CROSS REFERENCE TO RELATED APPLICATION (S)

[0001] The present application claims priority to U.S. provisional application No. 63/061,892 filed on August 6, 2020, the entire contents of which are incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The instant disclosure is directed to methods of treating refractory ascites as well as medicaments that may be used in these methods.

BACKGROUND OF THE INVENTION

[0003] The liver is a multifunctional vital organ responsible for maintenance of metabolic homeostasis, protein synthesis, and detoxification. Any condition that induces chronic inflammation in the liver may consequently progress to fibrosis and ultimately cirrhosis. In Denmark the majority of cases with cirrhosis is attributed to high alcohol consumption and chronic viral hepatitis C and B infections (see Fialla AD, De Muckadell OBS, Touborg Lassen A (2012) Incidence, etiology and mortality of cirrhosis: A population-based cohort study. Scand J Gastroenterol 47(6):702-709; Goldberg E, Chopra S (2018) Cirrhosis in adults: Etiologies, clinical manifestations, and diagnosis. UpToDate. Available at: www. uptodate. com).

[0004] Portal hypertension may develop in patients suffering from cirrhosis and is of utmost importance in the development of complications such as ascites and hepatorenal syndrome, variceal bleeding and hepatic encephalopathy, being responsible for the high morbidity and mortality in patients with liver cirrhosis (see Moller S, Henriksen JH, Bendtsen F (2009) Ascites: Pathogenesis and therapeutic principles. Scand J Gastroenterol 44(8):902- 911; Gines P, Cardenas A, Arroyo V, Rodes J (2004) Management of Cirrhosis and Ascites. N

Engl J Med 350(16): 1646-1654). Further, with the presence of ascites follows a 10-30 % risk of spontaneous bacterial peritonitis. Thus, the progression of liver cirrhosis with decompensation and ascites formation dictates a poor long-term prognosis, and presence of ascites is associated with discomfort and decreased quality of life (see Gines P, Cardenas A, Arroyo V, Rodes J (2004) Management of Cirrhosis and Ascites. N Engl J Med 350(16): 1646- 1654; Rimola A, G G-T, M N, Piddock L, Planas R BB (2000) Diagnosis, treatment and prophylaxis of spontaneous bacterial peritonitis: a consensus document. J Hepatol 32:142- 153).

[0005] Cirrhosis is associated with serious clinical problems such as sodium retention and ascites. Cirrhotic ascites accounts for about 75% of ascites patients with the remaining about 25% ascites occurrences being due to malignancies (10%), cardiac failure (3%), tuberculosis (2%), pancreatitis (1%) and other rarer causes (Moore, et al.; Hepatology 2003, 38:258-266 and Reynolds TB. Clin Liver Dis 2000,4:151-168). The prevalence of refractory ascites in patients with cirrhosis is 5-10% (Ring-Larsen H, Therapy of Ascites and Renal Dysfunction in Cirrhosis. Malden, Massachusetts: Blackwell Co. 1999, 480-491). Patients who suffer from cirrhosis and refractory ascites exhibit diuretic resistance (European Association for the Study of the Liver, J. Hepat. 2010, 53 :397-417).

[0006] The pathogenesis of ascites formation in cirrhosis is complex with involvement of numerous regulatory mechanisms. Portal hypertension induces vasodilation of the splanchnic vascular system with splanchnic blood pooling resulting in decreased effective blood volume manifested as arterial hypotension. To counteract this systemic hypotension, the organism responds with vasopressin secretion, activation of the sympathetic nervous system and RAAS as well as endothelin secretion to facilitate vasoconstriction along with salt and water retention (3, 4, 6). In cases of severe renal vasoconstriction, the patient may develop hepatorenal syndrome with impaired kidney function and elevated serum creatinine levels.

