than 90%.

[0076] This disclosure also features methods for treating fibrosis in general, such as pulmonary fibrosis, hepatic fibrosis, cirrhosis, and renal glomerulosclerosis, and methods for providing treatment for or protection against renal impairment. The method comprises administering to a subject a therapeutically effective bolus dose of a composition containing a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, and / or an ultra-long-acting NPRB agonist. The therapeutically effective bolus dose is a dose that does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement (baseline blood pressure measurement being the mean blood pressure before administration of the composition), and moreover, the above dose is administered within 1 to 12 hours (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 hour) The plasma cyclic GMP level after ~168 hours, 2~168 hours, 4~168 hours, or 12~168 hours) is increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the plasma level before administration of the bolus dose, i.e., the plasma level of a healthy subject, which is 4+ / -1 pmol / ml or approximately 1.4 mg / ml in humans (however, this may vary by species). For example, Shotan et al., Plasma cyclic guanosine monophosphate in chronic heart failure: hemodynamic and neurohormonal, which is incorporated in whole herein. See correlations and response to nitrate therapy. Clin Pharmacol Ther, 1993. 54(6): pp. 638-44. In a preferred embodiment, this baseline level is a pre-administration measurement level for the same subject receiving the treatment, and this level may vary from subject to subject. In the implementation of the Disclosure, any baseline parameter used as a reference parameter for evaluating the effect of the treatment is established by pre-treatment measurement. Typically, but not limited to, baseline plasma cyclic GMP levels fluctuate throughout the day, being relatively low during daytime wakefulness and relatively high immediately after bedtime, and in humans, they can fluctuate between 2 and 8 pmol / ml throughout the day. Thus, baseline plasma cyclic GMP levels measured before administration of the composition and plasma cyclic GMP levels measured after administration of the composition of the Disclosure can be obtained at the same predetermined time each day. Where a mean baseline is described, this mean baseline may be an mean baseline measurement obtained at least three times at intervals of at least 4 hours with respect to a given parameter within 24 hours for a given subject. This controls for inter-subject or inter-individual variability. In patients with congestive heart failure, baseline plasma cyclic GMP levels can be 2-3 times higher, and this baseline is established pre-treatment for each individual subject or group. Similarly, for blood pressure, the baseline is the measurement level before drug administration and is used as a criterion for evaluating the effectiveness of the treatment. Baseline cGMP levels in healthy mice with no known symptoms of any health condition are 20(3.7) pmol / mL [mean (SEM); n=8] or 7(1.3) ng / mL [mean (SEM); n=8]. Baseline cGMP levels in dogs with no known symptoms of any health condition are 5-12 ng / mL.

[0077] In some embodiments, administration of a therapeutically effective bolus dose of the composition to a subject further reduces the total number of cells and total protein in a BALF sample derived from the subject. In certain embodiments, administration of a therapeutically effective bolus dose of the composition to a subject further reduces MPO (activated neutrophil marker) in lung tissue derived from the subject compared to MPO before administration of the composition. In certain embodiments, administration of a therapeutically effective bolus dose of the composition to a subject further weakens inflammatory cytokine expression (e.g., IL-6, IL-1b, TNFα, MCP-1, and IFNg; these may be present, for example, in ARDS) compared to inflammatory cytokine expression before administration of the composition. In certain embodiments, administration of a therapeutically effective bolus dose of the composition to a subject reduces the fibrotic area (e.g., fibrotic area in pulmonary fibrosis, hepatic fibrosis, cirrhosis, and / or renal glomerulosclerosis) compared to the fibrotic area before administration of the composition, or provides treatment / protection against renal impairment. In certain embodiments, administration of a therapeutically effective bolus dose of the composition to a subject further reduces the fibrotic area of ​​the lungs in subjects with idiopathic pulmonary fibrosis compared to before administration of the composition. In some embodiments, administration of a therapeutically effective bolus dose of the composition to a subject further reduces cell count and protein levels, as well as the expression of one or any combination of IL-6, IL-1b, TNFα, MCP-1, and IFNg in subjects with idiopathic pulmonary fibrosis compared to before administration of the composition. In some embodiments, administration of a therapeutically effective bolus dose of the composition to a target reduces the expression of one or any combination thereof of the target AST, ALT, α-SMA, IL-6, IL-1b, TNFα, MCP-1, IFNg, iNOS, Elf-1, Tollip, IRAK-1, P-P38, P-P65, β-act, STAT1, P-STAT1, STAT2, STAT3, STAT6, fibrosis area, serum creatinine, urinary albumin / creatinine ratio, or hydroxyproline in the lungs.

[0078] In some embodiments, a therapeutically effective bolus dose does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement (baseline blood pressure measurement being the mean blood pressure before administration of the composition), and increases the plasma cyclic GMP level 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level in a healthy subject.

[0079] In some embodiments, the therapeutically effective bolus dose does not reduce blood pressure (or mean arterial pressure) by more than 15% of the baseline blood pressure measurement (baseline blood pressure measurement being the mean blood pressure before administration of the composition), and increases the plasma cyclic GMP level 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level in a healthy subject.

[0080] In some embodiments, a therapeutically effective bolus dose does not reduce blood pressure by more than 10% from the baseline blood pressure measurement (where the baseline blood pressure measurement is the mean blood pressure before administration of the composition), and the dose increases the plasma cyclic GMP level 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose or the mean plasma level in a healthy subject.

[0081] In some embodiments, the therapeutically effective bolus dose does not lower blood pressure by more than 5%, and the dose increases the plasma cyclic GMP level 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level in a healthy subject.

[0082] The methods of this disclosure are made possible by the remarkable discovery that CNP can be modified, derivatized, and / or formulated in such a manner that it can induce / induce an increase in cyclic GMP production and / or maximize it without associated adverse blood pressure reduction. In particular, the blood pressure effect of CNP can be minimized or eliminated with a therapeutic bolus dose that sustainably increases plasma cyclic GMP by more than 1.5 times baseline for more than 4 or 6 hours, depending on the administered peptide.

[0083] In some embodiments, the present disclosure features methods for treating subjects (e.g., mammalian subjects, patients in need thereof) who have lung, liver, and / or kidney disorders, or conditions or symptoms associated with lung, liver, and / or kidney disorders, such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pulmonary edema, elevated levels of inflammatory cells in the lungs, increased levels or expression of inflammatory cytokines in the lungs (compared to healthy lungs), elevated levels of alveolar proteins (compared to healthy lungs), hypoarterial oxygenation (hypoarterial oxygenation is defined as blood PaO2 less than 60 mmHg and / or blood hemoglobin oxygen saturation (SpO2) less than 90%), sepsis, bacteremia, pneumonia, fibrosis in general (e.g., pulmonary fibrosis, hepatic fibrosis, cirrhosis, and / or glomerulosclerosis), and / or kidney disorders. In some embodiments, lung, liver, and / or kidney disorders, or symptoms associated with lung, liver, and / or kidney disorders, include: acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pulmonary edema, elevated levels of inflammatory cells in the lungs, increased levels or expression of inflammatory cytokines in the lungs (compared to healthy lungs), elevated levels of alveolar proteins (compared to healthy lungs), hypoarterial oxygenation (hypoarterial oxygenation is defined as blood PaO2 less than 60 mmHg and / or blood hemoglobin oxygen saturation (SpO2) less than 90%), pneumonia, fibrosis in general (e.g., pulmonary fibrosis, hepatic fibrosis, cirrhosis, and / or glomerulosclerosis), and / or kidney disorders. In some embodiments, the subject may have hypoarterial oxygenation, defined as blood PaO2 less than 60 mmHg and / or blood hemoglobin oxygen saturation (SpO2) less than 90%. The method is described below.

[0084] In some embodiments, the method comprises administering a composition containing a long-acting CNP to a subject in a therapeutically effective bolus dose; wherein the therapeutically effective bolus dose does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement (the baseline blood pressure measurement being the mean blood pressure before administration of the composition), and does not cause such a decrease for 1 to 12 hours (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 hours) from the time of administration. The plasma cyclic GMP level after ~24 hours, 1 hour to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 hour to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) is sufficient to increase to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where baseline is the mean plasma level before administration of the composition or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level of the subject before administration of the composition). In some embodiments, the therapeutically effective bolus dose does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, or does not cause such a decrease (baseline blood pressure measurement being the mean blood pressure before administration of the composition), and moreover, 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours) The plasma cyclic GMP level after 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) can be increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0085] In some embodiments, the method comprises administering a composition containing an ultra-long-acting CNP to a subject in a therapeutically effective bolus dose; wherein the therapeutically effective bolus dose does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of a baseline blood pressure measurement (the baseline blood pressure measurement being the mean blood pressure before administration of the composition), and moreover, the effect lasts for 1 to 12 hours (e.g., 2 to 12 hours, 4 to 12 hours, 1 hour) from the time of administration. The plasma cyclic GMP level after ~24 hours, 2~24 hours, 4~24 hours, 1 hour~84 hours, 2~84 hours, 4~84 hours, 12~84 hours, 1 hour~168 hours, 2~168 hours, 4~168 hours, or 12~168 hours) is sufficient to increase the plasma cyclic GMP level to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where the cyclic GMP baseline is the mean plasma level before administration of the composition or the mean plasma level of a healthy subject. In some embodiments, the therapeutically effective bolus dose does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, or does not cause such a decrease (baseline blood pressure measurement being the mean blood pressure before administration of the composition), and moreover, 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours) The plasma cyclic GMP level after 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) can be increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0086] In some embodiments, the method comprises administering a composition comprising a long-acting CNP derivative to a subject in a therapeutically effective bolus dose; wherein the therapeutically effective bolus dose does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement (the baseline blood pressure measurement being the mean blood pressure before administration of the composition), and moreover, the effect lasts for 1 to 12 hours (e.g., 2 to 12 hours, 4 to 12 hours, 1 hour to) from administration. The composition is sufficient to increase plasma cyclic GMP levels after 24 hours, 2-24 hours, 4-24 hours, 1-84 hours, 2-84 hours, 4-84 hours, 12-84 hours, 1-168 hours, 2-168 hours, 4-168 hours, or 12-168 hours) to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where baseline is the mean plasma cyclic GMP level before administration of the composition or the mean plasma level for a healthy mammal. The therapeutically effective bolus dose is a dose that does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, or does not cause such a decrease (baseline blood pressure measurement is the mean blood pressure before administration of the composition); moreover, the above dose is effective within 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours). The plasma cyclic GMP level after 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) can be increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0087] In some embodiments, the method comprises administering a composition comprising an ultra-long-acting CNP derivative to a subject in a therapeutically effective bolus dose; wherein the therapeutically effective bolus dose does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement (the baseline blood pressure measurement being the mean blood pressure before administration of the composition), and moreover, the effect lasts for 1 to 12 hours (e.g., 2 to 12 hours, 4 to 12 hours, 1 hour to) from administration. The composition is sufficient to increase plasma cyclic GMP levels after 24 hours, 2-24 hours, 4-24 hours, 1-84 hours, 2-84 hours, 4-84 hours, 12-84 hours, 1-168 hours, 2-168 hours, 4-168 hours, or 12-168 hours) to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where baseline is the mean plasma cyclic GMP level before administration of the composition or the mean plasma level for a healthy mammal. The therapeutically effective bolus dose is a dose that does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement (baseline blood pressure measurement being the mean blood pressure before administration of the composition), and moreover, the above dose is effective within 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours). The plasma cyclic GMP level after 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) can be increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0088] In some embodiments, the method comprises administering a composition comprising a long-acting NPRB agonist to a subject in a therapeutically effective bolus dose; wherein the therapeutically effective bolus dose does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement (the baseline blood pressure measurement being the mean blood pressure before administration of the composition), and moreover, the effect lasts for 1 to 12 hours (e.g., 2 to 12 hours, 4 to 12 hours) from the time of administration. The treatment is sufficient to increase the plasma cyclic GMP level after 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where baseline is the mean plasma level before administration of the composition or the mean plasma level for a healthy mammal. The therapeutically effective bolus dose is a dose that does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, or does not cause such a decrease (baseline blood pressure measurement is the mean blood pressure before administration of the composition); moreover, the above dose is effective within 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours). The plasma cyclic GMP level after 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) can be increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0089] In some embodiments, the method comprises administering to a subject a therapeutically effective bolus dose of a composition comprising an ultra-long acting NPRB agonist; wherein the therapeutically effective bolus dose does not reduce blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, or cause such a reduction (the baseline blood pressure measurement is the mean blood pressure prior to administration of the composition), and moreover, the plasma cyclic GMP level 1 hour to 12 hours (e.g., 2 to 12 hours, 4 to 12 hours, 1 hour to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 hour to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 hour to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) after administration is increased to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where the baseline is the mean plasma level prior to administration of the composition, or the mean plasma level of a healthy mammalian subject. The therapeutically effective bolus dose is a dose that does not reduce blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, or cause such a reduction (the baseline blood pressure measurement is the mean blood pressure prior to administration of the composition); and moreover, the dose can increase the plasma cyclic GMP level 1 hour to 12 hours (e.g., 2 to 12 hours, 4 to 12 hours, 1 hour to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 hour to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 hour to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) after administration to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where the baseline plasma cyclic GMP level is defined as the mean plasma level prior to administration of the bolus dose, or the mean plasma level of a healthy subject (preferably, the mean plasma cyclic GMP level prior to administration of the composition to the subject).

[0090] The subjects may have hypoarterial oxygenation, which is defined as blood PaO2 less than 60 mmHg and / or blood hemoglobin oxygen saturation (SpO2) less than 90%. The method comprises administering a composition containing an ultra-long-acting NPRB agonist to the subjects in a therapeutically effective bolus dose; the therapeutically effective bolus dose does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement (baseline blood pressure measurement is the mean blood pressure before administration of the composition), and moreover, does not cause such a decrease for 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, The composition is sufficient to increase plasma cyclic GMP levels after 2–24 hours, 4–24 hours, 1–84 hours, 2–84 hours, 4–84 hours, 12–84 hours, 1–168 hours, 2–168 hours, 4–168 hours, or 12–168 hours) to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where baseline is the mean plasma cyclic GMP level before administration of the composition or the mean plasma level for a healthy mammal. The therapeutically effective bolus dose is a dose that does not lower blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, or does not cause such a decrease (baseline blood pressure measurement is the mean blood pressure before administration of the composition); moreover, the above dose is effective within 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours). The plasma cyclic GMP level after 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) can be increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0091] Long-acting NPRB agonists or ultra-long-acting NPRB agonists may contain polypeptides such as antibodies. In some embodiments, the long-acting NPRB agonist or ultra-long-acting NPRB agonist contains molecules having a molecular weight of less than 2 kDa.

[0092] In some embodiments, in any one of the methods described above, the composition has limited or no agonist activity toward NPRA and / or has a binding affinity to the NPRB receptor that is more than five times that of the NPRA receptor (or an EC50 that is five times lower).

[0093] In some embodiments, with respect to any one of the above methods, administration to the subject includes methods of administration such as oral or parenteral administration. Examples of parenteral administration include subcutaneous, intravenous, intramuscular, inhalation, nasal, or any combination thereof. In some embodiments, the above methods may include oral and / or subcutaneous administration. In certain embodiments, the above methods include intravenous administration. In some embodiments, the above methods include intramuscular administration. In some embodiments, the above methods include administration by inhalation (e.g., administration by intratracheal inhalation, in which the subject is exposed to a high aerosol concentration so that the pharmaceutically active ingredient is directly deposited in the lower respiratory tract). In certain embodiments, the above methods include nasal administration. In some embodiments, the above methods include oral administration.

[0094] In some embodiments, with respect to any one of the above methods, administration to a subject essentially consists of or comprises administering the composition of the Disclosure as a bolus dose. In some embodiments, with respect to any one of the above methods, administration to a subject does not involve administration of the composition of the Disclosure by prolonged infusion (e.g., by continuous infusion). In some embodiments, with respect to any one of the above methods, administration to a subject does not involve administering the composition of the Disclosure as a bolus dose followed by prolonged infusion. In some embodiments, with respect to any one of the above methods, administration to a subject does not involve oral administration of the composition of the Disclosure.

[0095] Medicinal active ingredients With respect to any of the methods described above, examples of long-acting or ultra-long-acting CNP derivatives include U-GLSKGCFGLKLDRIGSMSGLGC[SEQ ID NO: 2]; U-GLSKGCFGLK(U)LDRIGSMSGLGC[SEQ ID NO: 3]; GLSKGCFGLK(U)LDRIGSMSGLGC[SEQ ID NO: 4]; and / or U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue)[SEQ ID NO: 11], where U is attached to the N-terminal G and C, and / or to the epsilon-amino of the K residue.

[0096] In some embodiments, U in the above arrangement is part of formula (I) or (II), where formula (I) is (aliphatic) a -(X)- (I) and; During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is the linker (γE) m -(B) nAnd, During the ceremony, B is a sequence of 1 to 8 amino acid residues or peptides, where each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and The sum of m and n is at least 1. Equation (II) is, (polymer) a -(Y)- (II) and; During the ceremony, a is 0 or 1 (preferably a is 1); The polymers are cellulose, poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) (MPEG), poly(lactic acid-coglycolic acid), poly(N-vinylpyrrolidone), or derivatives thereof; Y is A sequence of 1 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); A non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; A linker containing amino acid residues, wherein the amino acid residues are (polymer) a Is it a linker containing amino acid residues that is covalently attached to it? or It is a peptide linker that is different from 1 to 10 amino acid residues or peptide sequences.

[0097] In some embodiments, in formula (II) above, Y is the linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0098] In this disclosure, the lowercase letter "x" refers to a native or non-native amino acid residue in the peptide sequence in which it appears. In formulas (I) and (II), the lowercase letter X refers to a linker. In some embodiments, x is not a methionine residue (M), not an asparagine residue (N), or neither methionine (M) nor asparagine (N). In some embodiments, x is not one of the 20 native amino acid residues encoded by the mammalian genome (e.g., amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y). In some embodiments, x is a non-native amino acid residue (i.e., an amino acid residue not encoded by the mammalian genome). In some embodiments, x is homoglutamine (also referred to herein as homo-Q).

[0099] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], where U is attached to the N-terminal G of GLSKGCFGLKLDRIGSMSGLGC, U is (aliphatic)a-(X)-, where a is 1, and the aliphatic is optionally substituted C. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 10~18 Chains, or optionally substituted C 12~18C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 X is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D).