[0007] Treatments of ascites can be classified as invasive or non-invasive (pharmacological) (see Moller S, Henriksen JH, Bendtsen F (2009) Ascites: Pathogenesis and therapeutic principles. Scand J Gastroenterol 44(8):902-911; Gines P, Cardenas A, Arroyo V, Rodes J (2004) Management of Cirrhosis and Ascites. N Engl J Med 350(16): 1646-1654; Moore KP, et al. (2003) The management of ascites in cirrhosis: Report on the consensus conference of The International Ascites Club. Hepatology 38(l):258-266; Descos L, et al. (1983) Comparison of six treatments of ascites in patients with liver cirrhosis. A clinical trial. Hepatogastroenterology 30(1): 15-20). Though the handling of ascites in cirrhosis with diuretics and a low-sodium diet predominantly is an effective treatment, 10 % of patients with ascites become resistant to pharmacological treatment and thus develop refractory ascites (see Moller S, Henriksen JH, Bendtsen F (2009) Ascites: Pathogenesis and therapeutic principles. Scand J Gastroenterol 44(8): 902-911; Arroyo V, et al. (1996) Definition and diagnostic criteria of refractory ascites and hepatorenal syndrome in cirrhosis. Hepatology 23(1): 164-176; Moore KP, Aithal GP (2006) Guidelines on the management of ascites in cirrhosis. Gut 55:Suppl. 6,vi 1-12; Laffi G, et al. (1993) Refractory ascites: definition, pathogenesis and treatment. Ann Ital di Med interna organo Uff della Soc Ital di Med interna 8(l):38-46). The generally accepted definition for refractory ascites consists of two distinct subgroups, diureticresistant where ascites does not resolve even on maximum dose of diuretics (400 mg spironolactone daily in combination with 160 mg furosemide daily) or diuretic-intractable due to diuretic-induced complications of high-dose diuretic treatments. With the development of refractory ascites follows a significantly poorer prognosis than for diuretic-responsive patients (see Salerno F, et al. (1993) Survival and prognostic factors of cirrhotic patients with ascites: a study of 134 outpatients. Am J Gastroenterol 88(4): 514-519). [0008] Currently, management of refractory ascites includes repeated large volume paracentesis, diuretics (Moore et al., Hepatology 2003, 38:258-266), transjugular intrahepatic portosystemic shunts (TIPS), peritoneovenous shunt, or liver transplantation (European Association for the Study of the Liver, J.Hepatol. 2010, 53:397-417 and Carstens et al., BMC Gastroenterology 2007, 7(1): 1-9).

[0009] For the last 30 years little has changed in the treatment of ascites and there is still no approved drug for the treatment of patients with liver cirrhosis and refractory ascites. At the debut of refractory ascites patients are facing a median survival of approximately 6 months, and most likely frequent hospitalizations as a result of complications or the need for paracentesis (see Arroyo V, et al. (1996) Definition and diagnostic criteria of refractory ascites and hepatorenal syndrome in cirrhosis. Hepatology 23(1): 164-176; Guardiola J, et al. (2002) External validation of a prognostic model for predicting survival of cirrhotic patients with refractory ascites. Am J Gastroenterol 97(9):2374-2378). Patients with refractory ascites have a survival rate as low as 50% at six to twelve months post-diagnosis (Cardenas and Arroyo, .Dig. Dis. 2005, 23:30-38 and Guardiola et al., Am. J. Gastroenterol. 2002; 97:2374- 2378). Thus, there is an unmet need for new and more effective treatments of refractory ascites in patients with liver cirrhosis.

[0010] A family of related peptides has been discovered that works in concert to achieve salt and water homeostasis in the body. These peptides, termed natriuretic peptides for their role in moderating natriuresis, diuresis and vasodilation, have varying amino acid sequences and originate from different tissues within the body. This family of natriuretic peptides consists of atrial natriuretic peptide (“ANP”), brain natriuretic peptide (“BNP”), C- type natriuretic peptide (“CNP”), Dendroaspis natriuretic peptide (“DNP”), urodilatin (“URO”, or ularitide), hybrid natriuretic peptides such as centeritide (a fusion between CNP and DNP). Their tissue-specific distribution of these peptides is as follows: heart (ANP, BNP, and DNP); brain (ANP, BNP, and CNP); endothelial cells (CNP); plasma (DNP); and kidney (CNP, URO). These peptides are constituents of a hormonal system that plays a critical role in maintaining an intricate balance of blood volume/pressure in the human body. For instance, urodilatin, a close analog of ANP secreted by kidney tubular cells, promotes excretion of sodium and water by acting directly on kidney cells in the collecting duct to inhibit sodium and water reabsorption.

[0011] Like other natriuretic peptides, such as ANP and BNP, urodilatin has been studied for use in treating various conditions, including renal failure, cardiovascular conditions such as congestive heart failure, bronchoconstrictive conditions, and cirrhosis (see, e.g., U.S. Pat. Nos. 5,571,789 and 6,831,064; Kentsch et al., Eur. J. Clin. Invest. 1992, 22(10):662-669; Kentsch et al., Eur. J. Clin. Invest. 1995, 25(4):281-283; Elsner et al., Am. Heart J. 1995, 129(4):766- 773; Forssmann et al., Clin. Pharmacol, and Therape. 1998, 64(3):322-330; Fluge et al., Eur. J. Clin. Invest. 1995, 25:728-736; Carstens et al., BMC Gastroenterology 2007, 7(1): 1-9, and Carstens et al., J. Am. Soc. Nephrol. 1998, 9:1489-1498).

[0012] There continues to be a need for new and more effective methods for treating refractory ascites.

[0013] All documents referenced herein are hereby incorporated by reference in their entireties for all purposes.

OBJECTS AND SUMMARY OF THE INVENTION

[0014] It is an object of the present invention to provide methods for the treatment of refractory ascites. [0015] The objects are met by the present invention which in certain embodiments is directed to a method of treating refractory ascites comprising administering a ularitide to a patient in need thereof continuously for a time period between about 2 hours and about 168 hours, between about 8 hours and about 84 hours, or between about 12 hours and about 48 hours.

[0016] In one embodiment, the methods of the present invention may result in the increase of the urine sodium excretion rate relative to baseline. In one embodiment, the methods of the present invention may result in the increase of urine flow rate and/or increase in urine volume relative to baseline. In another embodiment, the methods of the present invention may result in the decrease of a patient’s body weight post ularitide administration as compared relative to baseline. In one embodiment, the methods of the present invention may result in improvement upon ularitide administration to a patient (as compared relative to a baseline) in at least one of: serum creatinine, waist circumference, urine and plasma osmolality, plasma copeptin concentration, hematocrit, plasma cGMP, plasma aquaporin, eGFR (Cockroft) and GFR-24h- Crea, plasma renin, plasma angiotensin, plasma aldosterone, or a combination thereof. For any of the parameters above in certain embodiments, the increase or decrease for a beneficial or desired effect provided by the present invention may be at a ratio compared to baseline of at least 1.05, at least 1.1, at least 1.2, at least 1.5, at least 1.8, at least 2, at least 3, at least 4, at least 5 at least 8 or at least 10. In other embodiments, the increase or decrease for a beneficial or desired effect provided by the present invention may be at a ratio compared to baseline of from about 1.05 to about 10, about 1.1 to about 5, about 1.2 to about 8, about 1.5 to about 5 or from about 2 to about 4. In other embodiments, the increase or decrease for a beneficial or desired effect provided by the present invention may be a range from any of about 1.05, about 1.1, about 1.2, about 1.5, about 1.8, about 2, about 3, about 4, about 5 about 8 or about 10 to any of about 1.05, about 1.1, about 1.2, about 1.5, about 1.8, about 2, about 3, about 4, about 5 about 8 or about 10. [0017] In certain embodiments, the present invention is directed to a use of a ularitide for the treatment of refractory ascites on a patient in need thereof continuously for a time period between about 2 hours and about 48 hours.

[0018] In certain embodiments, the present invention is directed to a use of a ularitide in the preparation of a medicament for the treatment of refractory ascites on a patient in need thereof continuously for a time period between about 2 hours and about 48 hours. In certain embodiments, meaningful natriuresis and diuresis is observed in patients receiving continuous treatment with said Ularitide medicament for a time period between about 2 hours and about 48 hours.

[0019] In certain embodiments, the present invention is directed to a use of a ularitide to increase the urine sodium excretion rate, increase urine flow rate and/or urine volume, or decrease a patient’s body weight, and/or improve at least one of: serum creatinine, waist circumference, urine and plasma osmolality, plasma copeptin concentration, hematocrit, plasma cGMP, plasma aquaporin, eGFR (Cockroft) and GFR-24h-Crea, plasma renin, plasma angiotensin, plasma aldosterone, or a combination thereof, in a patient in need thereof.

[0020] In certain embodiments, the present invention is directed to a kit comprising a ularitide and instructions for use in the treatment of refractory ascites in a patient in need thereof continuously for a time period between about 2 hours and about 48 hours.