[0100] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 12], where x is a natural or non-natural amino acid residue, and U is a (aliphatic) amino acid residue. a The formula has -(X)-(Formula I), where 0 or 1 (preferably a is 1); and the aliphatic is optionally substituted C 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 10~18 Chains, or optionally substituted C 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is a linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue being independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0101] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 30], where x is a natural or non-natural amino acid residue, except that x is M (methionine); U is a compound of the formula (aliphatic). a The formula has -(X)-(Formula I), where 0 or 1 (preferably a is 1); and the aliphatic is optionally substituted C 4~24 Chain (for example, C which is optionally substituted) 10~24 Chains, or optionally substituted C 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is a linker (γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue being independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0102] In some embodiments, the long-acting or ultra-long-acting CNP derivative may include U-GLSKGCFGLKLDRIGSMSGLGC[SEQ ID NO: 2]; U-GLSKGCFGLK(U)LDRIGSMSGLGC[SEQ ID NO: 3]; GLSKGCFGLK(U)LDRIGSMSGLGC[SEQ ID NO: 4]; U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue)[SEQ ID NO: 12], or any combination thereof. U is part of equation (I), and equation (I) is, (aliphatic) a -(X)- (I) And, During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted. 10~24 Chain (for example, C which is optionally substituted) 12~28 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 10~24 It is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is the linker (γE) m -(B) n And, During the ceremony, B is a sequence of 1 to 8 amino acid residues or peptides, where each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and The sum of m and n is at least 1.

[0103] In some embodiments, x in U-CFGLKLDRIGSxSGLGC[SEQ ID NO: 12] is neither a methionine residue nor an asparagine residue, nor is it either a methionine residue or an asparagine residue. In some embodiments, x is not one of the 20 native amino acid residues encoded by the mammalian genetic code (e.g., amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y). In some embodiments, x is a non-native amino acid residue (i.e., an amino acid residue not encoded by the mammalian genetic code). In some embodiments, x is homoglutamine (also referred to herein as homo-Q).

[0104] In some embodiments, X is a sequence of 4 to 7 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G).

[0105] In some embodiments, the long-acting CNP derivative or ultra-long-acting CNP derivative includes U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U is (aliphatic) a -(X)- is; During the ceremony, a is 1; Aliphatic C is optionally substituted. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D).

[0106] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 13], where x is homoglutamine; U is (aliphatic) a -(X)-, where a is 0 or 1 (preferably a is 1), and the aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16C (=O) is the linker (γE) m -(B) n B is Gly; m is 0, 1, or 2; and n is 1.

[0107] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 14], where x is homoglutamine; U is (aliphatic) a -(X)-, where a is 0 or 1 (preferably a is 1), and the aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 Branched or linearly substituted C(=O) covalently bonded to X 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n B is Gly; m is 1; and n is 1.

[0108] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 15], where x is homoglutamine; U is (aliphatic) a-(X)-, wherein a is 0 or 1 (preferably, a is 1), and the aliphatic group is a branched or straight-chain optionally substituted C 16 chain covalently bonded to X through a carbonyl (e.g., CH3(CH2) 18 C(=O)), or an optionally substituted C 18 chain covalently bonded to X through a chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond with X, etc.); preferably, the aliphatic group is a branched or straight-chain optionally substituted C 16 chain covalently bonded to X through a carbonyl (e.g., CH3(CH2) 18 C(=O)) as part of an amide or ester linkage with X; more preferably, the aliphatic group is a branched or straight-chain optionally substituted C 16 chain covalently bonded to X through a carbonyl (e.g., CH3(CH2) 18 C(=O)) as part of an amide linkage with X, or the aliphatic group is HOC(=O)(CH2) 16 C(=O); X is a linker (γE) m -(B) n ; m is 1; and n is 0.

[0109] In some embodiments, the long-acting CNP derivative or ultra-long-acting CNP derivative is U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 12], and U is (aliphatic) a -(X)-; a is 0 or 1 (preferably, a is 1); the aliphatic group is an optionally substituted C 4~24 chain (e.g., an optionally substituted C 10~24 chain, an optionally substituted C 12~18C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n In the formula, B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. In some embodiments, x in U-CFGLKLDRIGSxSGLGC[SEQ ID NO: 12] is neither a methionine residue nor an asparagine residue, nor is it either a methionine residue or an asparagine residue. In some embodiments, x is not one of the 20 native amino acid residues encoded by the mammalian genetic code (e.g., amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y). In some embodiments, x is a non-natural amino acid residue (i.e., an amino acid residue not coded by the mammalian genetic code). In some embodiments, x is homoglutamine (also referred to herein as homo-Q).

[0110] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC (wherein x is homoglutamine (homo Q)) [SEQ ID NO: 16], where U is (aliphatic) a -(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n B is a 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; m is 0 and n is 2.

[0111] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC (wherein x is homoglutamine (homo Q)) [SEQ ID NO: 17], where U is (aliphatic) a -(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n B is a 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; m is 1 and n is 2.

[0112] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC (wherein x is homoglutamine (homo Q)) [SEQ ID NO: 18], where U is (aliphatic) a -(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16C (=O) is the linker (γE) m -(B) n B is (2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(Gly); m is 0 and n is 1.

[0113] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC (wherein x is homoglutamine (homo Q)) [SEQ ID NO: 19], where U is (aliphatic) a -(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n B is (2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(Gly); m is 1 and n is 1.

[0114] In some embodiments, the long-acting or ultra-long-acting CNP derivative U-CFGLKLDRIGSxSGLGC contains a disulfide bond between cysteine ​​residues in HOC(=O)(CH2) 16 C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGSHomoQSGLGC(HomoQ: Homoglutamine; Aeea: 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; HOC(=O)(CH2) 16 C(=O): Octadecadionic acid reacted with γE such that a carbonyl group (C(=O)) remains at one of the carboxylic acid terminals of the original octadecadionic acid; γE: Glutamic acid conjugated via a gamma-carboxyl group) [SEQ ID NO: 20]

[0115] In some embodiments, the long-acting or ultra-long-acting CNP derivative U-CFGLKLDRIGSxSGLGC contains a disulfide bond between cysteine ​​residues in HOC(=O)(CH2) 16 C(=O)-Aeea-Aeea-GCFGLKLDRIGSHomoQSGLGC(HomoQ: Homoglutamine; Aeea: 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; HOC(=O)(CH2) 16 (CO): Octadecadionic acid reacted with the amino terminus of Aeea such that a carbonyl (C(=O)) remains at the carboxylic acid terminus of the original octadecadionic acid) [SEQ ID NO: 21]

[0116] In some embodiments, in any of the definitions herein, the aliphatic is a linear or branched C, which is optionally substituted. 4~9 A chain (for example, a thioether, ether, thioether, carbamate moiety, bond, or similar linkage) covalently bonded to the peptide via a selectively substituted C 3~8 Chain-C(=O)-part and / or optionally substituted C 4~9It does not contain a chain. In certain embodiments, the aliphatic is not a linear or branched C8 chain (for example, a linear or branched C8 chain covalently bonded to the peptide via linkages such as carbonyl, thioether, ether, thioether, carbamate moiety, bond, or similar).

[0117] In some embodiments, U as described above is CH3(CH2) 14 C(=O)KKKKGGG-[Sequence ID 22];CH3(CH2) 16 C(=O)KKKKGGG-[Sequence ID 23];CH3(CH2) 18 C(=O)KKKKGGG-[Sequence ID 24];CH3(CH2) 20 C(=O)KKKKGGG-[Sequence ID 25]; or CH3(CH2) 22 C(=O)KKKKGGG[Sequence ID 26] is one example.

[0118] In some embodiments, the long-acting CNP derivative of the present disclosure is CH3(CH2) 14 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 5];CH3(CH2) 16 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 6];CH3(CH2) 18 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 7];CH3(CH2) 20 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 8];CH3(CH2) 22 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[SEQ ID NO: 9]; HOC(=O)(CH2) containing disulfide bonds between cysteine ​​residues 16 C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGS homoQSGLGC[SEQ ID NO: 20]; and / or HOC(=O)(CH2) containing disulfide bonds between cysteine ​​residues 16 One example is C(=O)-Aeea-Aeea-GCFGLKLDRIGS homo-QSGLGC [SEQ ID NO: 21].

[0119] In certain embodiments, the long-acting CNP derivative of the present disclosure is CH3(CH2) 16 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 6] is one example.

[0120] In some embodiments, the long-acting or ultra-long-acting CNP derivative includes U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 4], U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 27], or any combination thereof; During the ceremony, U is part of equation (II), and equation (II) is, (polymer) a -(Y)- (II) And, During the ceremony, a is 0 or 1 (preferably a is 1); The polymers are cellulose, poly(ethylene glycol)(PEG), methoxypoly(ethylene glycol)(MPEG), poly(lactic acid-coglycolic acid), or poly(N-vinylpyrrolidone); Y is A sequence of 4 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G); A non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; or Linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1, linker (γE) m -(B) n That is the case.

[0121] In some embodiments, the long-acting or ultra-long-acting CNP derivative includes U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], or any combination thereof; During the ceremony, U is part of equation (II), and equation (II) is, (polymer) a -(Y)- (II) And, During the ceremony, a is 0 or 1 (preferably a is 1); The polymers are cellulose, poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) (MPEG), poly(lactic acid-coglycolic acid), poly(N-vinylpyrrolidone), or derivatives thereof; Y is A sequence of 1 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); A non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; A linker containing amino acid residues, wherein the amino acid residues are (polymer) a Is it a linker containing amino acid residues that is covalently attached to it? It is a peptide linker that is different from 1 to 10 amino acid residues or peptide sequences.

[0122] In some embodiments, Y in formula (II) above is linker-(γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue being independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0123] In some embodiments, the polymer does not contain poly(ethylene glycol), does not contain MPEG, or does not contain both poly(ethylene glycol) and MPEG.

[0124] In some embodiments, Y is a sequence of 4 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G); or a linker (γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1, linker (γE) m -(B) n That is the case.

[0125] In some embodiments, Y is a sequence of 4 to 10 amino acid residues, where each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G).

[0126] In some embodiments, Y is the linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue being independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0127] In some embodiments, the CNPs of this disclosure or their derivatives do not include CNPs modified with polyalkylene glycol at the lysine residues at positions 4 and 10 of SEQ ID NO: 10 and / or at the N-terminus of the CNP of SEQ ID NO: 10.

[0128] In some embodiments, formulations comprising the long-acting CNP derivatives of the present disclosure comprise one or more CNPs or derivatives thereof, compounded with polymer excipients such as polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. The polymer is adapted to sequester the CNP derivative or to be non-covalently bonded to the CNP derivative.

[0129] In some embodiments, formulations comprising the ultra-long-acting CNP derivatives of the present disclosure comprise one or more long-acting CNP derivatives compounded with a polymer excipient comprising polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. The polymer is adapted to sequester the CNP derivative or to be non-covalently bonded to the CNP derivative.

[0130] In some embodiments, formulations comprising the long-acting NPRB agonist of the present disclosure comprise one or more CNPs or derivatives thereof, compounded with a polymer excipient comprising polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. The polymer is adapted to sequester the NPRB agonist or to be non-covalently bonded to the NPRB agonist.

[0131] In some embodiments, formulations comprising the ultra-long-acting NPRB agonist of the present disclosure comprise one or more long-acting CNP derivatives compounded with a polymer excipient comprising polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. The polymer is adapted to sequester the NPRB agonist or to non-covalently bond to the NPRB agonist.

[0132] Examples of poly(amino acids) grafted with polyethylene glycol, fatty acids, and / or anionic moieties include poly(amino acids) that may have D- or L-chirality, or both, and are linear homopolymers. In one specific embodiment, the linear homopolymer may be polylysine, polyornithine, polyarginine, polyglutamic acid, polyaspartic acid, polyserine, polytyrosine, or any other amide-linked homopolymer formed of amino acids. In another preferred embodiment, the linear hydrophobic homopolymer may include polyalanine, polyvaline, polyleucine, polyisoleucine, polyglycine, or polyphenylalanine. In some embodiments, the poly(amino acid) is polylysine.

[0133] Method for manufacturing the active ingredient of this pharmaceutical product The peptides of this disclosure (e.g., long-acting CNPs, long-acting CNP derivatives, and long-acting NPRB agonists) can be synthesized by solid-phase peptide synthesis (SPPS) using methods known to those skilled in the art. For example, a starting solid support such as H-Cys(Trt)-2-Cl-Trt Resin (BLDPharm, Shanghai, China) may be used in a peptide synthesizer such as an automated microwave peptide synthesizer (e.g., LibertyBlue HT12, CEM, Matthews, NC). Each amino acid, fatty acid, or protected alkylcarboxylic acid (diacid) can be sequentially immobilized on the peptide resin using Fmoc chemistry known to those skilled in the art, resulting in linearly protected peptides linked to the resin. The linear crude peptides can be deprotected and released from the resin by acid hydrolysis with trifluoroacetic acid and ether precipitation in the presence of a carbocation scavenger. The obtained linear peptides can be cyclized and reacted to form disulfide bonds, for example, by dissolving them in aqueous solutions of DMSO and acetonitrile. Finally, this peptide can be purified and characterized by reverse-phase HPLC (e.g., 1260 Infinity II Preparative LC Systems, Santa Clara, CA). The fraction with a purity of over 90% of the final peptide product is collected and dried as a white powder.

[0134] In some embodiments, formulations comprising the pharmaceutically active ingredient ("API") of the Disclosure have a weight ratio of polymer excipient to API such that the resulting mixture is long-acting or ultra-long-acting. For example, the weight ratio of polymer excipient to total API may be 5:1 to 100:1, 10:1 to 50:1, or 20:1 to 5:1. The polymer excipient may include polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. The polymer excipient is adapted to sequester the API or to non-covalently bond to the API. Examples of polymer excipients are described, for example, in Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18; Castillo et al., PLoS One, (2017) 12(2); e0171703; U.S. Patent No. 10,507,248; U.S. Patent No. 10,035,885; and U.S. Patent No. 10,010,613, each of which is incorporated herein by reference in whole. Polymer excipients may be, but are not limited to, polylysines grafted with epsilon amino to a level of 10-55% (e.g., 10-35% or 30-55%) of the total epsilon amino by PEG, with the remaining amino groups grafted with alkyl and / or anionic moieties (e.g., sulfuric acid, sulfonic acid, carboxyl, phosphoric acid, or phosphonic acid). Methods for producing polymer excipients are known in the art.

[0135] In short, in some embodiments, the polymer excipient is a polymer prepared by the following procedure: Poly-L-lysine (20PL) and hydrobromide (21 μmol or 1 g; Sigma, average Mw=26 kDa; dp126) were dissolved, and the amount of NH2 groups was determined by TNBS titration. Methoxypolyethylene glycol carboxymethyl (MPEG-CM; 10 g; Mw=5 kDa; 2 mmol; Laysan Bio) was conjugated to polylysine using NHSS and EDC to obtain a polymer excipient intermediate. The proportion of remaining amino groups was determined by TNBS. The hydrodynamic diameter was determined by size exclusion chromatography. The crude product can be freeze-dried. Stearyl-NHS (C18-NHS) was prepared by activating stearic acid with NHS. DCC coupling of stearyl-NHS to the polymer excipient intermediate can be performed. Excess reagents and by-products can be removed by standard techniques. Further C18-NHS (3.6 mmol) was added and reacted with the polymer intermediate overnight. The reaction mixture was concentrated by rotary evaporation under vacuum to remove volatile components until an oily substance was isolated. This oily substance can be dissolved in alcohol and water. This solution was filtered and repeatedly washed to obtain a holding solution containing the polymer excipient (polylysine with C18 hydrophobic side chains and MPEG hydrophilic side chains), which was then filtered through 0.2 μm (polysulfone filter, Nalgene, Rochester, NY) and freeze-dried to obtain the dry polymer excipient.

[0136] In the above, C 18 We have described polymer excipients having hydrophobic side chains, but other hydrophobic side chain lengths (for example, C 10~24 , C 12~20 , C 12~18 , C 14~18 , C 16~18 , or C 18 It is understood that hydrophobic and hydrophilic side chains (e.g., PEG, mPEG) can be adapted to produce polymer excipients having other hydrophobic and hydrophilic side chains.

[0137] Examples of poly(amino acids) grafted with polyethylene glycol, fatty acids, and / or anionic moieties include poly(amino acids) that may have D- or L-chirality or both, and are linear homopolymers. In one specific embodiment, the linear homopolymer may be polylysine, polyornithine, polyarginine, polyglutamic acid, polyaspartic acid, polyserine, polytyrosine, or any other amide-linked homopolymer formed of amino acids. In another preferred embodiment, the linear hydrophobic homopolymer may include polyalanine, polyvaline, polyleucine, polyisoleucine, polyglycine, or polyphenylalanine. In some embodiments, the poly(amino acid) is polylysine.

[0138] Examples of hydrophilic side chains include: poly(ethylene glycol) which can be esterified by a dicarbon chain to form a poly(ethylene glycol) monoester; methoxypoly(ethylene glycol) monoester (MPEG); or a copolymer of poly(ethylene glycol) and poly(propylene glycol) monoester, in the form of an ester with a dicarboxylic acid which provides a carboxyl group at the end of the copolymer that can be used to covalently link to a poly(amino acid). Other forms include: poly(ethylene glycol)-carboxyl; methoxypoly(ethylene glycol)-carboxyl; poly(ethylene glycol)-carboxymethyl; methoxypoly(ethylene glycol)-carboxymethyl; poly(ethylene glycol)-monamine; methoxypoly(ethylene glycol)-monamine; poly(ethylene glycol)-hydrazide; methoxypoly(ethylene glycol)-hydrazide; and methoxypoly(ethylene glycol)imidazolide block copolymers of poly(ethylene glycol) with one or more polymers represented by polyamino acids, polysaccharides, polyamidoamines, and polyethyleneimines (these blocks are arranged alternately to obtain linear block copolymers). In one embodiment, the total molecular weight of the protective chain may be greater than 300 daltons but not greater than 10,000 daltons. In one embodiment, one or more protective chains are linked to the poly(amino acid) backbone by a single linkage.

[0139] While we do not wish to be bound by theory, it is thought that when an API composition is administered, the higher the weight ratio of the polymer excipient to the API, the longer the presence in plasma will persist, and the longer the elevation of plasma cyclic GMP above baseline will persist.