[0021] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an active agent" includes a single active agent as well as a mixture of two or more different active agents; and reference to an “excipient” includes a single excipient as well as a mixture of two or more different excipients, and the like.

[0022] As used herein, the term “about” in connection with a measured quantity, refers to the normal variations in that measured quantity, as expected by one of ordinary skill in the art in making the measurement and exercising a level of care commensurate with the objective of measurement and the precision of the measuring equipment. In certain embodiments, the term “about” includes the recited number ±10%, such that “about 10” would include from 9 to 11.

[0023] As used herein, the terms "active agent" refer to any material that is intended to produce a therapeutic, prophylactic, pharmacologic, immunologic, metabolic, physical or other intended effect, whether or not approved by a government agency for that purpose. These terms with respect to specific agents include all pharmaceutically active agents, all pharmaceutically acceptable salts thereof, stereoisomers, crystalline forms, co-crystals, ether, esters, hydrates, solvates, and mixtures thereof, where the form is pharmaceutically active.

[0024] The term "administrate" or "administration," as used herein (unless the mode of administration is explicitly specified), encompasses various methods of delivering a composition containing ularitide to a patient. Modes of administration may include, but are not limited to, methods that involve delivering the composition intravenously, intraperitoneally, intranasally, transdermally, topically, subcutaneously, parentally, intramuscularly, orally, or systemically, and via injection, ingestion, implantation, intraarterial, intracictemal, intradermal, nasal (aerosol or inhalation), buccal, intralesional, intracranial, intraprostatic, intrapleural, intratracheal, intravitreal, intravaginal, intrarectal, intratumoral, intraocular, subconjunctival, intravesicular, mucosal, intrapericardial, intrumbilical, local, by infusion, by continuous infusion, by bolus infusion, by absorption, by immersion, be localized perfusion, via catheter, via a lavage, by adsorption by any other means. The preferred means of administering a composition comprising a ularitide is intravenous injection, where the composition is formulated as a sterile solution. Another route of administration is oral ingestion, where the ularitide can be formulated as a pharmaceutical composition in the form of a syrup, an elixir, a suspension, a powder, a granule, a tablet, a capsule, a lozenge, a troche, an aqueous solution, a cream, an ointment, a lotion, a gel or an emulsion. In some embodiments, the pharmaceutical composition for oral ingestion is formulated for sustained release over a period of at least 24 hours, for immediate release, or for immediate release followed by a sustained release. Furthermore, administration of ularitide can be achieved by subcutaneous injection of a ularitide-containing composition, where the composition is formulated as a sterile solution and which is prepared as a sustained release system comprising microspheres or biodegradable polymers, such that the ularitide can be released into a patient's body at a controlled rate over a period of time, e.g., at least 24 hours or 48 hours.

[0025] An "effective amount" or a “therapeutically effective amount” or a “pharmacologically effective amount” refers to the amount of an active ingredient, e.g., ularitide, in a pharmaceutical composition that is sufficient to produce a beneficial or desired effect at a level that is readily detectable by a method commonly used for detection of such an effect. In some embodiments, such an effect results in a change of at least 10% from the value of a basal level where the active ingredient is not administered. In other embodiments, the change is at least 20%, 50%, 80%, or an even higher percentage from the basal level. As will be described below, the effective amount of an active ingredient may vary from subject to subject, depending on age, general condition of the subject, pre-existing or concomitant diseases of the subject, medical history of the subject, the severity of the condition being treated, and the particular biologically active agent administered and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

[0026] The term "natriuretic peptide" refers to a peptide that has the biological activity of bronchodilation promoting natriuresis, diuresis and vasodilation. Assays for testing such activity are known in the art, e.g., as described in U.S. Pat. Nos. 4,751,284 and 5,449,751.

Examples of natriuretic peptides include, but are not limited to, atrial natriuretic peptide (ANP(99-126)), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), Dendroaspis natriuretic peptide (DNP), hybrid natriuretic peptides such as centeritide (a fusion between CNP and DNP), urodilatin (URO, or ularitide), and any fragments of the prohormone ANP(1-126) or BNP precursor polypeptide that retains the vasodilating, natriuretic or diuretic activity, long-acting natriuretic peptide (LANP), kaliuretic peptide (KP), vessel dilator (VD), and combinations thereof. For further description of exemplary natriuretic peptides and their use or preparation, see, e.g., U.S. Pat. Nos. 4,751,284, 4,782,044, 4,895,932, 5,449,751, 5,461,142, 5,571,789, and 5,767,239. See also, Ha et al., Regul. Pept. 2006, 133(1-3): 13-19. The invention can also be practiced with peptides and proteins which may cause a diuresis or vasodilation effect such as relaxin.

[0027] As used in this application, the term "urodilatin" refers to a 32-amino acid peptide hormone that is described by U.S. Pat. No. 5,449,751 and has the amino acid sequence set forth in GenBank Accession No. 1506430A. Urodilatin, the 95-126 fragment of atrial natriuretic peptide (ANP), is also referred to as ANP(95-126). The term "atrial natriuretic peptide" or "ANP(99-126)" refers to a 28-amino acid peptide hormone, which is transcribed from the same gene and derived from the same polypeptide precursor, ANP(1-126), as urodilatin but without the first four amino acids at the N-terminus of urodilatin. For a detailed description of the prohormone, see, e.g., Oikawa et al., Nature 1984, 309:724-726 ; Nakayama et al., Nature 1984, 310:699-701; Greenberg et al., Nature 1984, 312:656-658; Seidman et al., Hypertension 1985, 7:31-34; and GenBank Accession Nos. 1007205A, 1009248A, 1101403A and AAA35529.

[0028] Conventionally, the term urodilatin (URO) is more often used to refer to the naturally occurring peptide, whereas the term ularitide is often used to refer to the recombinantly produced or chemically synthesized identical peptide. In this application, the term "urodilatin" and "ularitide" are used interchangeably to broadly encompass both a naturally occurring peptide and a recombinant or synthetic identical peptide. The terms also encompass any peptide of the above-cited amino acid sequence containing chemical modification (e.g., linearization, deamination, phosphorylation, PEGylation, etc.) at one or more residues or substitution by the corresponding D-isomer(s), so long as the peptide retains the biological activity as a natriuretic peptide. Furthermore, a chemically modified ularitide may contain one or two amino acid substitutions for the purpose of facilitating the desired chemical modification (e.g., to provide a reactive group for conjugation). "Urodilatin" or "ularitide" of this application, regardless of whether it contains chemical modifications, retains a substantial portion, i.e., at least 50%, preferably at least 80%, and more preferably at least 90%, of the biological activity of the naturally-occurring wild-type urodilatin or ANP(95-126).

[0029] The term "pharmaceutically acceptable excipient or carrier" refers to any pharmaceutically inert ingredient in a composition that may act, for example, to stabilize the active ingredient. A pharmaceutically acceptable excipient can include, but is not limited to, carbohydrates (such as glucose, sucrose, or dextrans), antioxidants (such as ascorbic acid or glutathione), chelating agents, low-molecular weight proteins, high-molecular weight polymers, gel-forming agents or other stabilizers and additives. Other examples of a pharmaceutically acceptable carrier include wetting agents, emulsifying agents, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 18th ed. (1990).

[0030] As used herein, a "patient" refers to a subject, particularly a human (but could also encompass a non-human), who has presented a clinical manifestation of a particular symptom or symptoms suggesting the need for treatment, who is treated prophylactically for a condition, or who has been diagnosed with a condition to be treated. [0031] The term “subject” encompasses the definition of the term “patient” and does not exclude individuals who are otherwise healthy.

[0032] The terms “treatment of’ and “treating” include the administration of an active agent(s) with the intent to lessen the severity of or prevent a condition, e.g., refractory ascites.

[0033] The terms “prevention of’ and “preventing” include the avoidance of the onset of a condition, e.g., refractory ascites.

[0034] The term “condition” or “conditions” refers to those medical conditions, such as refractory ascites, that can be treated, mitigated or prevented by administration to a subject of an effective amount of an active agent (such as ularitide or pharmaceutically acceptable salt thereof).

[0035] The term “basal level” or “baseline” may be used interchangeably and refer to the parameters collected from a subject prior to administration of an active agent.

[0036] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to illuminate certain materials and methods and does not pose a limitation on scope. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosed materials and methods.