[0140] In some embodiments, formulations comprising the long-acting CNP, long-acting CNP derivatives, and / or long-acting NPRB agonists of the present disclosure have a weight ratio of a polymer excipient to the CNP, CNP derivative, and / or NPRB agonist such that the resulting mixture is the long-acting CNP, the long-acting CNP derivative, and / or the long-acting NPRB agonist. For example, the weight ratio of this polymer excipient to the CNP, CNP derivative, and / or NPRB agonist may be 5:1 to 100:1, 10:1 to 50:1, or 20:1 to 5:1. This polymer excipient may include polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. See, for example, Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18; Castillo et al., PLoS One, (2017) 12(2); e0171703; U.S. Patent No. 10,507,248; U.S. Patent No. 10,035,885; U.S. Patent No. 10,010,613 (each of which is incorporated herein by reference in its entirety). This polymer excipient is adapted to sequester or non-covalently bond to CNP, CNP derivatives, and / or NPRB agonists. The polymer excipient may be, but is not limited to, polylysine grafted with epsilon amino to a level of 30-55% or 10-35% of the total epsilon amino by PEG, with the remaining amino groups grafted with alkyl and / or anionic moieties (e.g., sulfuric acid, sulfonic acid, carboxyl, phosphoric acid, or phosphonic acid). Methods for producing the polymer excipient are known in the art. While we do not wish to be bound by theory, it is thought that when CNP, CNP derivatives, and / or NPRB agonist compositions are administered to a subject, a higher weight ratio of the polymer excipient to the CNP, CNP derivative, and / or NPRB agonist will result in longer-lasting presence of the CNP, CNP derivative, or NPRB agonist in plasma, and a longer-lasting elevation of plasma cyclic GMP above baseline.

[0141] In some embodiments, ultralong-acting CNP, ultralong-acting CNP derivatives, and / or ultralong-acting NPRB agonist formulations comprise CNP, CNP derivatives, and / or NPRB agonists, and a polymer excipient, in a weight ratio of the polymer excipient to the CNP, CNP derivatives, and / or NPRB agonist such that the resulting mixture is an ultralong-acting CNP, an ultralong-acting CNP derivative, and / or an ultralong-acting NPRB agonist. For example, the weight ratio of the polymer excipient to the CNP, CNP derivatives, and / or NPRB agonist may be 5:1 to 100:1, 10:1 to 50:1, or 20:1 to 5:1. Examples of such polymer excipients include polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. See, for example, Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18; Castillo et al., PLoS One, (2017) 12(2); e0171703; U.S. Patent No. 10,507,248; U.S. Patent No. 10,035,885; U.S. Patent No. 10,010,613 (each of which is incorporated herein by reference in its entirety). This polymer excipient is adapted to sequester or non-covalently bond to CNP, CNP derivatives, and / or NPRB agonists. The polymer excipient may be, but is not limited to, polylysine grafted with epsilon amino to a level of 30-55% or 10-35% of the total epsilon amino by PEG, with the remaining amino groups grafted with alkyl and / or anionic moieties (e.g., sulfuric acid, sulfonic acid, carboxyl, phosphoric acid, or phosphonic acid). Methods for producing the polymer excipient are known in the art. While we do not wish to be bound by theory, it is thought that when CNP, CNP derivatives, and / or NPRB agonist compositions are administered to a subject, a higher weight ratio of the polymer excipient to the CNP, CNP derivative, and / or NPRB agonist will result in longer-lasting presence of the CNP, CNP derivative, and / or NPRB agonist in plasma, and longer-lasting elevation of plasma cyclic GMP above baseline.

[0142] Disease state In some embodiments, any of the methods of the present disclosure includes treating ALI. In certain embodiments, any of the methods of the present disclosure includes treating ARDS. In some embodiments, any of the methods of the present disclosure includes treating pulmonary edema. In some embodiments, any of the methods of the present disclosure includes treating hypoarterial oxygenation. In certain embodiments, any of the methods of the present disclosure includes treating elevated inflammatory cell levels in the lungs. In some embodiments, any of the methods of the present disclosure includes treating sepsis. In some embodiments, any of the methods of the present disclosure includes treating bacteremia. In some embodiments, any of the methods of the present disclosure also includes treating pulmonary fibrosis. In some embodiments, any of the methods of the present disclosure includes treating fibrosis in general (e.g., pulmonary fibrosis, cirrhosis, and / or glomerulosclerosis), and / or renal impairment.

[0143] In some embodiments, if the disease to be treated is ALI or ARDS, ALI or ARDS is (i) a systemic injury selected from trauma, sepsis (i.e., systemic infection), bacteremia (i.e., bacteria in the blood), pancreatitis, shock, frequent transfusions, disseminated intravascular coagulation, burns, drug overdose or toxicity, opioids, aspirin, phenothiazines, tricyclic antidepressants, amiodarone, chemotherapeutic agents, nitrofurantoin, protamine, thrombotic thrombocytopenic purpura, head trauma, and / or paraquat; And / or (ii) lung injury caused by or associated with any one of the following: aspiration of gastric contents, pulmonary intubation, embolism (e.g., of thrombus, fat, air, or amniotic fluid origin), tuberculosis, viral pneumonia (e.g., SARS caused by coronavirus or influenza virus), bacterial pneumonia, cytogenic organizing pneumonia, airway obstruction, smoking of free basic cocaine, drowning, inhalation of toxic gases, oxygen toxicity, pulmonary contusion, radiation exposure, exposure to high altitude, lung reinflation, and / or reperfusion.

[0144] In some embodiments, when the disease to be treated is ALI or ARDS, ALI or ARDS may be caused by an infection, where the infection is caused by coronavirus or influenza virus, pulmonary fibrosis, sepsis; bacteremia; intubation; and / or a toxic gas selected from the group consisting of chlorine gas, fumes, phosgene, and / or concentrated oxygen.

[0145] In certain embodiments, if the disease being treated is ALI or ARDS, then ALI or ARDS is caused by an infection (for example, in this case, the infection is caused by a coronavirus or influenza virus).

[0146] In certain embodiments, when the disease being treated is ALI or ARDS, ALI or ARDS is caused by pulmonary fibrosis.

[0147] In certain embodiments, if the disease being treated is ALI or ARDS, then ALI or ARDS is caused by sepsis.

[0148] In another embodiment, if the disease being treated is ALI or ARDS, then ALI or ARDS is caused by bacteremia.

[0149] In another embodiment, if the disease being treated is ALI or ARDS, then ALI or ARDS is caused by intubation.

[0150] In another embodiment, if the disease to be treated is ALI or ARDS, the ALI or ARDS is caused by toxic gases, such as chlorine gas, fumes, phosgene, concentrated oxygen, or any combination thereof.

[0151] Acute lung injury (damage) / Acute respiratory distress syndrome (ALI / ARDS) refers to a life-threatening clinical pulmonary syndrome with a 28-day mortality rate of 30-50%. The incidence of ALI / ARDS in the United States is approximately 200,000 cases per year. For example, Johnson ER, and See Matthay MA, J Aerosol Med Pulm Drug Deliv. 23(4):243-52, 2010. ALI is a syndrome or condition characterized by disruption of the endothelial and epithelial barriers, neutrophilic inflammatory response, pulmonary edema, and alveolar damage resulting from marked dysfunction of pulmonary blood oxygenation, lung compliance, and airway resistance. Acute respiratory distress syndrome (ARDS) is a more severe form of ALI. As used herein, ALI includes both ALI and ARDS. For clinical diagnostic purposes in humans, ALI is defined by the North American-European consensus classification (e.g., Henru et al., Intensive Care Med (2013) 39:2161-2170) based on the outcome or summation of the aforementioned condition or syndrome (which is reduced blood oxygenation in patients without heart failure). This decrease is consistent with pulmonary edema, along with the presence of bilateral infiltrates (the term "infiltrates" is used by the physician interpreting the chest X-ray) on the X-ray (determined by the X-ray), and a PaO2:FiO2 (defined below) ratio of 200-300 mgHg at a wedge pressure setting of less than 18 mgHg (i.e., not due to cardiovascular causes). Patients with ARDS have very low blood oxygen levels with a sudden onset of shortness of breath and a PaO2 of less than 63 mmHg or a PaO2:FiO2 ratio of less than 300 mgHg, where PaO2 is the partial pressure of oxygen in the arterial blood and FiO2 is the fractional concentration of inhaled oxygen. Alternatively, or in addition, blood oxygenation can be determined in terms of hemoglobin oxygen saturation, or SpO2, using pulse oximetry, a finger or ear-mounted sensor using light. A blood SpO2 of 96-99% indicates adequately oxygenated blood; a SpO2 below 90% indicates a PaO2:FiO2 ratio of less than 300 mgHg. Other blood tests may be performed, including tests for renal function, thyroid function, blood cell counts, and tests to rule out heart attack (ECG / echocardiogram) as a cause of pulmonary edema rather than ALI.

[0152] ALI / ARDS can result from a wide variety of injuries, including sepsis (the most common cause of ALI exacerbations worldwide; see, e.g., Leonard D. Hudson, Arthur S. Slutsky, in Goldman's Cecil Medicine (Twenty-Fourth Edition), 2012), gastric aspiration, shock, infection, pulmonary contusion, non-chest trauma, toxic inhalation, drowning, and / or frequent blood transfusions. Lung injury caused by influenza or coronaviruses (e.g., H1N1, SARS-CoV-1 and 2) can lead to life-threatening ALI / ARDS. Mechanistically, these injuries result in the destruction of the alveolar endothelium and epithelial barrier, and leakage of fluid (pulmonary edema), plasma proteins, and inflammatory cells (neutrophils, macrophages) into the lung / alveolar space, restricting red blood cell access to oxygen from alveolar air, leading to severe hypoxemia, or very low hemoglobin oxygen saturation and dyspnea. The accumulation of inflammatory cells is thought to be central to promoting and perpetuating damage through the generation of large amounts of oxygen-derived free radicals by these cells. Cytokines, growth factors, and degrading enzymes are also produced by inflammatory cells and released into the extracellular environment. These molecules and proteins can damage parenchymal cells in inflamed tissue, potentially leading to cell death. The severity of ALI is positively correlated with the number of inflammatory cells (activated neutrophils) in the alveolar space. The early activation state of neutrophils in ALI patients determines the clinical course of the disease. See, for example, Yang et al., Am.J.Respir.Crit.CareMed. 167:15671574, 2003. Many animal models of ALI are associated with the presence of high concentrations of neutrophils. For example, see Abraham et al., Am.J.Physiol.Lung Cell.Mol.Physiol.279:1137-1145,2000; Flick et al., Circ.Res.48:344-351,1981; Heflin AC Jr and Brigham K LJClin.Invest.68:1253-1260,1981; Shasby et al., Am.Rev.Respir.Dis.125:443-447,1982.

[0153] Therefore, "acute lung injury" (ALI) refers to lung injury in mammals or humans that causes bilateral infiltration consistent with pulmonary edema on radiography, in the absence of clinical evidence of heart failure, and with very low blood oxygen levels, such as a PaO2 (arterial oxygen partial pressure) of less than approximately 60 mmHg or a PaO2:FiO2 ratio of less than 300 mgHg (if the PaO2:FiO2 ratio is significantly below 300 mgHg, this condition is also characterized by shortness of breath). The term ALI also includes acute respiratory distress syndrome (ARDS), a more severe form of ALI. The PaO2:FiO2 ratio is the ratio of the fractional concentration of oxygen in arterial blood (PaO2) to the fractional concentration of inhaled oxygen (FiO2), in the absence of clinical evidence of heart failure. FiO2 may be a fractional concentration of atmospheric oxygen of 0.21 (or 21%), but in a hospital setting where oxygen is used as supportive therapy, this can be as high as 0.60. Since FiO2 can fluctuate depending on the oxygen used in the inhaled air, only PaO2 below 60 mmHg is an indicator of ALI, and a PaO2 below 60 mmHg indicates a more severe ALI / ARDS.

[0154] Because PaO2 requires blood sampling, pulse oximetry is also used to measure blood oxygenation. Pulse oximetry is a non-invasive method for monitoring human blood hemoglobin oxygen saturation (SO2), now known as the "fifth vital sign." It determines SO2 using the absorbance of light at two wavelengths (one for oxygenated hemoglobin and the other for deoxygenated hemoglobin) at an extremity such as a finger or ear. Specifically, deoxyhemoglobin absorbs light most maximally in the red band of the spectrum (600-750 nm), and oxyhemoglobin absorbs light most maximally in the infrared band (850-1000 nm). The ratio of absorbance between oxyhemoglobin and the sum of the absorbances of oxyhemoglobin and deoxyhemoglobin is calculated and compared to a pre-calibrated direct measurement. ALI patients with SO2 less than 90% correlate well with PaO2 less than 60 mmHg and are diagnosed with ALI. All animal models of ALI are associated with the presence of high concentrations of neutrophils, and these measurements indicate the severity and resolution of ALI. See, for example, Abraham et al., Am.J.Physiol.Lung Cell.Mol.Physiol.279:1137-1145,2000; Flick et al., Circ.Res.48:344-351,1981; Heflin ACJr.and Brigham K LJClin.Invest.68:1253-1260,1981; Shasby et al., Am.Rev.Respir.Dis.125:443-447,1982). This model is widely used by those skilled in the art to diagnose the severity of ALI in mammalian subjects.

[0155] ALI is diagnosed or determined when a patient presents evidence of vital signs indicating hypooxygenation. These signs include shortness of breath, tachypnea, bluish / cherry-red skin, cough, wheezing, and sweating. Hypooxygenation is confirmed by measuring arterial oxygen partial pressure (PaO2) by measuring blood oxygenation using either pulse oximetry or blood sampling and blood gas analysis. Blood gas analysis indicating hypooxygenation is a PaO2 of 60 mmHg or less, or an arterial oxygen partial pressure:fractionated inspired oxygen ratio (PaO2 / FiO2 ratio) of less than 300 mmHg. Using oximetry, hypooxygenation is indicated by a blood hemoglobin oxygen saturation (SpO2) of less than 90%. Pulse oximetry can also be used to monitor a person's blood hemoglobin oxygen saturation (SO2). In ALI patients, SO2 is less than 90%, which correlates well with a PaO2 of less than 300 mmHg, leading to a diagnosis of ALI. The hypoxia is due to pulmonary edema (which is also a symptom of ALI) and can be determined by chest X-ray, which can confirm the diagnosis of pulmonary edema and rule out other possible causes of shortness of breath.

[0156] ALI is also associated with elevated levels of inflammatory cells in the lungs, as determined from bronchoalveolar lavage fluid (BALF). BALF is obtained and analyzed for the levels and size of inflammatory cells. Bronchoalveolar lavage (BAL), performed during flexible bronchoscopy, is widely accepted as a minimally invasive technique that provides important information about immunological, inflammatory, and infectious processes occurring at the alveolar level. See, for example, Harbeck RJ, Clin Diagn Lab Immunol. 1998, 5(3):271-7. Briefly, the BAL technique generally involves introducing a flexible fiber optic bronchoscope transnasally with the patient in a semi-lateral position. The bronchoscope enters the trachea through the pharynx and vocal cords and reaches the appropriate area of ​​the lung. Aliquots of sterile saline (generally 30-40 ml) are infused through the bronchoscope, and the bronchoscope is gently withdrawn immediately afterward. With 100 ml of saline solution, it is possible to sample approximately 1 million alveoli or about 1.5-3% of the lung's components, and approximately 1 ml of epithelial lining fluid can be recovered. The entire procedure takes less than 15 minutes. Cells recovered from the lung by lavage are far more heterogeneous than cells obtained from peripheral blood. Flow cytometry reveals that the major cell population includes normal-sized macrophages, neutrophils, eosinophils, erythrocytes, and lymphocytes. During inflammation and ALI, the number of epithelial cells increases significantly. In addition to the increase in number, lung macrophage size can be 8-30 mm or more, and BAL fluid lymphocytes can be larger than their peripheral blood counterparts, depending on the state of the lung, especially if they are activated.

[0157] Sepsis is defined as a life-threatening organ dysfunction resulting from a dysregulated host response to infection. The consensus document describes organ dysfunction as a sharp increase of 2 points in the Total Sequential Organ Failure Assessment (SOFA) score resulting from infection. See, for example, Gul et al., Turk J Anaesthesiol Reanim. 2017 Jun;45(3):129-138. Septic shock occurs in a subset of sepsis patients and involves underlying circulatory and cellular / metabolic abnormalities associated with increased mortality. Septic shock is defined as persistent hypotension requiring vasopressors to maintain mean arterial blood pressure of ≥65 mmHg and serum lactate levels greater than 2 mmol / L (18 mg / dL) despite adequate fluid resuscitation. See, for example, Singer et al., JAMA 2016;315(8):801-810. This definition, also known as Sepsis-3, eliminates the requirement of systemic inflammatory response syndrome (SIRS) in defining sepsis and removes the definition of severe sepsis. What was previously called severe sepsis is now the new definition of sepsis. Severe sepsis is the most common cause of exacerbation of ALI worldwide. See, for example, Leonard D. Hudson, Arthur S. Slutsky, in Goldman's Cecil Medicine (Twenty-Fourth Edition), 2012. Since inflammation caused by sepsis leads to ALI, there is no treatment for ALI other than supportive care, so it is necessary to reduce the progression to ALI as early as possible. Endotoxins, or more precisely bacterial lipopolysaccharides (LPS), are recognized as the most potent microbial vectors involved in the pathogenesis of sepsis and septic shock. See, for example, Opal SM. Contrib Nephrol. 2010;167:14-24. Therefore, the use of LPS to simulate sepsis in animal models is widely used when testing the efficacy of a treatment before use in humans. Sepsis is known to be associated with a drop in blood pressure, and since natriuretic peptides are known to lower blood pressure, using them as a treatment is contrary to common sense.Nevertheless, as discussed above, this disclosure features the use of C-type natriuretic peptide (CNP) derivatives, long-acting CNP, long-acting CNP derivatives, ultra-long-acting CNP, ultra-long-acting CNP derivatives, long-acting NPRB agonists, and / or ultra-long-acting NPRB agonists for the treatment of sepsis, and has beneficial effects.

[0158] Bacteremia is the presence of bacteria in the bloodstream and is well known to those skilled in the art. When bacteria are present in the bloodstream for a sufficiently long period and in sufficient numbers, especially in people with a weakened immune system, bacteremia can lead to other infections and sometimes a severe systemic reaction called sepsis. Bacteremia can result from normal activities (such as vigorous brushing of teeth), dental or medical procedures, or infections (such as pneumonia or urinary tract infections). Usually, bacteremia (especially when it occurs during normal activities) does not lead to infection because typically only a small number of bacteria are present and are rapidly removed from the bloodstream by the immune system.

[0159] Acute respiratory distress syndrome (ARDS) is a more severe form of ALI. It is a rapidly progressing disease that occurs in critically ill patients, in which case fluid leakage into the lungs progresses to a stage where breathing becomes difficult or impossible.

[0160] Pulmonary edema is a condition characterized by the presence of excess fluid in the lungs themselves, and is defined and diagnosed by the presence of bilateral infiltrates ("infiltrates" is a term used by physicians and those skilled in the art to interpret chest X-rays) consistent with pulmonary edema. This fluid accumulates in numerous air sacs in the lungs, making breathing difficult. The presence of bilateral infiltrates ("infiltrates" is a term used by physicians who interpret chest X-rays) consistent with pulmonary edema is evident in the radiograph (X-ray). In most cases, heart problems cause pulmonary edema. However, fluid accumulation can also occur for other reasons, including pneumonia, exposure to certain toxins or medications, chest wall trauma, and visits or exercise at high altitudes. Sudden onset of pulmonary edema (acute pulmonary edema) is a medical emergency requiring emergency medical attention. Pulmonary edema can sometimes be fatal, but the prognosis improves with prompt treatment. Treatment for pulmonary edema varies depending on the cause, but generally includes oxygen supplementation and medication.

[0161] Hypoxemia, or hypoxicemia, is a subnormal level of oxygen in the blood, where the partial pressure of oxygen is less than 60 mmHg, or the pulse oximeter reading is less than 90%. Normal arterial oxygen partial pressure is approximately 75–100 millimeters of mercury (mmHg) or 10.5–13.5 kilopascals (kPa). Normal pulse oximeter readings typically range from 95–100%, representing blood hemoglobin saturation. Hypoxemia is a sign of an underlying disorder that can cause various symptoms, including shortness of breath.

[0162] Elevated inflammatory cell levels in the lungs refer to an increase of at least three times the level, quantity, or number of inflammatory cells (macrophages, neutrophils, eosinophils, and lymphocytes) in the lungs compared to normal levels in healthy controls, as similarly measured. These levels can be up to ten times higher than those in healthy controls. This level is determined from bronchoalveolar lavage fluid (BALF) by flow cytometry. The major cell populations in BALF include normal-sized macrophages, neutrophils, eosinophils, erythrocytes, and lymphocytes. During inflammation and ALI, the number and size of lung macrophages (8–30 μm or larger) increase, along with a significant increase in the number of epithelial cells. Similarly, lymphocytes can be larger than their peripheral blood counterparts, depending on the state of the lung, especially if they are activated.

[0163] In some embodiments, administration of a therapeutically effective bolus dose of the composition to a subject reduces the total number of cells and total protein in a BALF sample derived from the subject. In certain embodiments, administration of a therapeutically effective bolus dose of the composition to a subject reduces MPO (activated neutrophil marker) in lung tissue derived from the subject. In certain embodiments, administration of a therapeutically effective bolus dose of the composition to a subject weakens (i.e., reduces) the expression of inflammatory cytokines (e.g., IL-6, IL-1b, TNFα, MCP-1, and IFNg) in the subject. In certain embodiments, administration of a therapeutically effective bolus dose of the composition to a subject reduces the fibrotic area of ​​the lung in subjects with idiopathic pulmonary fibrosis. In certain embodiments, administration of a therapeutically effective bolus dose of the composition to a subject reduces the fibrotic area in the lung, liver, or kidney. In some embodiments, administration of a therapeutically effective bolus dose of the composition to a subject results in a decrease in cell count and protein levels, as well as a reduction in the expression of one or any combination of IL-6, IL-1b, TNFα, MCP-1, and IFNg in subjects with idiopathic pulmonary fibrosis. In some embodiments, administration of a therapeutically effective bolus dose of the composition to a subject results in a reduction in the expression of one or any combination of AST, ALT, α-SMA, IL-6, IL-1b, TNFα, MCP-1, IFNg, iNOS, Elf-1, Tollip, IRAK-1, P-P38, P-P65, β-act, STAT1, P-STAT1, STAT2, STAT3, STAT6, fibrosis area, serum creatinine, urinary albumin / creatinine ratio, and hydroxyproline in the lungs.

[0164] The following embodiments are provided for illustrative purposes only, not to limit the present disclosure. [Examples]

[0165] All peptides used in the examples were synthesized using solid-phase peptide synthesis (SPPS) (BLDPharm, Shanghai, China) in an automated microwave peptide synthesizer (LibertyBlue HT12, CEM, Matthews, NC) with H-Cys(Trt)-2-Cl-Trt Resin (0.54 mmol / g) as the starting solid support. Each component molecule of the peptide, such as an amino acid, fatty acid, or protected alkyl diacid, was sequentially immobilized on this peptide resin using Fmoc chemistry known to those skilled in the art, to obtain linearly chained protected peptides linked to this resin. The linear crude peptides were deprotected and released from the resin by acid decomposition with trifluoroacetic acid and ether precipitation in the presence of a carbocation scavenger. The obtained linear peptides were cyclized by dissolving them in 10% DMSO and 20% acetonitrile aqueous solution and reacted for at least 2 days to form disulfide bonds. Finally, this peptide is mixed with 10% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA) and 0.1% The peptides were purified and characterized by reverse-phase HPLC (1260 Infinity II Preparative LC Systems, Santa Clara, CA) using a gradient with acetonitrile containing TFA. This gradient was run at 40 mL / min over 24 minutes at room temperature using a Waters 30×150 mm XBridge C18 column (P / N 186003284) and a Waters C18 prep column (P / N 186006893), and acquired at 214 nm. The peptide fraction with a purity of over 90% was collected and dried as a white powder to obtain the final peptide product.

[0166] Example 1: Superior in vivo performance of long-acting CNP compared to native CNP when administered as a bolus. All mice used in this study were maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF Feed, Oriental Yeast Co., Ltd., Tokyo, Japan, or PicoLab Rodent Diet 20, LabDiet Corp., St. Louis, Missouri).

[0167] For pharmacokinetic studies, female CD-1 mice (6-8 weeks old from the Charles River laboratory) were treated with 2.0 mg / kg of native human CNP (Chempep Inc. Wellington, FL), a long-acting CNP derivative (dCNP, Chempep Inc. Wellington, FL), or an ultra-long-acting CNP derivative (VLA-dCNP) via subcutaneous administration between the scapulae. All test products were formulated or dissolved in 100 mM sorbitol, 100 mM methionine, 20 mM histidine, pH 6.0. Blood samples were taken at various time points (0, 0.5, 1, 2, 3, 4, 5, and 24 for native CNP; 0, 1, 2, 4, 8, 12, 24, 48, and 72 for dCNP and VLA-dCNP) due to retroorbital hemorrhage, with each animal hemorrhaging twice at two different time points. Blood samples were processed in K2EDTA tubes to obtain plasma. The plasma was analyzed using a commercially available CNP ELISA kit from Phoenix Pharmaceuticals (catalog # EKE-012-03). The CNP was native human CNP (GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 10]), and the dCNP was the following sequence CH3(CH2). 16 VLA-dCNP is one of the addition derivatives of human CNP having C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[SEQ ID NO: 6]. VLA-dCNP is a co-formulation of dCNP and a PK-expanding polymer excipient in a dCNP:excipient weight ratio of 1:10. Details of this polymer are described in Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18, which are incorporated herein by reference in their entirety.

[0168] For pharmacokinetic studies of the cyclic GMP reaction test, male C57BL / 6J mice (6 weeks old, Kyudo Co., Ltd.; Saga, Japan) were treated with 1.0 mg / kg of native human CNP, long-acting CNP derivative (dCNP), and ultra-long-acting CNP derivative (VLA-dCNP) via subcutaneous bolus administration between the scapulae. All test products were formulated or dissolved in 100 mM sucrose, 100 mM methionine, 50 mM histidine, pH 7.4. Blood samples were taken at various time points (0, 1, 4, 8, 12, and 24 hours for native CNP and dCNP; 0, 1, 2, 4, 8, 5, 24, and 48 hours for dCNP and VLA-dCNP) by abdominal aortic blood sampling after laparotomy, with one bleeding dose per animal per time point. To obtain plasma, EDTA (final concentration 1.5 mg / mL, Dojin Chemical Laboratories, Kumamoto, Japan) and aprotinin (final concentration 500 KIU / mL, Sigma Aldrich, St. Louis MO) were added to blood and centrifuged (×2,000 g; 15 min, 4C). After collecting the supernatant, the plasma samples were stored at -80°C. The plasma samples were analyzed using a commercially available cyclic GMP kit from CisBio (Codolet, France). The CNP was native human CNP (GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 10]), and the dCNP was the following sequence: CH3(CH2) 16(C=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 6] is one of the addition derivatives of human CNP. VLA-dCNP is a co-formulation of dCNP and a PK-expanding polymer excipient in a dCNP:excipient weight ratio of 1:10. Details of this polymer are described in Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18, which are incorporated herein by reference in their entirety. Specifically, this PK expander excipient was prepared using N-hydroxysulfosuccinimide reagent and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to activate the carboxyl groups of polyethylene glycol (PEG) for attachment of linear polylysine skeletons with molecular weights ranging from 15 to 40 kDa (average molecular weight of polylysine of 25 kDa, determined by multi-angle laser scattering or MALLS) to epsilon-amino groups of 5 kDa polyethylene glycol (PEG) (in an epsilon-amino:NHSS:EDC:PEG carboxyl group molar ratio of 0.2:1:1:0.3). The product was characterized by trinitrobenzenesulfonic acid (TNBS)amino in process analysis. It is estimated that 55% of the epsilon-amino groups were depleted during the PEG addition reaction, and the remaining epsilon-amino groups were depleted during the stearic acid addition reaction using NHS-stearic acid. When measured by TNBS, the amount of measurable amino groups present at the end of stearic acid addition was very small (less than 5%). This PK volume-expanding excipient was purified by an ultrafiltration process known to those skilled in the art. The buffering agents used for bolus administration with and without the PK volume-expanding excipient were 100 mM sucrose, 100 mM methionine, and 50 mM histidine.

[0169] Referring to Figure 1A, plasma CNP levels [mean (SD); n=5] for CD-1 mice after subcutaneous administration of native CNP, CNP derivative (dCNP), and ultra-long-acting CNP derivative (VLA-dCNP) at a dose of 2.0 mg / kg are shown. The inset is an enlarged scale in the lower left corner showing the low plasma CNP levels (diamond) after administration of native CNP. Error bars represent the standard deviation of the n=5 plasma samples. The baseline CNP level before administration was 1.74 (0.6) ng / mL [mean (SD); n=15]. Figure 1B is a plot showing plasma cyclic GMP in male C57BL / 6J mice measured using the CisBio (Codolet, France) cyclic GMP kit after subcutaneous administration of native CNP, long-acting CNP derivative (dCNP), and ultra-long-acting CNP derivative (VLA-dCNP) at a dose of 1.0 mg / kg. Baseline plasma cyclic GMP levels were 20(3.7) pmol / mL [mean (SEM); n=8] or 7(1.3) ng / mL [mean (SEM); n=8]. Subcutaneous administration of native CNP did not show a significant increase in plasma cyclic GMP compared to baseline after 2 hours, but similar administration of long-acting CNP (dCNP and VLA-dCNP) showed a significant increase in cyclic GMP for at least 24 hours.

[0170] Example 2: High-dose bolus administration of an ultra-long-acting CNP derivative (VLA-dCNP) can surprisingly increase plasma cyclic GMP without a corresponding decrease in blood pressure. For this study, the following three different long-acting natriuretic peptides were used (ultra-long-acting ANP derivatives or VLA-dANP; modified ANP similar to dCNP, where VLA-ANP is CH3(CH2)16C(=O)KKKKGGG-SLRRSSCFGGRMDRIGAQSGLGCNSFRY[SEQ ID NO: 28] + PK volume expander excipient, and dANP is CH3(CH2) 16The cardiovascular and hemodynamic effects were evaluated for C(=O)KKKKGGG-SLRRSSCFGGRMDRIGAQSGLGCNSFRY [SEQ ID NO: 28] alone. The PK volume-expanding excipient was a polymer excipient described in Example 1 above and incorporated herein by reference in whole by Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18. The ultra-long-acting BNP or VLA-dBNP was CH3(CH2) 16 C(=O)KKKKGGG-SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH(dBNP)[SEQ ID NO: 29] + the PK volume expander excipient described above. dBNP, CH3(CH2) 16C(=O)KKKKGGG-SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH [SEQ ID NO: 29] does not contain the PK expanding excipient described above. VLA-dCNP is dCNP as described in Example 1 + the PK expanding polymer excipient described above. dCNP is as described in Example 1 and does not contain the PK expanding excipient. These formulations (in 100 mM sucrose, 100 mM methionine, and 50 mM histidine buffer) were administered to beagle dogs [n=12 animals / test product; the same animals were used for other test products after a washout period of at least one week]. These test products were administered by a single subcutaneous injection containing 25 μg / Kg of peptide and 1 mg / Kg of PK expanding polymer (2.5% load). Twelve animals were pre-equipped with telemetry transmitters at Data Sciences International (St. Paul, MN), and heart rate, mean arterial pressure, systolic arterial pressure, diastolic arterial pressure, PR interval, QRS duration, QT interval, and body temperature were continuously recorded. All animals were monitored for 7 days for each dose. At 4, 6, 8, 16, 20, 24, 28, 32, 40, 48, 66, 78, 90, 102, 114, 126, 138, 150, 162, and 174 hours after each dose, 3 mL blood samples were collected in K3EDTA collection tubes and stored on wet ice until spun in a refrigerated centrifuge. Plasma was collected and treated with plasma preservation reagent (phosphate (in deionized water), 15:85, v / v). The samples were inverted several times and then frozen with dry ice. This sample was stored in a freezer (-80°C) and then transported on dry ice for LC-MS analysis of cyclic GMP.

[0171] All natriuretic peptides are thought to act by increasing cytoplasmic cyclic GMP production, thereby causing a corresponding decrease in blood pressure. However, when comparing bolus doses of the long-acting forms of the three major natriuretic peptides, it was surprisingly found that high bolus doses of the ultra-long-acting CNP derivative of this disclosure (sufficient to increase blood cyclic GMP over 3 days) could increase plasma cyclic GMP without causing a dangerous drop in blood pressure. In contrast, the similarly developed ultra-long-acting ANP and BNP derivatives caused a significant drop in blood pressure when administered as bolus doses (sufficient to increase blood cyclic GMP over 3 days). In the case of the ultra-long-acting ANP derivative, the blood pressure reduction was as high as 45%, and in the case of the ultra-long-acting BNP derivative, it was as high as 20%. For all three long-acting natriuretic peptide derivatives, the increase in cyclic GMP was more than 1.5 times and 6 times from baseline. The cyclic GMP AUCs are 3,483 ng*h / mL for VLA-dANP, 2,585 ng*h / mL for VLA-dBNP, and 2,627 ng*h / mL for VLA-dCNP.

[0172] Figure 2A shows the corresponding increase in plasma cyclic GMP [mean (SEM); n=12] after monitoring following bolus administration of 25 ug / kg ultra-long-acting CNP derivative (VLA-dCNP), ultra-long-acting BNP derivative (VLA-dBNP), and ultra-long-acting BNP derivative (VLA-dANP). The baseline plasma cyclic GMP level was 8 (0.2) ng / mL [mean (SEM); n=12], which is similar to that of healthy individuals. See, for example, Igaki, et al., Hypertens Res 1998;21:7-13. All ultra-long-acting formulations of natriuretic peptides increased cyclic GMP above the baseline of 8 ng / mL. The cyclic GMP AUC values ​​were 3,483 ng*h / mL for VLA-dANP, 2,585 ng*h / mL for VLA-dBNP, and 2,627 ng*h / mL for VLA-dCNP. The ultra-long-acting CNP derivative (VLA-dCNP) increased plasma cyclic GMP over 3 days without associated blood pressure decreases.

[0173] Figure 2B shows the mean arterial pressure in dogs [mean (SEM); n=12] monitored after bolus administration of 25 ug / kg ultra-long-acting CNP derivative (VLA-dCNP), ultra-long-acting BNP derivative (VLA-dBNP), and ultra-long-acting BNP derivative (VLA-dANP). VLA-dCNP did not cause a significant decrease in blood pressure from baseline (0 hours) after administration at very high doses. In contrast, other ultra-long-acting natriuretic peptides, such as VLA-dBNP and VLA-dANP derivatives, caused a decrease in blood pressure of more than 15%. This was particularly true for VLA-dANP, where a decrease in blood pressure of up to 50% could occur in the case of a similar increase in cyclic GMP. The ultra-long-acting CNP derivative (VLA-dCNP) increased plasma cyclic GMP over 3 days without associated decreases in blood pressure.

[0174] Example 3: Bolus administration of an ultra-long-acting CNP derivative suppresses lung damage. In ALI and ARDS, an increase in cells, particularly neutrophils, is observed in bronchoalveolar lavage fluid (BALF). Therefore, the number of cells and total protein count in neutrophil markers (Figure 3) and MPO (Figure 4) are measured. A decrease in MPO-positive cells (neutrophils) and total protein count indicates the resolution of ALI / ARDS. Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were treated with lipopolysaccharide (LPS) (Sigma-Aldrich; 0.05 mg / kg intratracheally) and then treated with various test substances. The test substances were: ultra-long-acting CNP derivative or VLA-dCNP (described in Example 1) (low (L) 0.1 mg / kg sc; medium (M) 0.3 mg / kg sc; high (H) 1.0 mg / kg sc), native C-type natriuretic peptide or CNP (high 1.0 mg / kg sc), long-acting CNP derivative or dCNP (described in Example 1) (high 1.0 mg / kg sc), atrial natriuretic peptide (ANP) (high 1.0 mg / kg sc), B-type natriuretic peptide or BNP (high 1.0 mg / kg sc), the anti-inflammatory drug antitumor necrosis factor α antibody or TNFα ab (clone XT3.11; BioXcell West Lebanon, NH) (1.0 mg / kg sc), and a cyclic GMP degradation inhibitor or PDE5 inhibitor called vardenafil (VDN, Cayman Chemicals Ann Arbor, MI) (1.0 mg / kg The study was conducted using sc. The test substance was administered immediately after LPS administration. The study included a normal control group (NC) that did not receive LPS treatment and an LPS-treated group (control) that did not receive the test substance. 24 hours after treatment, mice were sacrificed under isoflurane anesthesia, and bronchoalveolar lavage fluid (BALF) was collected. The total number of cells in the BALF was counted in a counting chamber. The total protein concentration in the BALF was measured using the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific).Statistical analysis was performed based on Dunnett's test using GraphPad InStat 3 (n=15, 23, 7, 7, 7, 7, 7, 7 and 9; NC, control, CNP(H), dCNP(H), ANP(H), BNP(H), anti-TNFα ab, VDN, VLA-dCNP(H). *P<0.01 (against VLA-dCNP(H))). In ALI and ARDS, an increase in cells (especially neutrophils) in the BALF is observed. Therefore, the number of cells in the BALF and MPO, an activated neutrophil marker, were measured.

[0175] Figure 3A is a timeline of the protocol for evaluating dCNP-suppressed LPS-induced acute lung injury. Figure 3B shows the increase in cells, particularly neutrophils, in BALF in ALI and ARDS according to the protocol shown in Figure 3A. A decrease in cells indicated the resolution of ALI / ARDS. Statistical analysis was performed based on Dunnett's test using GraphPad InStat 3 (n=15, 23, 7, 7, 7, 7, 7, 7, and 9; NC, control, CNP(H), dCNP(H), ANP(H), BNP(H), TNFα ab, VDN, VLA-dCNP(H). *P<0.01 (against VLA-dCNP(H))). Figure 3C shows the total protein in BALF in ALI and ARDS according to the protocol shown in Figure 3A. A decrease in total protein indicated the resolution of ALI / ARDS. Statistical analysis was performed based on Dunnett's test using GraphPad InStat 3 (n=15, 23, 7, 7, 7, 7, 7, 7, and 9; NC, control, CNP(H), dCNP(H), ANP(H), BNP(H), TNFα ab, VDN, VLA-dCNP(H). *P<0.01 (for VLA-dCNP(H))).