DETAILED DESCRIPTION

[0037] The present invention is directed to a method of treating refractory ascites comprising administering ularitide to a patient in need thereof continuously for a time period between about 2 hours and about 168 hours, or between about 8 hours and about 84, or between about 12 hours and 48 hours. In other embodiments, the ularitide is administered to a patient in need thereof continuously for a time period between about 12 hours and about 72 hours, or between about 24 hours and about 60 hours, or between about 36 hours and about 54 hours or about 46 hours. Continuous infusion is meant to include intervals where the infusion is temporarily stopped (e.g., due to lack of tolerability caused e.g. by a hypotensive state, to provide a bolus dose, change bags, provide maintenance to the delivery system, etc.) as long as the interval does not result in a sub-therapeutic condition.

[0038] By virtue of the present invention, the continuous administration of ularitide may result in improved outcomes, e.g., at about 24 hours, about 36 hours, or about 48 hours post infusion start, in at least one of: urine sodium excretion rate, urine flow rate and/or urine volume, a patient’s absolute bodyweight, serum creatinine, waist circumference, urine and plasma osmolality, plasma copeptin concentration, hematocrit, plasma cGMP, plasma aquaporin, eGFR (Cockroft) and GFR-24h-Crea, plasma renin, plasma angiotensin, plasma aldosterone, or a combination thereof, compared to short treatment, outside of the time frame and/or compared to baseline. For instance, in one embodiment, administration of ularitide (continuous and/or bolus) may increase urine sodium excretion rate and/or increase urine flow rate and/or increase urine volume, and/or decrease a patient’s body weight, compared to short treatment, outside of the time frame and/or compared to baseline.

[0039] The continuous administration of the present invention may occur for a time period ranging between about 2 hours and about 168 hours, between about 4 hours and about 144 hours, between about 8 hours and about 120 hours, between about 12 hours and about 96 hours, between about 16 hours and about 72 hours, between about 20 hours and about 48 hours, between about 24 hours and about 36 hours, about 2 hours and about 48 hours, between about

4 hours and about 48 hours, between about 8 hours and about 48 hours, between about 12 hours and about 48 hours, between about 16 hours and about 48 hours, between about 20 hours and about 48 hours, between about 24 hours and about 36 hours, or for about 30 hours.

[0040] Upon initiation of therapy, the administration may be an immediate administration (e.g. by a parenteral bolus) or continuous over a time period of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 30 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours or at least about 48 hours. In certain embodiments, the duration is from about 2 hours to about 168 hours, from about 2 hours to about 120 hours, from about 2 hours to about 48 hours, from about 2 hours to about 24 hours, from about 12 hours to about 120 hours, and in other embodiments, from about 12 hours to about 96 hours, from about 24 hours to about 96 hours, or from about 24 hours to about 72 hours, from about 24 hours to about 36 hours, or from about 36 hours to about 60 hours, or from about 40 hours to about 56 hours, from about 44 hours to about 52 hours, from about 46 hours to about 50 hours, about 48 hours, from about 4 hours to about 144 hours, from about 8 hours to about 120 hours, from about 16 hours to about 72 hours, from about 20 hours to about 48 hours, or for about 30 hours. A preferred means for administering the ularitide is by parenteral (e.g., intravenous) administration.

[0041] In some embodiments, the administration may further comprise a bolus dose of ularitide (e.g. by a parenteral bolus). The bolus dose may occur over a duration of about 5 minutes to about 90 minutes, about 15 minutes to about 75 minutes, about 30 minutes to about 60 minutes, or about 45 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, or any range in between. [0042] The rate of bolus administration may range from about 300 ng/kg/minute to about 5000 ng/kg/minute, from about 1000 ng/kg/minute to about 4000 ng/kg/minutes, from about 2000 ng/kg/minute to about 3000 ng/kg/minute, or about 300 ng/kg/minute, about 500 ng/kg/minute, about 1000 ng/kg/minute, about 2000 ng/kg/minute, about 3000 ng/kg/minute, about 4000 ng/kg/minute, or any range in between.