[0176] Example 4. Lung therapy using dCNP and VLA-dCNP A) dCNP and VLA-dCNP reduce neutrophil infiltration in the lungs, indicating the resolution of ALI / ARDS. Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mice were treated with LPS (Sigma-Aldrich; 0.05 mg / kg intratracheal administration), and then with ultra-long-acting CNP derivatives or VLA-dCNP (described in Example 1) (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivatives or dCNP (described in Example 1) (1.0 mg / kg sc), atrial natriuretic peptide (ANP) (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), antitumor necrosis factor α antibody or TNFα ab (clone XT3.11; BioXcell West Lebanon, NH) (1.0 mg / kg sc), and a PDE5 inhibitor called a cyclic GMP degradation inhibitor or vardenafil (VDN) (Cayman Chemicals Ann Arbor, MI) (1.0 mg / kg sc). The test substances were administered immediately after LPS administration. Twenty-four hours after the procedure, mice were sacrificed under isoflurane anesthesia, lung tissue was collected, and fixed with 4% paraformaldehyde. Paraffin sections of the fixed lung tissue were immunostained with anti-MPO rabbit polyclonal antibody (Agilent Technologies Santa Clara, CA), horseradish peroxidase (HRP)-labeled anti-rabbit IgG goat polyclonal antibody (Nichirei Bioscience Corporation, Tokyo, Japan), and 3,3'-diaminobenzidine-4HCl (DAB) (Agilent Technologies Santa Clara, CA). The number of MPO-positive cells per field of view was counted. Statistical analysis was performed based on Dunnett's test using GraphPad InStat 3 (n=18, 6, 6, 6, 6, 6, and 6; control, CNP, dCNP, ANP, BNP, anti-TNFα ab, VDN, VLA-dCNP; *P<0.01 (for VLA-dCNP) and **P<0.05 (for VLA-dCNP)).In cases of ALI and ARDS, an increase in cells (especially neutrophils) in the BALF is observed. Therefore, the number of cells in the BALF (Figure 3B) and the activated neutrophil marker MPO (Figure 4A) are measured. Inflammatory cell infiltration observed with HE staining indicates inflammation of the lung.

[0177] Figure 4A shows that VLA-dCNP treatment reduced the number of MPO-positive neutrophils, where MPO is a marker pro-inflammatory neutrophil granulocyte. Statistical analysis was performed based on Dunnett's test using GraphPad InStat 3 (n=18, 6, 6, 6, 6, 6, and 6; control, CNP, dCNP, ANP, BNP, TNFα ab, VDN, VLA-dCNP; *P<0.01 (for VLA-dCNP) and **P<0.05 (for VLA-dCNP)).

[0178] B) dCNP and VLA-dCNP reduced inflammatory cell infiltration or inflammation in the lungs. Increased H&E staining indicates inflammatory cell infiltration or inflammation in the lungs. Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). After treating the mice with LPS (Sigma-Aldrich; 0.05 mg / kg intratracheal administration), they were administered ultra-long-acting CNP derivative or VLA-dCNP (described in Example 1) (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivative or dCNP (described in Example 1) (1.0 mg / kg sc), atrial natriuretic peptide (ANP) (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), antitumor necrosis factor α antibody or TNFα ab (1.0 mg / kg The mice were treated with a PDE5 inhibitor (1.0 mg / kg sc) called a cyclic GMP degradation inhibitor or vardenafil (VDN). The test substance was administered immediately after LPS administration. Twenty-four hours after treatment, the mice were sacrificed under isoflurane anesthesia, lung tissue was collected, and fixed with 4% paraformaldehyde. Paraffin sections of the fixed lung tissue were stained with hematoxylin and eosin. Hematoxylin and eosin (H&E) stains are essential for recognizing various tissue types and morphological changes. These stains present a wide variety of cytoplasmic, nuclear, and extracellular matrix features. Hematoxylin stains cell nuclei blue, representing the number of cells or multinucleated cells, while eosin generally stains proteins pink, indicating cytoplasmic and extracellular matrix proteins. Increased H&E staining indicated inflammatory cell infiltration or inflammation in the lungs.

[0179] Figure 4B shows micrographs of paraffin sections of lung tissue stained with hematoxylin and eosin (HE), revealing increased nucleated cells, cell count, extracellular matrix and overall protein, scarring, and / or increased protein permeability into the alveolar space. Inflammatory cell infiltration observed by HE staining indicates inflammation of the lung (the right panel shows darker staining as cell count and protein increase). For these studies, mice were treated with LPS (Sigma-Aldrich; 0.05 mg / kg intratracheally) and then with ultra-long-acting CNP derivatives or VLA-dCNP (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivatives or dCNP (1.0 mg / kg sc), atrial natriuretic peptide (ANP) (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), antitumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a PDE5 inhibitor called a cyclic GMP degradation inhibitor or vardenafil (VDN) (1.0 mg / kg sc). The test substances were administered immediately after LPS administration. 24 hours after treatment, mice were sacrificial under isoflurane anesthesia, lung tissue was excised, and fixed with 4% paraformaldehyde. Paraffin sections of fixed lung tissue were stained with anti-MPO antibody (brown to dark brown) and hematoxylin-eosin stain (nuclei are blue-purple, proteins are pink).

[0180] Example 5: VLA-dCNP and dCNP treatment weakened LPS-induced upregulation of inflammatory cytokines in BALF. Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mice were treated with LPS (Sigma-Aldrich; 0.05 mg / kg intratracheal administration), and then with ultra-long-acting CNP derivatives or VLA-dCNP (described in Example 1) (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivatives or dCNP (described in Example 1) (1.0 mg / kg sc), atrial natriuretic peptide (ANP) (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), antitumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a PDE5 inhibitor called a cyclic GMP degradation inhibitor or vardenafil (VDN) (1.0 mg / kg sc). The test substances were administered immediately after LPS administration. 24 hours after treatment, mice were sacrificed under isoflurane anesthesia, and bronchoalveolar lavage fluid (BALF) was collected. The concentrations of each cytokine, specifically interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), were measured using commercially available Time Resolution FRET Kits (Cisbio, Bedford MA). Macrophage chemoattractant protein-1 (MCP-1) was measured using an ELISA kit (R&D SYSTEMS, Minneapolis MN). Previous studies have revealed the role of TNFα (PLoS One, 2014 Jul22;9(7):e102967), as well as elevated TNFα and IL-6 levels in non-survivors (Chest, 1997:111:1306-21), and elevated MCP-1 levels in patients who developed ARDS / ALI (International Journal of Molecular Sciences, 2019:20(9):2218). Statistical analysis was performed based on Student's t-test using GraphPad.

[0181] Figure 5A shows that VLA-dCNP and dCNP treatment weakened LPS-induced upregulation of inflammatory cytokines (IL6) in BALF and promoted the resolution of ARDS / ALI. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration) and then with ultra-long-acting CNP derivatives or VLA-dCNP (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivatives or dCNP (1.0 mg / kg sc), atrial natriuretic peptide (ANP) (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), antitumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a PDE5 inhibitor called a cyclic GMP degradation inhibitor or vardenafil (VDN) (1.0 mg / kg sc). 24 hours after treatment, bronchoalveolar lavage fluid (BALF) was collected and IL-6 cytokines were measured. Statistical analysis was performed based on Student's t-test. (n=15, 23, 7, 7, 7, 7, 7, 7 and 9; NC, control, CNP, dCNP, ANP, BNP, TNFα ab, VDN, and VLA-dCNP. *P<0.01 (for VLA-dCNP) and **P<0.05 (for VLA-dCNP)).

[0182] Figure 5B shows that VLA-dCNP and dCNP treatment weakened LPS-induced upregulation of inflammatory cytokines (TNFα) in BALF, thereby promoting the resolution of ARDS / ALI. The protocol was the same as that described in Figure 5A, except that bronchoalveolar lavage fluid (BALF) was collected and TNFα cytokine levels were measured.

[0183] Figure 5C shows that VLA-dCNP and dCNP treatment weakened LPS-induced upregulation of inflammatory cytokine (MCP-1) in BALF, thereby promoting the resolution of ARDS / ALI. The protocol was the same as that described in Figure 5A, except that bronchoalveolar lavage fluid (BALF) was collected and MCP-1 cytokine levels were measured.

[0184] Example 6: VLA-dCNP treatment weakened LPS-induced upregulation of inflammatory cytokines in lung tissue. Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were treated with LPS (Sigma-Aldrich; 0.05 mg / kg intratracheal administration) and then with VLA-dCNP (1.0 mg / kg sc). VLA-dCNP (described in Example 1) was administered immediately after LPS administration. 24 hours after treatment, the mice were anesthetized with isoflurane and then sacrificed. Proteins from collected lung tissue were extracted using a cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton® X-100, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7) supplemented with 1 mM PMSF, 2 μg / ml aprotinin, and 1 mM pervanadate. The concentrations of each cytokine in the extracted lung proteins, namely interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and macrophage chemoattractant protein-1 (MCP-1), were measured using an ELISA kit (R&D SYSTEMS, Minneapolis MN). Statistical analysis was performed based on Student's t-test using GraphPad Prism 6.

[0185] Figures 6A-6D show that VLA-dCNP treatment weakened LPS-induced upregulation of inflammatory cytokines in lung tissue, promoting the resolution of ARDS / ALI. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration) and then with VLA-dCNP (1.0 mg / kg sc). Lung tissue was collected 24 hours after treatment. The concentrations of each cytokine in the extracted lung proteins, namely interleukin-6 (IL-6) (Figure 6A), tumor necrosis factor α (TNF-α) (Figure 6B), interleukin-1β (IL-1β) (Figure 6C), and macrophage chemoattractant protein-1 (MCP-1) (Figure 6D), were measured using an ELISA kit. Statistical analysis was performed based on Student's t-test (n=10, 10, 9; NC, control, VLA-dCNP; *P<0.05 (compared to control)).

[0186] Example 7: VLA-CNP weakened the expression of LPS-induced inflammatory cytokines such as IL-6, TNFα, and IL-1b, which are generally regulated by the NFκb system, the master regulator of the inflammatory system. This suggests that VLA-dCNP broadly suppresses the inflammatory response in the target body and dissipates ARDS / ALI (Figure 7). Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mice were treated with LPS (Sigma-Aldrich; 0.05 mg / kg intratracheal administration), and then with ultra-long-acting CNP derivatives or VLA-dCNP (described in Example 1) (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivatives or dCNP (described in Example 1) (1.0 mg / kg sc), atrial natriuretic peptide or ANP (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), tumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a PDE5 inhibitor called a cyclic GMP degradation inhibitor or vardenafil (VDN) (1.0 mg / kg sc). The test substances were administered immediately after LPS administration. Twenty-four hours after treatment, mice were anesthetized with isoflurane and sacrificed. Lung tissue was then collected, shredded in TRI reagent (Molecular Research Center, Inc. Cincinnati, OH), and kept at -80°C until analysis. Total RNA was extracted from the collected lung tissue using the chloroform-phenol method. Complementary DNA (cDNA) was synthesized from the extracted mRNA using a cDNA kit (Qiagen, Hilden, Germany). Quantitative RT-PCR analysis was performed using a premix kit (Takara Bio, Shiga, Japan). Several studies showed that the use of specific iNOS inhibitors and / or iNOS knockout animals supported the claim that NO / iNOS is the cause of oxidative stress and endothelial damage in endotoxin-induced ARDS / ALI (World Journal of Critical Care Medicine, 2012 1(2):50-60). Statistical analysis was performed based on Student's t-test using GraphPad.

[0187] Figure 7A shows that VLA-dCNP reduced the expression of LPS-induced inflammatory cytokines such as IL-6, which are generally regulated by the NFκb system, the master regulator of the inflammatory system. This suggests that VLA-dCNP broadly suppressed the inflammatory response in the subject's body, thereby promoting the resolution of ARDS / ALI. Measurement of inflammation-related gene expression in ALI lung tissue. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration), and then treated with ultra-long-acting CNP derivatives or VLA-dCNP (1.0 mg / kg sc), native C-type natriuretic peptide or CNP (1.0 mg / kg sc), CNP derivatives or dCNP (1.0 mg / kg sc), atrial natriuretic peptide or ANP (1.0 mg / kg sc), B-type natriuretic peptide or BNP (1.0 mg / kg sc), tumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a cyclic GMP degradation inhibitor or a PDE5 inhibitor called vardenafil (VDN) (1.0 mg / kg sc). Lung tissue was collected 24 hours after treatment. Total RNA was extracted from the collected lung tissue. Statistical analysis was performed based on Student's t-test. (n=15, 22, 6, 6, 6, 6, 6, 5 and 9; NC, control, CNP, dCNP, ANP, BNP, TNFα ab, VDN, and VLA-dCNP. *P<0.01 (for VLA-dCNP) and **P<0.05 (for VLA-dCNP)).

[0188] Figure 7B shows that VLA-dCNP attenuated the expression of LPS-induced inflammatory cytokines such as iNOS, suggesting that VLA-dCNP broadly suppressed the target inflammatory response and promoted the resolution of ARDS / ALI. The protocol was as described in Figure 7A.

[0189] Referring to Figure 7C, VLA-dCNP reduced the expression of LPS-induced inflammatory cytokines such as MCP-1, suggesting that VLA-dCNP broadly suppressed the inflammatory response in the subjects' bodies and promoted the resolution of ARDS / ALI. The protocol was as described in Figure 7A.

[0190] Referring to Figure 7D, VLA-dCNP reduced the expression of LPS-induced inflammatory cytokines such as IL1b, suggesting that VLA-dCNP broadly suppressed the inflammatory response in the subjects' bodies and promoted the resolution of ARDS / ALI. The protocol was as described in Figure 7A.

[0191] Referring to Figure 7E, the bar graph shows that VLA-dCNP attenuated the expression of LPS-induced inflammatory cytokines such as IFNg, suggesting that VLA-dCNP broadly suppressed the inflammatory response in the subjects' bodies and promoted the resolution of ARDS / ALI. The protocol was as described in Figure 7A.

[0192] Example 8: VLA-dCNP suppressed inflammation levels in lung tissue. Tollip is a negative regulator of the TLR-dependent inflammatory pathway. This data showed that VLA-dCNP (described in Example 1) upregulates negative regulators of the TLR-dependent inflammatory pathway, which may contribute to the anti-inflammatory effect of the compound (Journal of Biological Chemistry, 2002;227:7059-7065). IRAK1, P-P38, and P-P65 were measured and are known important mediators in the Toll-like receptor 4 (TLR-4)-dependent inflammatory pathway, which is essential for lipopolysaccharide (LPS)-induced ALI. TLR-4 is the receptor for LPS and plays a crucial role in LPS-induced inflammatory responses such as ALI and sepsis. Tollip is an endogenous negative regulator that can attenuate TLR4-dependent signaling, and ELF-1 suppresses Tollip expression in cells. When Elf-1 is downregulated, Tollip expression is upregulated, which may suppress LPS-induced inflammation. Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mice were treated with LPS (Sigma-Aldrich; 0.05 mg / kg intratracheal administration) and then with an ultra-long-acting CNP derivative or VLA-dCNP (1.0 mg / kg sc). VLA-dCNP was administered immediately after LPS administration. 24 hours after treatment, mice were anesthetized with isoflurane and sacrificed. Lung tissue was dissolved in a cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton® X-100, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7) supplemented with 1 mM PMSF, 2 μg / ml aprotinin, and 1 mM pervanadate. The sample was then added together with 2-mercaptoethanol (Fujifilm, Tokyo, Japan) and sodium dodecyl sulfate (SDS) solution and boiled. Western blot analysis was performed using SDS gel (Bio-rad, Hercules CA) and PVDF membrane (Merck Millipore, Burlington MA).After a 2.5% BSA blocking step, the membrane was detected using antibodies against Elf-1 (Santa Cruz Biotechnology, Dallas, Texas), Tollip (Protein Tech, Tokyo, Japan), IRAK-1 (Cell Signaling Technology, Danvers, MA), P-P38 (Cell Signaling Technology, Danvers, MA), P-P65 (Santa Cruz Biotechnology, Dallas, Texas), and β-actin (Sigma-Aldrich, St. Louis, MO). Subsequently, after incubation with a secondary antibody (Abcam, Cambridge, UK), the membrane was washed with 1% Tween® TBS. The membrane was then detected using an image analysis system (Vilber Lourmat, Collegien France). Statistical analysis was performed based on Student's t-test using GraphPad Prism 6.

[0193] Referring to Figure 8, VLA-dCNP suppressed inflammation levels in lung tissue and promoted the resolution of ARDS / ALI. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration) and then with VLA-dCNP (1.0 mg / kg sc). Lung tissue was collected 24 hours after treatment. Western blot analysis was performed using antibodies against Elf-1, Tollip, IRAK-1, P-P38, P-P65, and β-actin (internal standard). Statistical analysis was performed based on Student's t-test (n=5, *P<0.05 (against control)).

[0194] Example 9: VLA-dCNP suppressed STAT levels in lung tissue, indicating a reduction in inflammation. Furthermore, STAT1, P-STAT1, STAT2, and STAT3 are also involved in iNOS expression, which contributes to ARDS / ALI. Several studies have shown that the use of specific iNOS inhibitors and / or iNOS knockout animals supports the claim that NO / iNOS is responsible for oxidative stress and endothelial damage in endotoxin-induced ARDS / ALI (World Journal of Critical Care Medicine, 2012 1(2):50-60). In addition, STAT6-deficient mice show reduced airway inflammation (Journal of Immunology, 2013, 190:904-912). Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mice were treated with LPS (Sigma-Aldrich; 0.05 mg / kg intratracheal administration), and then with VLA-dCNP (described in Example 1) (1.0 mg / kg sc). VLA-dCNP was administered immediately after LPS administration. 24 hours after treatment, the mice were anesthetized with isoflurane and sacrificed. Lung tissue was dissolved in cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton® X-100, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7) supplemented with 1 mM PMSF, 2 μg / ml aprotinin, and 1 mM pervanadate. The samples were then added together with 2-mercaptoethanol (Fujifilm, Tokyo, Japan) and sodium dodecyl sulfate solution and brought to a boil. Western blot analysis was performed using SDS gel (Bio-Rad, Hercules CA) and PVDF membrane (Merck Millipore, Burlington MA).After a 2.5% BSA blocking step, the membrane was detected using antibodies against STAT-1 (Cell Signaling Technology (CST), Danvers MA), P-STAT-1 (CST), STAT-2 (CST), STAT-3 (CST), STAT-6 (CST), and β-actin (Sigma-Aldrich, St. Louis MO). Subsequently, after incubation with a secondary antibody (Abcam, Cambridge, UK), the membrane was washed with 1% Tween® TBS. The membrane was then detected using an image analysis system (Vilber Lourmat, Collegien France). Statistical analysis was performed based on Student's t-test using GraphPad Prism 6.