[0043] In some embodiments, the bolus administration may precede the continuous administration of ularitide. In other embodiments, the bolus administration may come after the completion of the continuous administration of ularitide. In yet other embodiments, the bolus administration may intervene the continuous administration of ularitide. For instance if a total amount of ularitide (ULAtotaic) is to be administered continuously, 50% of ULAtotaic may be administered continuously, followed by a break in continuous administration to allow for a bolus administration of ularitide, which may then be followed by completion of the remaining 50% of ULAtotaic to be administered continuously. Similar breaks to allow for bolus administration in the middle of the continuous administration may occur, e.g., after about 10% of ULAtotaic is administered, after about 20% of ULAtotaic is administered, after about 30% of

ULAtotaic is administered, after about 40% of ULA _to taic is administered, after about 50% of

ULAtotaic is administered, after about 60% of ULAtotaic is administered, after about 70% of

ULAtotaic is administered, after about 80% of ULAtotaic is administered, after about 90% of

ULAtotaic is administered, or any range in between.

[0044] In some embodiments, the continuous administration may be stopped once to allow for bolus administration of ularitide. In other embodiments, the continuous administration may be stopped a plurality of times, e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, etc. to allow for a plurality of bolus administrations of ularitide. [0045] When the peptide (e.g., the natriuretic peptide or ularitide) is administered parenterally, the administration can be, e.g., by injection or infusion. When the parenteral administration is by injection, the route can be intravenous (into a vein), subcutaneous (under the skin), intramuscular (into muscle), intraperitoneal, intravitreal (intraocular), intracerebral or intraspinal. When the parenteral administration is by infusion, it is typically by an intravenous route. The parenteral administration can be by a sterile dosage form that is a solution, suspension or emulsion. For the present invention, the ularitide can be formulated for administration by a variety of techniques, including, for example, intravenous, intra-arterial, subcutaneous, intramuscular, intracictemal, intraperitoneal, intradermal, transdermal, nasal (inhalation or aerosol), buccal, topical, intralesional, intracranial, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intrathecal, intravaginal, intrarectal, intratumoral, intraocular, subconjunctival, intravesicular, transmucosal, intramedullary, intestinal, parenteral, intrapericardial, intrumbilical, direct intraventricular, intraperitoneal, oral, local, by injection, by infusion, by continuous infusion, by absorption, by adsorption, by immersion, by localized perfusion, via a catheter, or via a lavage, among others.

[0046] In another embodiment, one or more different medicines (e.g., diuretics and aldosterone antagonists) are administered to the patient. These one or more different medicines may be administered in combination with the ularitide, for example, by the same route (e.g., intravenously), with the option of being in one single pharmaceutical composition or two or more separate compositions; or these one or more different medicines may be administered separately by a different means (e.g., by oral ingestion). In some embodiments, the ularitide may be administered to a patient along with spironolactone and/or furosemide.

[0047] The composition used in the method of this invention optionally further comprises a pharmaceutically acceptable excipient or carrier. For example, mannitol may be used in such a pharmaceutical composition. In an exemplary embodiment, the concentration of mannitol is two times, three times, four times, five times, six times, 7 times, 8 times, 9 times, 10 times the concentration of the ularitide (or any range within these values). In another exemplary embodiment, the composition is an aqueous solution of 0.9% NaCl in which the ularitide is dissolved. In one particular embodiment of the method, the composition is an aqueous solution of 0.9% NaCl in which ularitide and mannitol are dissolved.

[0048] In another aspect, the present invention provides the use of ularitide for the manufacture of a medicament for the treatment of refractory ascites. The medicament may contain, in addition to an effective amount of the active agent (e.g., ularitide), a pharmaceutically acceptable excipient or carrier. In one embodiment, the medicament is formulated for continuous intravenous administration over a time period of at least about 2 hours. In other embodiments, the medicament is formulated for continuous intravenous administration over a time period between about 2 hours and about 168 hours. In some cases, the medicament is formulated for a sustained release of the active agent over a period of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 30 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours or at least about 48 hours. In certain embodiments, the duration is from about 2 hours to about 168 hours, from about 2 hours to about 120 hours, from about 2 hours to about 48 hours, from about 2 hours to about 24 hours, from about 12 hours to about 120 hours, and in other embodiments, from about 12 hours to about 96 hours, from about 24 hours to about 96 hours, or from about 24 hours to about 72 hours, from about 24 hours to about 36 hours, or from about 36 hours to about 60 hours, or from about 40 hours to about 56 hours, from about 44 hours to about 52 hours, from about 46 hours to about 50 hours, about 48 hours, from about 4 hours to about 144 hours, from about 8 hours to about 120 hours, from about 16 hours to about 72 hours, from about 20 hours to about 48 hours, or for about 30 hours. For example, the administration of the ularitide-containing medicament may last about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 96 hours, about 120 hours or any desirable time duration within this range.