[0195] Referring to Figure 9, VLA-dCNP suppressed STAT levels in lung tissue and promoted the resolution of ARDS / ALI. Male C57BL / 6J mice (6 weeks old) were treated with LPS (0.05 mg / kg intratracheal administration) and then with VLA-dCNP (1.0 mg / kg sc). Lung tissue was collected 24 hours after treatment. Western blot analysis was performed using antibodies: anti-STAT-1, P-STAT-1, STAT-2, STAT-3, STAT-6 and β-actin (internal standard). Statistical analysis was performed based on Student's t-test (n=5, *P<0.05 (compared to control)).

[0196] Example 10. VLA-dCNP suppressed Elf-1 expression in human umbilical vein endothelial cells, demonstrating suppression of TLR-dependent inflammation, including LPS, or TLR-dependent injury-associated molecular pattern (DAMP) / pathogen-associated molecular pattern (PAMP)-induced inflammation. Toll-like receptors (TLR4) are receptors for lipopolysaccharide (LPS) and play a crucial role in LPS-induced inflammatory responses such as ALI and sepsis. Tollip is an endogenous negative regulator that can weaken TLR4-dependent signaling, and ELF-1 suppresses Tollip expression in cells. When Elf-1 is downregulated, Tollip expression is upregulated, thereby suppressing LPS-induced inflammation.

[0197] Human umbilical vein endothelial cells (HUVECs) were purchased from Takara Bio (Shiga, Japan). The cells were maintained in HuMedia-EG2 medium purchased from Kurabo. The cells were placed in 12-well plates (Nunc, Roskilde, Denmark) in 2 mL of HuMedia-EG2, with 1 × 10⁶ cells per plate. 5Cells were seeded at a cell / well density. After 24 hours, the cells were treated for 6 hours with various concentrations of VLA-dCNP (described in Example 1) in M199 (Thermo Fisher Scientific, Waltham MA) supplemented with 1% BSA (Sigma-Aldrich, St. Louis MO). The cells were then lysed in cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton® X-100, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7) supplemented with 1 mM PMSF, 2 μg / ml aprotinin, and 1 mM pervanadate. The samples were then added together with 2-mercaptoethanol and sodium dodecyl sulfate (SDS) solutions and brought to a boil. Western blot analysis was performed using SDS gel (Bio-rad, Hercules CA) and PVDF membrane (Merck Millipore, Burlington MA). After a 2.5% BSA blockage step, the membrane was detected using an antibody against Elf-1 (Santa Cruz Biotechnology, Dallas TX) and β-actin (Sigma-Aldrich, St. Louis MO). Subsequently, after incubation with a secondary antibody (Abcam, Cambridge, UK), the membrane was washed with 1% Tween® TBS. The membrane was detected using an image analyzer (Vilber Lourmat, Collegien France). Statistical analysis was performed based on Student's t-test using GraphPad Prism 6.

[0198] Referring to Figure 10, VLA-dCNP suppressed Elf-1 expression in human umbilical vein endothelial cells. Human umbilical vein endothelial cells (HUVECs) were maintained in HuMedia-EG2 and inoculated into 12-well plates (1 × 10⁵ cells / well in 2 mL of HuMedia-EG2). After 24 hours, cells were treated with each concentration of VLA-dCNP (0.07 μM (0.21 μg / mL) or 0.7 μM (2.1 μg / mL)) (in M199 1% BSA) for 6 hours. Protein levels were evaluated by Western blotting analysis using antibodies against Elf-1 and β-actin (internal standard). Statistical analysis was performed based on Student's t-test (n=4, *P<0.05 (compared to control)).

[0199] Example 11. VLA-dCNP suppressed Elf-1 levels in the nuclei of human umbilical vein endothelial cells. Human umbilical vein endothelial cells (HUVECs) were purchased from Takara Bio (Shiga, Japan). The cells were maintained in HuMedia-EG2 medium purchased from Kurabo (Osaka, Japan). 1 × 10⁶ cells were placed in 2 mL of HuMedia-EG2. 5 Cells were seeded in glass-bottom dishes at a cell / well density. After 24 hours, the cells were treated for 6 hours with various concentrations of VLA-dCNP (described in Example 1) in M199 (Thermo Fisher Scientific, Waltham MA) supplemented with 1% BSA (Sigma-Aldrich, St. Louis MO). The cells were fixed with 4% paraformaldehyde (Fujifilm, Tokyo, Japan), treated with anti-Elf-1 Ab (Santa Cruz Biotechnology, Dallas TX), and then treated with Alexa Fluor 488-labeled secondary antibody (Thermo Fisher Scientific, Waltham MA). The cells were incubated with MA and Hoechst 33342. Photographs were taken using a fluorescence microscope (Keyence, Osaka, Japan). The superposition of green (Elf-1) and blue (nucleus) fluorescence, and the average fluorescence intensity of green relative to blue, were evaluated. Statistical analysis was performed using GraphPad. The analysis was based on Student's t-test performed using Prism 6.

[0200] Referring to Figure 11, VLA-dCNP suppressed Elf-1 levels in the nuclei of human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells (HUVECs) were maintained in HuMedia-EG2. 1 × 10⁶ cells were kept in 2 mL of HuMedia-EG2. 5 Cells were seeded in glass-bottom dishes at a cell / well density. After 24 hours, cells were treated for 6 hours with VLA-dCNP (0.07 μM (0.21 μg / mL)) or CNP 0.1 μM (0.21 μg / mL) at various concentrations in M199 (Thermo Fisher Scientific, Waltham MA) supplemented with 1% BSA (Sigma-Aldrich, St. Louis MO). Cells were fixed with 4% paraformaldehyde, treated with antibody against Elf-1 Ab (Santa Cruz Biotechnology, Dallas TX), and then incubated with Alexa Fluor 488-labeled secondary antibody (Thermo Fisher Scientific, Waltham MA) and Hoechst 33342.

[0201] Example 12: VLA-dCNP induces Tollip expression in the human lung fibroblast cell line HFL1. Tollip is a negative regulator of the TLR-dependent inflammatory pathway. This data shows that VLA-dCNP (described in Example 1) upregulates the negative regulator of the TLR-dependent inflammatory pathway, which may contribute to the anti-inflammatory effect in vivo. Human lung fibroblast cell line HFL1 was purchased from ATCC (Old Town Manassas, VA). Cells were maintained in Dulbecco's modified Eagle medium (Fujifilm, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) purchased from Sigma Aldrich (St. Louis MO). Cells were placed in 12-well plates (Nunc, Roskilde, Denmark) in 2 mL of 10% FBS DMEM, 1 × 10⁶ cells per well. 5Cells were seeded at a cell / well density. After 16 hours, cells were treated for 12 hours with various concentrations of VLA-dCNP in M199 (Thermo Fisher Scientific, Waltham MA) supplemented with 1% BSA (Sigma-Aldrich, St. Louis MO), and LPS (final concentration 1.0 μg / mL) was added over 2 hours. The cells were lysed in cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton® X-100, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7) supplemented with 1 mM PMSF, 2 μg / ml aprotinin, and 1 mM pervanadate. The samples were then added together with 2-mercaptoethanol and sodium dodecyl sulfate (SDS) solution and brought to a boil. Western blot analysis was performed using SDS gel (Bio-rad, Hercules CA) and PVDF membrane (Merck millipore Burlington MA). After a 2.5% BSA blocking step, the membrane was detected using antibodies against Tollip (Proteintech) and β-actin (Sigma-Aldrich, St. Louis MO), followed by incubation with a secondary antibody (Abcam, Cambridge, UK), and then washing with 1% Tween® TBS. Image analysis was performed using a Vilber image analyzer. The membrane was detected using Lourmat (Collegien France). Statistical analysis was performed based on Student's t-test using GraphPad Prism 6.

[0202] Referring to Figure 12, VLA-dCNP induces Tollip expression in the human lung fibroblast cell line HFL1. Human lung fibroblast cells HFL1 (1.0 × 10⁵ cells / well) were cultured in DMEM medium for 16 hours, and then incubated with 1% BSA-M199 medium containing 0.21 μM (0.66 ug / mL) VLA-dCNP and (NC) without VLA-dCNP. After 12 hours of incubation, the cells were stimulated with LPS (final concentration 1.0 μg / mL). After a further 2 hours of incubation, the cells were harvested and lysed. Intracellular protein expression levels were evaluated by Western blotting using antibodies against Tollip and β-actin (internal standard). Statistical analysis was performed based on Student's t-test (n=4, *P<0.05 (against control)).

[0203] Example 13: VLA-dCNP had a protective effect against the lethality of LPS-induced sepsis (Table 1). Male Balb / c mice (11 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mice were treated with LPS (Sigma-Aldrich; 10 mg / kg ip) and then with various doses of VLA-dCNP (described in Example 1) (low 0.1 mg / kg sc; medium 0.3 mg / kg sc; high 1.0 mg / kg sc), native C-type natriuretic peptide or CNP (high 1.0 mg / kg sc), CNP derivative or dCNP (high 1.0 mg / kg sc), B-type natriuretic peptide or BNP (high 1.0 mg / kg sc), antitumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a PDE5 inhibitor called a cyclic GMP degradation inhibitor or vardenafil (VDN) (1.0 mg / kg sc). The test substances were administered immediately after LPS administration. Survival was observed every 2 hours.

[0204] Referring to Figure 13A, VLA-dCNP demonstrated a protective effect against LPS-induced sepsis. Balb / c (11-week-old male) mice were treated with LPS (10 mg / kg ip) and then with VLA-dCNP at various doses (low 0.1 mg / kg sc; medium 0.3 mg / kg sc; high 1.0 mg / kg sc). Survival was observed every 2 hours. Statistical analysis was performed using the log-rank test based on Graphpad Prism 6.0 (n=10, 10, 10, 11).

[0205] Referring to Figure 13B, C57BL / 6J (6-week-old male) mice were treated with LPS (15 mg / kg ip) and then with prescribed doses of VLA-dCNP (low 0.1 mg / kg sc; medium 0.3 mg / kg sc; high 1.0 mg / kg sc). Survival was observed every 2 hours. Statistical analysis was performed by log-rank test (n=11, 10, 11, 11). VLA-dCNP showed a protective effect against LPS-induced sepsis.

[0206] Table 1. VLA-dCNP had a protective effect against LPS-induced sepsis mortality. Survival rate per hour (%) is shown. Balb / c (11-week-old male) mice were treated with LPS (10 mg / kg ip) and then with various doses of VLA-dCNP (L; 0.1 mg / kg sc; M; 0.3 mg / kg sc; H; 1.0 mg / kg sc), natural C-type natriuretic peptide or CNP (high 1.0 mg / kg sc), CNP derivative or dCNP (high 1.0 mg / kg sc), B-type natriuretic peptide or BNP (high 1.0 mg / kg sc), antitumor necrosis factor α antibody or TNFα ab (1.0 mg / kg sc), and a cyclic GMP degradation inhibitor or a PDE5 inhibitor called vardenafil (VDN) (1.0 mg / kg sc). Survival was observed every 2 hours (Figures 13A and 13B). In the table, the first observed mortality rate is shown in italics and bold, and the last observed survival rate (%) is shown in bold.

[0207] [Table 1]

[0208] Example 14. VLA-dCNP reduced the fibrotic area of ​​the lung, indicating the resolution of idiopathic pulmonary fibrosis (IPF). Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed; Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were treated with bleomycin (Nippon Kayaku, Tokyo, Japan; 1.0 mg / kg intratracheal administration). VLA-dCNP (described in Example 1) (0.3 or 0.1 mg / kg, 5 times / week, subcutaneous bolus administration) was administered from day 7 after bleomycin administration. 21 days after bleomycin treatment, the mice were sacrificed under isoflurane anesthesia, lung tissue was collected, and fixed with 4% paraformaldehyde (Fujifilm, Tokyo, Japan). Paraffin sections of the fixed lung tissue were stained with Masson's trichrome staining reagent (Kyodo Pathology, Kobe, Japan) (B). Masson's trichrome staining showed a reduction in the fibrotic area of ​​the lung tissue. The fibrosis area was measured using Image J (NIH, Bethesda, Maryland, USA) (A). Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc.). The test was performed based on Dunnett's multiple comparison test, conducted using Diego, CA, USA.

[0209] Referring to Figure 14A, VLA-dCNP reduced the fibrotic area of ​​the lung, indicating resolution of idiopathic pulmonary fibrosis (IPF) or interstitial lung disease (ILD). Male C57BL / 6J mice (6 weeks old) were treated with bleomycin (1.0 mg / kg intratracheal administration) and then with VLA-dCNP at different doses (0.1 mg / kg sc and 0.3 mg / kg sc). VLA-dCNP was administered 7 days after bleomycin administration (5 times / week). On day 21, mice were sacrificed, lung tissue was collected, and Masson's trichrome staining was performed. Statistical analysis was performed based on Dunnett's test using GraphPad Prism 6. (n=5, 8, 9, 7; negative control, control, VLA-dCNP 0.1, and VLA-dCNP 0.3. *P<0.05 (compared to control)). Figure 14B shows the lung tissue sample stained with Masson's trichrome from Figure 14A.

[0210] Example 15: VLA-dCNP reduced cell count and protein levels in BALF from an idiopathic pulmonary fibrosis acute exacerbation (IPF-AE) model, and attenuated TNFα and IL-6. Considering that IL-6 is upregulated in IPF-AE patients (American Journal of Physiology; Lung Cellular and Molecular Physiology, 2010 299:L3-L7) and that TNFα showed a trend toward statistical significance in IPF-AE patients (PLoS One, 2015 10(1):e0116775), collectively, VLA-dCNP has a potentially beneficial effect in IPF-AE patients. Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed; Oriental Yeast Co., Ltd., Tokyo, Japan). Mice were treated with bleomycin (Nippon Kayaku, Tokyo, Japan; 1.0 mg / kg intratracheal administration). Three weeks later, mice were treated with LPS (0.05 mg / kg intratracheal administration, Sigma Aldrich, St. St. Louis, MO, USA) and then with VLA-dCNP (described in Example 1) at various doses (medium 0.3 mg / kg sc; high 1.0 mg / kg subcutaneous bolus). VLA-dCNP was administered immediately after LPS administration. Twenty-four hours after treatment, mice were sacrificed under isoflurane anesthesia, and bronchoalveolar lavage fluid (BALF) was collected. The total number of cells in the BALF was counted in a counting chamber. The total protein concentration in the BALF was measured using the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific). The concentrations of each cytokine, namely interleukin-6 (IL-6) and tissue necrosis factor α (TNF-α), were measured using a commercially available Time Resolution FRET Kit (Cisbio, Bedford MA).

[0211] Referring to Figure 15A, VLA-dCNP reduced the number of cells in BALF from an idiopathic pulmonary fibrosis acute exacerbation (IPF-AE) model. Referring to Figure 15B, VLA-dCNP reduced protein levels in BALF from an idiopathic pulmonary fibrosis acute exacerbation (IPF-AE) model. Referring to Figure 15C, VLA-dCNP attenuated IL-6 in BALF from an idiopathic pulmonary fibrosis acute exacerbation (IPF-AE) model. Referring to Figure 15D, VLA-dCNP reduced both the number of cells and protein levels in BALF from an idiopathic pulmonary fibrosis acute exacerbation (IPF-AE) model, as well as attenuating TNFα in BALF.

[0212] Example 16. VLA-dCNP reduced tubular damage in cisplatin (CDDP)-induced acute kidney injury (AKI). Referring to Figures 16A and 16B, VLA-dCNP reduced tubular damage in cisplatin (CDDP)-induced acute kidney injury (AKI).

[0213] C57BL / 6J mice (8 weeks old, male, n=8, 7, 8 / group) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed; Oriental Yeast Co., Ltd., Tokyo, Japan). On days 2, 9, and 16, the mice were treated with CDDP (TCI, Tokyo, Japan; 6 mg / kg bwIP saline (Otsuka Pharmaceutical, Tokushima, Japan)), followed by VLA-dCNP (described in Example 1) (0.3 mg / kg) or buffer (for the control group) in buffer (100 mM methionine (Tokyo Chemical Industries, Ltd., Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries, Ltd.); 50 mM histidine (Tokyo Chemical Industries, Ltd.); H2O (Otsuka Pharmaceutical, Tokushima, Japan)) (subcutaneous injection under isoflurane anesthesia, 5 times / week). On day 19, mice were sacrificed under isoflurane gas. The kidneys were fixed with paraformaldehyde. Deparaffinized tissue sections were immersed in 0.5% orthoperiodate at room temperature for 7 minutes, washed twice with purified water for 2 minutes each, and stained with Schiff's reagent at room temperature for 15 minutes. Subsequently, the sections were immersed three times in sulfurous acid solution (10 mL of 10% sodium bisulfite, 10 mL of 1N hydrochloric acid, 180 mL of purified water) at room temperature for 2 minutes each, and washed with running water for 5 minutes. Finally, the sections were stained with Meyer hematoxylin solution at room temperature for 4 minutes, washed with running water for 5 minutes, and then renal damage was evaluated using a bright-field fluorescence microscope (BZ-X700, Keyence, Tokyo, Japan) at a magnification of ×20.

[0214] Example 17. VLA-dCNP and long-acting CNP suppress liver enzymes and inflammation / fibrosis markers in diet-induced hepatic fibrosis. In this example, a choline-deficient, amino acid-restricted, and high-fat diet model is used, which is known to rapidly induce fibrosis. For example, Matsumoto et al., Int See J Exp Pathol. 2013 Apr;94(2):93-103 (the entire article is incorporated herein by reference). Elevated aspartate transaminase (AST) indicates liver or other organ damage that may be subject to inflammatory and fibrotic processes. Elevated alanine transaminase (ALT) indicates liver damage that may be subject to inflammatory and fibrotic processes in the liver. Activated hepatic stellate cells, the major collagen-producing cells in hepatic fibrosis, exhibit increased α-smooth muscle actin (α-SMA) during fibrosis. Furthermore, liver tissue also shows increased levels of inflammatory markers such as tumor necrosis factor α (TNFα) and monocyte chemotactic protein 1 (MCP-1) during the process of fibrosis. All of these markers (AST, ALT, α-SMA, TNFα, and MCP1) were suppressed when administered to a high bolus dose (1 mg / kg) of a long-acting CNP derivative, as well as to medium bolus doses (0.3 mg / kg) and high bolus doses (1 mg / kg) of VLA-dCNP (as described in Example 1). Overall, long-acting CNP derivatives and VLA-dCNP suppressed tissue damage, inflammation, and fibrosis processes.

[0215] Referring to Figures 17A-17E, VLA-dCNP and long-acting CNP suppressed liver enzymes and inflammation / fibrosis markers in diet-induced hepatic fibrosis. Figure 17A shows a significant decrease in the liver enzyme aspartate aminotransferase (AST); Figure 17B shows a significant decrease in the liver enzyme alanine aminotransferase (ALT); Figure 17C shows a significant decrease in alpha-smooth muscle actin (α-SMA), a marker of fibrotic cells; Figure 17D shows a significant decrease in tumor necrosis growth factor α (TNF-α), a marker of inflammation that induces fibrosis; and Figure 17E shows a significant decrease in monocyte chemotactic protein 1 (MCP-1), a mediator of macrophage-induced inflammation in liver tissue, when administered to subjects with long-acting CNP derivatives and / or VLA-dCNP.

[0216] In this study, C57BL / 6J mice (6 weeks old, male, n=10 / group) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed; Oriental Yeast Co., Ltd., Tokyo, Japan) or choline-deficient, amino acid-restricted, high-fat feed (CDAHFD) (Research Diet, New Brunswick, NJ). Starting on day 5 and continuing for two weeks, patients were treated with VLA-dCNP (0.1, 0.3, or 1.0 mg / kg), dCNP (0.1, 0.3, or 1.0 mg / kg), and CNP (0.1, 0.3, or 1.0 mg / kg) or buffer (for the control group) in 100 mM buffer (Tokyo Chemical Industries, Ltd.); 50 mM histidine (Tokyo Chemical Industries, Ltd.); and H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) (subcutaneous bolus injection under isoflurane anesthesia for less than 30 seconds, 5 times / week (administered only on weekdays)). Blood / plasma samples were collected from cardiac puncture under isoflurane, and the liver was removed after puncture on day 17 (8.5 weeks of age). AST and ALT were evaluated using an enzyme substrate assay (Fujifilm, Wako, Japan).

[0217] Example 18. Long-acting CNP derivatives (dCNP) and VLA-dCNP alleviate renal fibrosis and improve renal function in cisplatin-induced acute kidney injury. Cisplatin is a common and potent chemotherapy drug for cancer treatment, but its dose-dependent side effect is nephrotoxicity, which can lead to acute kidney injury. Serum creatinine provides an indicator of how well the kidneys are functioning. The urinary albumin / creatinine ratio can provide a more accurate indicator of how much albumin is being released into the urine. The presence of small amounts of albumin in the urine can be an early indicator of kidney disease.

[0218] Renal tissue injury initiates inflammatory and fibrotic processes that occur to promote regeneration and repair. Following renal injury, damaged tissue releases cytokines (TNF-α, tumor necrosis factor-α; one or more ILs, one or more interleukins; and TGF-β, transforming growth factor-β) and chemokines (SDF-1, stromal cell-derived factor-1; MCP-1, monocyte chemotactic protein-1; CCL2; CX3CL1, fractalkines; and CXCL10, CXC motif chemokine 10) into the kidney, which stimulate the activity and infiltration of inflammatory cells (neutrophils; monocytes; Mφ, macrophages; NK cells, natural killer cells; T cells; B cells). Normal tissue repair processes occur simultaneously with myofibroblast activation, collagen deposition, and wound healing responses; however, prolonged activation of pro-inflammatory and pro-fibrotic cell types (fibroblasts / fibrocytes, myofibroblasts / pericytes) leads to excessive extracellular matrix deposition (see, for example, Black et al., Renal Inflammation and Fibrosis: A Double-edged Sword, Journal of Histochemistry & Cytochemistry 2019, Vol.67(9)663-681, the whole of which is incorporated herein by reference), and can lead to chronic kidney disease (CKD). See, for example, Eoghainin O hAinmhire, Benjamin D. Humphreys; Fibrotic Changes Mediating Acute Kidney Injury to Chronic Kidney Disease Transition, Nephron 2017;137:264-267, the whole of which is incorporated herein by reference.

[0219] Therapies used in cancer treatment may cause damage to major organ systems such as the heart (i.e., cardiotoxicity), lungs (e.g., pulmonary fibrosis), and bones (e.g., myelosuppression). Cancer and its treatment may increase the likelihood of acute kidney injury, which can lead to fibrosis and chronic kidney disease. Cancer cells can cause urinary tract obstruction leading to acute kidney injury, which can result in inflammation, as well as fibrosis (e.g., prostate or urothelial carcinoma, uterine or ovarian cancer, compression of the urinary tract due to retroperitoneal nodule hypertrophy, tumor masses, and / or retroperitoneal fibrosis). Systemic anti-cancer treatment can damage the kidney directly (e.g., cisplatin-induced necrosis of the proximal tubules) or indirectly (e.g., methotrexate-induced crystalline nephropathy and tumor lysis syndrome), both of which can cause inflammation, fibrosis, and chronic kidney disease. Acute kidney injury is a serious side effect of conventional cytotoxic chemotherapy drugs and can affect the effectiveness of cancer treatment and patient survival. See, for example, Perazella. MA,Onco-nephrology:renal toxicities of chemotherapeutic agents.Clin J Am Soc Nephrol 2012;7:1713-21;Malyszko et al.,Kozlowska K,Kozlowski L,Malyszko J.Nephrotoxicity of anticancer treatment.Nephrol Dial Transplant 2017;32:924-36. Cisplatin, used as part of chemotherapy regimens for various cancers, can cause acute kidney injury in 20-30% of patients due to mitochondrial damage caused by reactive oxygen species. See, for example, Miller et al., Mechanisms of cisplatin nephrotoxicity. Toxins (Basel) 2010;2:2490-518; and Brooks et al., Regulation of mitochondrial dynamics in acute kidney injury in cell culture and rodent models. J Clin Invest 2009;119:1275-85. Cisplatin accumulates in the S3 segment of the proximal tubule, promoting glutathione depletion and a large amount of mitochondrial reactive oxygen species. This accumulation may be related to the selective uptake of cisplatin via active basolateral-apical transporters such as CTR1 and SLC22A2 (formerly OCT2), both expressed on the basolateral membrane of the S3 segment.

[0220] Another notable side effect of cisplatin is hearing loss or ototoxicity. Ototoxicity arises from similar mitochondrial damage caused by reactive oxygen species (ROS) generated in the inner ear upon exposure to cisplatin, which leads to inflammation. See, for example, Yu et al., Current. See "Strategies to Combat Cisplatin-Induced Ototoxicity Front. Pharmacol., 03 July 2020." Studies have shown that ROS can stimulate cochlear duct inflammation. Inner ear inflammation can trigger inner ear cell death via endoplasmic reticulum stress, autophagy, and necroptosis, which induce apoptosis. See, for example, Sheth et al., "Mechanisms of Cisplatin-Induced Ototoxicity and Otoprotection," Frontiers in Cellular Neuroscience, 27 Oct., Vol 11, 2017.

[0221] Figure 18A shows a significant improvement in renal function based on a decrease in serum creatinine; Figure 18B shows a significant improvement in renal function based on a decrease in urinary albumin levels, calculated by the albumin / creatinine ratio; Figure 18C shows a significant decrease in fibrotic area (%) in the kidney; fibrotic area was measured using Image J (NIH, Bethesda, Maryland, USA); Figure 18D is a series of representative images of the kidney stained with Masson's trichrome (MT). The magnification is ×20. With this Masson's trichrome stain, the nucleus is stained with iron hematoxylin (brown / black in the image), the cytoplasm with acid fuchsin (pink / red in the image), and the collagen fibrillation area is stained with aniline blue (blue in the image).

[0222] In this study, mice were given CDDP (TCI, Tokyo, Japan; 10 mg / kg) on ​​days 0, 7, 14, and 21. The cells were treated with bwIP saline (Otsuka Pharmaceutical, Tokushima, Japan), and then with CNP (low dose (L): 0.1 mg / kg; and high dose (H): 1.0 mg / kg), dCNP (as described in Example 1) (L: 0.1 mg / kg; and H: 1.0 mg / kg), VLA-dCNP (as described in Example 1) (L: 0.1 mg / kg; and H: 1.0 mg / kg), or buffer (for the control group) in a buffer (in 100 mM methionine (Tokyo Chemical Industries, Ltd., Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries, Ltd.); 50 mM histidine (Tokyo Chemical Industries, Ltd.); and H2O (Otsuka Pharmaceutical, Tokushima, Japan)). In this study, negative controls were not subjected to CDDP induction and received buffer five times a week for four weeks. Blood / serum samples were collected via cardiac puncture under isoflurane on day 28. Serum creatinine, BUN, and urinary creatinine were measured by colorimetric methods (Arbor Assays, Ann Arbor MI), (Thermo Fisher Scientific, Waltham MA), and (R&D Systems, Detroit, MN), respectively. Urine samples were collected and the kidneys were fixed with paraformaldehyde. Kidney sections were stained with Masson's trichrome stain, and the fibrosis area (%) was evaluated using a bright-field fluorescence microscope (BZ-X700, Keyence, Tokyo, Japan) at a magnification of ×20. In the case of this Masson trichrome stain, the nucleus is stained with iron hematoxylin (brown / black in the image), the cytoplasm is stained with acid fuchsin (pink / red in the image), and the collagen fibrillation area is stained with aniline blue (blue in the image). The fibrillation area (%) was calculated as follows: First, the tissue area was calculated by (total number of pixels - number of pixels in the empty area (highest brightness area)). Next, the difference between the blue light intensity and the red light intensity was calculated using ImageJ and converted to pixels. Finally, the fibrillation area (%) = (fibrillation area / total tissue area) × 100. Statistical analysis was performed based on Student's t-test using GraphPad Prism 6.*P<0.05 or **P<0.01 (compared to the control).

[0223] Example 19. Both the long-acting CNP derivative (dCNP) and VLA-dCNP suppressed fibrosis in a mouse model of bleomycin-induced idiopathic pulmonary fibrosis acute exacerbation (AE-IPF). Acute exacerbation of IPF (AE-IPF) is defined as a rapid acceleration of a disease of unknown etiology or a progressive lung disease. See, for example, J Thorac Dis 2015 7(3)499-519. Hydroxyproline is a major component of collagen and plays an important role in the stability of the collagen triple helix. In this study, it was used to assess the collagen content of lung tissue.

[0224] Figure 19A shows a significant reduction in fibrosis based on a decrease in hydroxyproline, a major component of collagen in lung tissue; Figure 19B shows a significant reduction in fibrotic area (%) in the lung based on quantification of histological Masson trichrome staining of lung tissue sections. Fibrotic area was measured using Image J (NIH, Bethesda, Maryland, USA); Figure 19C shows a representative image of a Masson trichrome (MT) stained kidney at 20x magnification.

[0225] In this study, male C57BL / 6J mice (6 weeks old, male, n=6 / group) were purchased from Kyudo (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed; Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were treated with bleomycin (Nippon Kayaku, Tokyo, Japan; 1.0 mg / kg intratracheal administration). Two weeks later, mice were treated with LPS (0.05 mg / kg intratracheally administered Sigma Aldrich, St. Louis, MO, USA) and then with bolus doses of CNP (0.3 mg / kg), dCNP (described in Example 1) (0.3 mg / kg), or VLA-dCNP (described in Example 1) (0.3 mg / kg) in a buffer (100 mM methionine (Tokyo Chemical Industries, Ltd., Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries, Ltd.); 50 mM histidine (Tokyo Chemical Industries, Ltd.); in H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan)), or buffer (for the control group) (subcutaneous bolus injection under isoflurane anesthesia). The test substance and control were administered for three consecutive days one day prior to LPS administration. The day after the last treatment, the animals were euthanized, a portion of the lung was collected, and the rest was fixed with paraformaldehyde. A portion of the lung (20 mg) was homogenized, and the extract was measured for hydroxyproline (Abcam Cambridge, UK). Fixed lung sections were stained with Masson's trichrome stain and evaluated using a bright-field fluorescence microscope (BZ-X700, Keyence, Tokyo, Japan) at a magnification of ×20. With Masson's trichrome stain, the nucleus was stained with iron hematoxylin (brown / black in the image), the cytoplasm with acid fuchsin (pink / red in the image), and the collagen fibrillation area was stained with aniline blue (blue in the image). The fibrosis area (%) was calculated as follows: First, the tissue area was calculated as (total number of pixels - number of pixels in the empty area (highest brightness area)). Next, the difference between the blue light intensity and the red light intensity was calculated using ImageJ and converted to pixels. Finally, the fibrosis area (%) = (fibrosis area / total tissue area) × 100. Statistical analysis was performed based on Student's t-test using GraphPad Prism 6. *P<0.05 (compared to control).

[0226] Example 20: The pharmacokinetic profiles of long-acting CNP derivatives s1 (dCNP-s1) and s2 (dCNP-s2) after bolus administration showed continuous presence in the blood over time. Referring to Figure 20, the graph shows plasma CNP levels [mean (SEM); n=5] in CD-1 mice after subcutaneous bolus administration of 2.0 mg / kg CNP derivatives s1 (dCNP-s1) and s2 (dCNP-s2). For comparison, the inset shows the lower plasma CNP levels (diamond) when native CNP is administered. Error bars represent the standard error of the mean of the n=5 plasma samples. The baseline CNP level before administration was 0.391 (0.02) ng / mL [mean (SEM); n=10]. The long-acting dCNP-s1 and dCNP-s2 provide 10 times higher blood CNP levels compared to native CNP when administered at similar dose weight / kg doses, for a sustained period (at least 8 hours).

[0227] For this pharmacokinetic study, all test animals (mice) were maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (Lab Pico Rodent #5053; Animal Specialties, Woodburn, OR). Male CD-1 mice (6-8 weeks old; Charles River, Hollister, CA) were treated with 2.0 mg / kg of CNP derivative s1 (dCNP-s1; PharmaIN Corp, Bothell, WA) and CNP derivative s2 (dCNP-s2; PharmaIN Corp, Bothell, WA) by subcutaneous administration between the scapulae. All test samples were compounded or dissolved in 100 mM sucrose, 100 mM methionine, 50 mM histidine, pH 7.4. Blood samples were taken at various time points (0, 0.5, 1, 2, 4, 6, 8, and 24 hours) due to retroorbital hemorrhage, and bleeding was induced twice per animal at two different time points. Blood samples were processed in K2EDTA tubes to obtain plasma. Plasma was analyzed using a commercially available CNP ELISA kit from Phoenix Pharmaceuticals (catalog # EKE-012-03). The CNP was native human CNP (GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 10]), and dCNP-s1 and dCNP-s2 were the following sequence: HOC(=O)(CH2) 16 C(=O)-Aeea-Aeea-GCFGLKLDRIGS homo QSGLGC [SEQ ID NO: 21] and HOC(=O)(CH2) 16 C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGS HomoQSGLGC [SEQ ID NO: 20] is a derivative of human CNP, each having a disulfide bond between two cysteine ​​residues: HomoQ: homoglutamine residue; Aeea: 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue (wherein the formula, the amino and carboxylic acid groups are used to form an amide bond to obtain the CNP derivative); HOC(=O)(CH2) 16 C(=O)- is derived from octadecadionic acid; γE: gamma glutamic acid residue.

[0228] Examples, rather than limitations, are disclosed in accordance with the following enumerated paragraphs: A1. Disorders of the lungs, liver, and / or kidneys; or methods for treating subjects having symptoms related to disorders of the lungs, liver, and / or kidneys, the following: The treatment involves administering to a subject a therapeutically effective bolus dose of a composition comprising a long-acting CNP, a long-acting CNP derivative, a long-acting NPRB agonist, an ultra-long-acting CNP, an ultra-long-acting CNP derivative, an ultra-long-acting NPRB agonist, a long-acting CNP agonist, an ultra-long-acting CNP agonist, or any combination thereof. Here, the composition does not lower blood pressure by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement obtained before administration of the therapeutically effective bolus dose of the composition. Here, the composition increases the plasma cyclic GMP level 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times the baseline plasma cyclic GMP level, where the baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level of the subject before administration of the composition). Here, lung, liver, and / or kidney disorders, or symptoms associated with lung, liver, and / or kidney disorders, are as follows: i) Acute lung injury (ALI), ii) acute respiratory distress syndrome (ARDS); iii) pulmonary edema; iv) Elevated levels of inflammatory cells in the lungs, v) Increased levels or expression of inflammatory cytokines in the lungs compared to healthy lungs, vi) Increased protein levels in the alveolar space compared to healthy lungs, vii) Hypoarterial oxygenation (Hypoarterial oxygenation is defined as a blood PaO2 of less than 60 mmHg and / or a blood hemoglobin oxygen saturation (SpO2) of less than 90%), viii) pneumonia; ix) Fibrosis, x) renal failure; and any combination thereof (for example, combinations of 2, 3, 4, 5, 6, 7, 8, 9, or 10 of i) to x). A2. Long-acting or ultra-long-acting CNP derivatives include U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 4], U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 11], or any combination thereof. During the ceremony, U is a part of equation (I) or (II), where equation (I) is: (aliphatic) a -(X)- (I) and; During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is the linker (γE) m -(B) n And, During the ceremony, B is a sequence of 1 to 8 amino acid residues, where each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and The sum of m and n is at least 1. Equation (II) is, (polymer) a -(Y)- (II) and; During the ceremony, a is 0 or 1 (preferably a is 1); The polymers are cellulose, poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) (MPEG), poly(lactic acid-coglycolic acid), poly(N-vinylpyrrolidone), or derivatives thereof; Y is A sequence of 1 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); A non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; A linker containing amino acid residues, wherein the amino acid residues are (polymer) a Is it a linker containing amino acid residues that is covalently attached to it? or It is a peptide linker that is different from 1 to 10 amino acid residues or peptide sequences. The method described in paragraph A1. A3.Y is linker (γE) m -(B) n The method according to paragraph 2, wherein B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. A4. Long-acting CNP derivatives or ultra-long-acting CNP derivatives include U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 4], U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 12], or any combination thereof. During the ceremony, U is part of equation (I), and equation (I) is, (aliphatic) a -(X)- (I) and; During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted. 10~24 Chain (for example, C which is optionally substituted) 12~28 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc., preferably covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably covalently bonded to X via carbonyl as part of an amide linkage with X. 10~24 It is a chain, X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is the linker (γE) m -(B) n And, During the ceremony, B is a sequence of 1 to 8 amino acid residues or peptides, where each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and The sum of m and n is at least 1. The method described in any one of paragraphs A1 through A3. A5.X is a sequence of 4 to 7 amino acids, where each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G). or X is the linker (γE) m -(B) n The formula is such that B is a sequence of 1 to 8 amino acid residues, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. The method described in any one of paragraphs A2 through A4. A6. Long-acting CNP derivatives or ultra-long-acting CNP derivatives include U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], During the ceremony, U is (aliphatic) a -(X)- is; During the ceremony, a is 1; Aliphatic C is optionally substituted. 4~24Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain, X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is the linker (γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, and each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. The method described in any one of paragraphs A1 through A5. A7. Aliphatic C is optionally substituted in linear or branched chains. 4~9 A chain (for example, a thioether, ether, thioether, carbamate moiety, bond, or similar linkage) covalently bonded to the peptide via a selectively substituted C 3~8 The alkyl-C(=O)- moiety and / or optionally substituted C 4~9 The method described in any one of paragraphs A2 to A6, excluding alkyl groups. A8. Long-acting CNP derivatives are CH3(CH2) 14 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 5]; CH3(CH2) 16 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 6]; CH3(CH2) 18 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 7]; CH3(CH2) 20 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 8]; CH3(CH2) 22 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 9]; HOC(=O)(CH2) containing disulfide bonds between cysteine ​​residues 16 C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGS homo QSGLGC[Sequence ID 20]; and HOC(=O)(CH2) containing disulfide bonds between cysteine ​​residues 16 C(=O)-Aeea-Aeea-GCFGLKLDRIGS Homo QSGLGC [Sequence ID 21] A method that selects one of the following from paragraphs A1 to A7. A9.The long-acting CNP derivative is CH3(CH2) 14 The method described in any one of paragraphs A1 to A8, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 5]. A10. Long-acting CNP derivatives are CH3(CH2) 16 The method described in any one of paragraphs A1 to A8, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 6]. A11. Long-acting CNP derivatives are CH3(CH2) 18 The method described in any one of paragraphs A1 to A8, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 7]. A12. Long-acting CNP derivatives are CH3(CH2) 20The method described in any one of paragraphs A1 to A8, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 8]. A13. Long-acting CNP derivatives are CH3(CH2) 22 The method described in any one of paragraphs A1 to A8, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 9]. A14. Long-acting CNP derivatives include HOC(=O)(CH2) with disulfide bonds between cysteine ​​residues. 16 The method described in any one of paragraphs A1 to A8, which is C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGS homo QSGLGC [Sequence ID 20]. A15. Long-acting CNP derivatives include HOC(=O)(CH2) with disulfide bonds between cysteine ​​residues. 16 The method described in any one of paragraphs A1 to A8, which is C(=O)-Aeea-Aeea-GCFGLKLDRIGS homo QSGLGC [Sequence ID 21]. A16. Long-acting or ultra-long-acting CNP derivatives include U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 4], U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 27], or any combination thereof; During the ceremony, U is part of equation (II), and equation (II) is, (polymer) a -(Y)- (II) And, During the ceremony, a is 0 or 1 (preferably a is 1); The polymers are cellulose, poly(ethylene glycol)(PEG), methoxypoly(ethylene glycol)(MPEG), poly(lactic acid-coglycolic acid), or poly(N-vinylpyrrolidone); Y is A sequence of 4 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G); A non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; or Linker (γE) m -(B) n The formula is such that B is a sequence of 1 to 8 amino acid residues, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1, linker (γE) m -(B) n That is, The method described in any one of paragraphs A1 through A3. A17. Long-acting or ultra-long-acting CNP derivatives include U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], or any combination thereof; During the ceremony, U is part of equation (II), and equation (II) is, (polymer) a -(Y)- (II) And, During the ceremony, a is 1; The polymers are cellulose, poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) (MPEG), poly(lactic acid-coglycolic acid), poly(N-vinylpyrrolidone), or derivatives thereof; Y is A sequence of 1 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); A non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; A linker containing amino acid residues, wherein the amino acid residues are (polymer) a Is it a linker containing amino acid residues that is covalently attached to it? Is it a peptide linker that differs from 1 to 10 amino acid residues or the peptide sequence? or Linker (γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1, linker (γE) m -(B) n That is, The method described in any one of paragraphs A1-A3 and A16. A18. The polymer is a polymer that does not contain poly(ethylene glycol), does not contain MPEG, or does not contain both poly(ethylene glycol) and MPEG, according to any one of paragraphs A1-A3, A14, and A15. A19.Y is A sequence of 4 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G); or Linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1, linker (γE) m -(B) n That is, The method described in either paragraphs A1-A3 or A16-A18. A20. Bolus doses may be administered up to twice daily, via subcutaneous, intravenous, intramuscular, nasal, inhalation, enteral, or any combination thereof; or The route of administration is subcutaneous; or The route of administration is intravenous; or The route of administration is intramuscular or The route of administration is by inhalation; or The route of administration is via the nose; or The enteral route of administration is orally. The method described in any one of paragraphs A1 through A19. A21. The method according to any one of paragraphs A1 to A20, wherein the subject has pulmonary edema; hypo-arterial oxygenation; elevated levels of inflammatory cells in the lungs; increased levels or expression of inflammatory cytokines in the lungs; sepsis; bacteremia; ALI or ARDS associated with pneumonia, pulmonary fibrosis, or any combination thereof. A22. The method according to any one of paragraphs A1 to A21, wherein the inflammatory cytokine comprises IL-6, IL-1b, TNFα, MCP-1, IFNg, or any combination thereof. A23. The method according to any one of paragraphs A1 to A22, wherein pneumonia includes bacterial pneumonia, viral pneumonia, and sterile pneumonia. A24. ALI or ARDS, as follows: (i) systemic injury selected from trauma, sepsis (i.e., systemic infection), bacteremia (i.e., bacteria in the blood), pancreatitis, shock, frequent blood transfusions, disseminated intravascular coagulation, burns, drug overdose or toxicity, opioids, aspirin, phenothiazines, tricyclic antidepressants, amiodarone, chemotherapeutic agents, nitrofurantoin, protamine, thrombotic thrombocytopenic purpura, head trauma, paraquat, and any combination thereof; or (ii) Aspiration of gastric contents, pulmonary intubation, embolism, tuberculosis, viral pneumonia, bacterial pneumonia, cytogenic tissue pneumonia, airway obstruction, smoking of free basic cocaine, drowning, inhalation of toxic gases, oxygen toxicity, pulmonary contusion, radiation exposure, exposure to high altitude, lung reinflation, reperfusion, and lung injury selected from any combination thereof. The method described in any one of paragraphs A1 to A23, resulting from the above. A25. The method described in paragraph A24, wherein the embolism is caused by a blood clot, fat, air, or amniotic fluid. A26. The method described in paragraph A23 or A24, wherein the viral pneumonia is SARS caused by a coronavirus or influenza virus. A27. Below: ALI or ARDS caused by infection, ALI or ARDS caused by PF, or ALI or ARDS caused by sepsis; or ALI or ARDS caused by bacteremia; or ALI or ARDS caused by intubation: or ALI or ARDS caused by toxic gases selected from the group consisting of chlorine gas, fumes, phosgene, concentrated oxygen, and any combination thereof. The method described in any one of paragraphs A1 through A26. A28. The method described in paragraph A27, wherein the infectious disease is caused by a coronavirus or influenza virus. A29. The method according to any one of paragraphs A1 to A21, wherein fibrosis includes pulmonary fibrosis, hepatic fibrosis, cirrhosis, and glomerulosclerosis. A30. The method according to any one of paragraphs A1 to A29, comprising a long-acting CNP composition or an ultra-long-acting CNP composition comprising CNP, a CNP derivative, or a long-acting CNP derivative and a polymer excipient comprising a polymer excipient containing polyethylene glycol, a fatty acid, and / or poly(amino acids) grafted with an anionic moiety, wherein the polymer excipient is adapted to sequester either CNP or a CNP derivative or to non-covalently bond to either CNP or a CNP derivative. A31. The method according to any one of paragraphs A1 to A30, comprising an ultra-long-acting CNP derivative composition comprising a long-acting CNP derivative and a polymer excipient comprising a polymer excipient grafted with polyethylene glycol, a fatty acid, an anionic moiety, or any combination thereof, wherein the polymer excipient is adapted to sequester the long-acting CNP derivative or to be non-covalently bonded to the long-acting CNP derivative. A32. A long-acting NPRB agonist or an ultra-long-acting NPRB agonist comprising a polypeptide, as described in any one of paragraphs A1 and A20-A30. A33. The polypeptide is the method described in paragraph A32, comprising an antibody. A34. A long-acting NPRB agonist or an ultra-long-acting NPRB agonist is the method according to any one of paragraphs A1 and A20-A32, comprising a molecule with a molecular weight of less than 2 kDa. A35. A method for treating subjects who have ALI or ARDS, or who are at risk of developing ALI or ARDS, The procedure involves administering a therapeutically effective bolus dose to a subject of a composition containing a long-acting CNP derivative or an ultra-long-acting CNP derivative, including U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], or GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 4], U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 11], or any combination thereof. During the ceremony, U is a part of equation (I) or (II), where equation (I) is: (aliphatic) a -(X)- (I) and; During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain, X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is the linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, and each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. Equation (II) is, (polymer) a -(Y)- (II) and; During the ceremony, a is 0 or 1 (preferably a is 1); The polymers are cellulose, poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) (MPEG), poly(lactic acid-coglycolic acid), poly(N-vinylpyrrolidone), or derivatives thereof. Y is The sequence consists of 1 to 10 amino acid residues or peptide sequences, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); It is a non-amino acid linker, and is a non-amino acid linker containing an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; It is an amino acid residue-containing linker, and the amino acid residue is (polymer) a Is it a linker containing amino acid residues that is covalently attached to it? or It is a peptide linker that is different from 1 to 10 amino acid residues or peptide sequences; The composition does not lower blood pressure by more than 15% of the baseline blood pressure measurement, where the baseline blood pressure measurement is the mean blood pressure before administration of the composition; and The composition increases plasma cyclic GMP levels 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is either the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject). method. A36.Y is linker (γE) m -(B) n The method according to paragraph A35, wherein B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. A37. Long-acting CNP derivatives are CH3(CH2) 14 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 5]; CH3(CH2) 16 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 6]; CH3(CH2) 18 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 7]; CH3(CH2) 20 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 8]; CH3(CH2) 22 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 9]; HOC(=O)(CH2) containing disulfide bonds between cysteine ​​residues16 C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGS homo QSGLGC[Sequence ID 20]; and HOC(=O)(CH2) containing disulfide bonds between cysteine ​​residues 16 C(=O)-Aeea-Aeea-GCFGLKLDRIGS Homo QSGLGC [Sequence ID 21] The method described in paragraph A35 or A36, selected from the options provided. A38. Long-acting CNP derivatives are CH3(CH2) 14 The method described in any one of paragraphs A35 to A37, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 5]. A39. Long-acting CNP derivatives are CH3(CH2) 16 The method described in any one of paragraphs A35 to A37, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 6]. A40. Long-acting CNP derivatives are CH3(CH2) 18 The method described in any one of paragraphs A35 to A37, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 7]. A41. Long-acting CNP derivatives are CH3(CH2) 20 The method described in any one of paragraphs A35 to A37, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 8]. A42. Long-acting CNP derivatives are CH3(CH2) 22 The method described in any one of paragraphs A35 to A37, which is C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 9]. A43. Long-acting CNP derivatives include HOC(=O)(CH2) with disulfide bonds between cysteine ​​residues. 16 The method described in any one of paragraphs A35 to A37, wherein the drug is C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGS homo QSGLGC [Sequence ID 20]. A44. Long-acting CNP derivatives include HOC(=O)(CH2) with disulfide bonds between cysteine ​​residues. 16 The method described in any one of paragraphs A35 to A37, wherein C(=O)-Aeea-Aeea-GCFGLKLDRIGS homo QSGLGC [Sequence ID 21]. A45. The method according to any one of paragraphs A35 to A44, comprising an ultra-long-acting CNP derivative composition comprising a long-acting CNP derivative and a polymer excipient comprising a polymer excipient grafted with polyethylene glycol, a fatty acid, an anionic moiety, or any combination thereof, wherein the polymer excipient is adapted to sequester the long-acting CNP derivative or to be non-covalently bonded to the long-acting CNP derivative. A46. The method according to any one of paragraphs A1 to A45, wherein administration of a therapeutically effective bolus dose composition to a subject reduces the total number of cells and total protein in a BALF sample derived from the subject. A47. The method according to any one of paragraphs A1 to A46, wherein administration of a therapeutically effective bolus dose of the composition to a subject reduces MPO levels in the lung tissue derived from the subject. A48. The method according to any one of paragraphs A1 to A47, wherein administration of a therapeutically effective bolus dose of the composition to a subject reduces the expression of inflammatory cytokines (e.g., IL-6, IL-1b, TNFα, MCP-1, and / or IFNg) in the subject. A49. The method according to any one of paragraphs A1 to A48, wherein administration of a therapeutically effective bolus dose of the composition reduces the fibrotic area of ​​the lung in a subject with idiopathic pulmonary fibrosis. A50. The method according to any one of paragraphs A1 to A49, wherein, when administered to a subject in a therapeutically effective bolus dose, the cell count and protein levels decrease, and the expression of one or any combination of IL-6, IL-1b, TNFα, MCP-1, IFNg is reduced. A51. The method according to any one of paragraphs A1 to A49, wherein, when administered to a subject in a therapeutically effective bolus dose, the expression of one of IL-6, IL-1b, TNFα, MCP-1, IFNg, or any combination thereof is reduced, and the mortality rate is reduced in subjects with sepsis. A52. The method according to any one of claims A1 to A51, wherein administration of a therapeutically effective bolus dose of the composition to a subject reduces the expression of AST, ALT, α-SMA, IL-6, IL-1b, TNFα, MCP-1, IFNg, iNOS, Elf-1, Tollip, IRAK-1, P-P38, P-P65, β-act, STAT1, P-STAT1, STAT2, STAT3, STAT6, fibrosis area, serum creatinine, urinary albumin / creatinine ratio, lung hydroxyproline, or any combination thereof. A53. A composition comprising a long-acting CNP derivative containing formula U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 30], During the ceremony, x is a natural or unnatural amino acid residue, except that x is not a methionine residue: U is given by equation (I): (aliphatic) a -(X)- (I) This is part of it; During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is the linker (γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, and each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. composition. A54.x is homoglutamine, and aliphatic is carbonyl (e.g., CH3(CH2)) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 C is either in a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 The composition described in paragraph A53, wherein C (=O); a is 1; B is Gly; m is 0, 1, or 2, and n is 1. A55.x is homoglutamine, and aliphatic is carbonyl (e.g., CH3(CH2)) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 C is either in a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 The composition described in paragraph A53, wherein C (=O); a is 1; B is Gly; m is 1; and n is 1. A56.x is homoglutamine, and the aliphatic compound is a branched or linear C optionally substituted with X via a carbonyl group. 18 Chain (for example, CH3(CH2)) 16 C(=O) or C covalently bonded to X via chemical bonds such as thioethers, ethers, thioethers, or carbamate moieties, or equivalents. 18 The chain is; preferably, the aliphatic compound is a branched or linear C optionally substituted with X covalently via a carbonyl as part of an amide or ester bond with X. 18 Chain (for example, CH3(CH2)) 16 C(=O) is either C(=O) or, more preferably, the aliphatic compound is a branched or linear C(=O) covalently bonded to X via a carbonyl group as part of the amide bond with X. 18 Chain (for example, CH3(CH2)) 16 It is either C(=O) or, if it is an aliphatic compound, HOC(=O)(CH2) 16 The composition described in paragraph A53, where C(=O); a is 1; m is 1; and n is 0. A57.x is homoglutamine (homo Q) [SEQ ID NO: 16], and U is (aliphatic) a-(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 C is either in a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n The composition according to paragraph A53, wherein B is a 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; m is 0 and n is 2. A58.x is homoglutamine (homo Q) [SEQ ID NO: 17], and U is (aliphatic) a -(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 C is either in a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n The composition according to paragraph A53, wherein B is a 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; m is 1 and n is 2. A59.x is homoglutamine, and its aliphatic component is CH3(CH2). 16 C(=O) or HOC(=O)(CH2) 16 The composition described in paragraph A53, wherein C (=O); B is a 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, m is 0, and n is 2. A60.x is homoglutamine, and its aliphatic component is CH3(CH2). 16 C(=O) or HOC(=O)(CH2) 16 The composition described in paragraph A53, wherein C (=O); B is a 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, m is 1, and n is 2. A61.x is homoglutamine, and its aliphatic component is CH3(CH2). 16 C(=O) or HOC(=O)(CH2) 16 The composition described in paragraph A53, wherein C is O; B is (2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(Gly), m is 1, and n is 1. A62. Long-acting CNP derivatives include HOC(=O)(CH2) with disulfide bonds between cysteine ​​residues. 16 The composition described in paragraph A53 is C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGS homoQSGLGC [SEQ ID NO: 20]. A63. Long-acting CNP derivatives include HOC(=O)(CH2) with disulfide bonds between cysteine ​​residues. 16 The composition described in paragraph A53, which is C(=O)-Aeea-Aeea-GCFGLKLDRIGS homoQSGLGC [SEQ ID NO: 21]. A64.a is the method described in any one of paragraphs A2 to A52, which is 1. A65.a is a composition described in any one of paragraphs A53 to A63, wherein A65.a is 1.

[0229] While exemplary embodiments are illustrated and described, it will be understood that various modifications can be made to these embodiments without departing from the spirit and scope of this disclosure.

[0230] Embodiments of this disclosure in which exclusive ownership or privilege is asserted are defined as follows: In certain embodiments, for example, the following are provided: (Item 1) A method for treating a subject having lung, liver, and / or kidney disorders, or symptoms related to lung, liver, and / or kidney disorders, the following: The treatment involves administering to the subject a therapeutically effective bolus dose of a composition comprising a long-acting CNP, a long-acting CNP derivative, a long-acting NPRB agonist, an ultra-long-acting CNP, an ultra-long-acting CNP derivative, an ultra-long-acting NPRB agonist, a long-acting CNP agonist, an ultra-long-acting CNP agonist, or any combination thereof. The composition does not reduce blood pressure by more than 20% of the baseline blood pressure measurement obtained before administration of the therapeutically effective bolus dose of the composition. The composition increases the plasma cyclic GMP level 1 to 12 hours after administration to more than 1.5 times the baseline plasma cyclic GMP level, wherein the baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject. The aforementioned lung, liver, and / or kidney disorders, or symptoms associated with such lung, liver, and / or kidney disorders, are as follows: i) Acute lung injury (ALI), ii) acute respiratory distress syndrome (ARDS); iii) pulmonary edema; iv) Elevated levels of inflammatory cells in the lungs, v) Increased levels or expression of inflammatory cytokines in the lungs compared to healthy lungs, vi) Increased protein levels in the alveolar space compared to healthy lungs, vii) Hypoarterial oxygenation (Hypoarterial oxygenation is defined as a blood PaO2 of less than 60 mmHg and / or a blood hemoglobin oxygen sa...

Claims

[Claim 1] The invention described in the specification.

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