[0049] In some embodiments, the medicament is administered in a manner such that the patient is receiving the active agent (e.g., ularitide) at a rate of at least about 1 ng/kg/minute, of at least about 2 ng/kg/minute, of at least about 5 ng/kg/minute, of at least about 7.5 ng/kg/minute, of at least about 10 ng/kg/minute, of at least about 15 ng/kg/minute, of at least about 20 ng/kg/minute, of at least about 30 ng/kg/minute, of at least about 45 ng/kg/minute, of at least about 60 ng/kg/minute, of at least about 75 ng/kg/minute, of at least about 100 ng/kg/minute, of at least about 200 ng/kg/minute, or of at least about 250 ng/kg/minute, or any desirable administration rate within this range such as for instance from about 1 ng/kg/minute to about 250 ng/kg/minute. In other embodiments, the administration rate is about 7.5 ng/kg/ minute, about 15 ng/kg/minute, about 20 ng/kg/minute, about 30 ng/kg/minute, about 45 ng/kg/minute, about 60 ng/kg/minute, about 100 ng/kg/minute, or about 200 ng/kg/minute. In one preferred example, ularitide is administered at the rate of about 15 ng/kg/minute. In another preferred example, ularitide is administered at the rate of about 30 ng/kg/minute. In yet another preferred example, ularitide is administered at the rate of about 45 ng/kg/minute.

[0050] The methods of the present invention can be utilized to treat refractory ascites. The refractory ascites may originate from one or more of cirrhosis, malignancy, cardiac failure, tuberculosis, pancreatitis, or other rare causes.

[0051] The administration of a ularitide according to the present invention is preferably achieved by intravenous injection, subcutaneous injection or oral ingestion. For intravenous administration, the composition comprising, e.g., ularitide, may be formulated with an aqueous diluent, suitably mixed with other optional additives such as a surfactant and/or a preservative for proper fluidity, stability and sterility of the composition, necessary for easy storage and injection. The injectable solution containing the ularitide may be prepared using a solvent or dispersion medium including water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the proliferation of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. The injectable solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Lastly, the injectable solution, once prepared by incorporating the active agents in the required amount in the appropriate solvent with optional excipients, is sterilized using a method that does not inactivate the active ingredient(s) of the composition, e.g., by filtered sterilization.

[0052] As disclosed herein, the ularitide can be formulated with mannitol. Non-limiting examples of other sugars that may be used in embodiments of the present invention include abequose, allose, allulose, altrose, apiose, arabinose, beet oligosaccharides, bifurcose, deoxyribose, dextrose(D-glucose), erlose, erythrose, erythrulose, fructose (levulose), fucose, fuculose, galactose, gentiobiose, gentiotriose, gentiotetraose, gulose, hamamelose, inulobiose, inulotriose, inulotetraose, isomaltose, isomaltotriose, isomaltotetraose, isomaltopentaose, isomaltulose (palatinose), kestose, kojibiose, lactose, lactulose, laminaribiose, lyxose, mannose, maltose, maltotriose, maltotetraose, maltulose, meletzitose, melibiose, methose, nigerose, nystose, panose, paratose, primeverose, psicose, raffinose, rhamnose, ribose, ribulose, rutinose, sorbinose, sorbose, soybean oligosaccharides, stachyose, sucrose, tagatose, talose, theanderose, threose, trehalose, turanose, xylobiose, xylotriose, xylose or xylulose. The carbohydrates used in embodiments of the present invention may be of their respective D- or L-configurations.

[0053] In certain embodiments, non-limiting examples of sugar alcohols that may be used include allitol, arabitol, erythritol, galactitol, glycerol, glycol, iditol, inositol, isomalt, lactitol, maltotetraol, maltotriol, ribitol, sorbitol, talitol, threitol and xylitol. The sugar alcohols used in embodiments according to the present invention may be of their respective the D- or L- configurations. These sugar alcohols have the benefits of having low glycemic indices. Mannitol, for example, has been used to treat increased intracranial pressure.

[0054] For oral administration, the composition comprising the ularitide may be formulated with an inert diluent or other pharmaceutically acceptable excipient, or it may be enclosed in a hard- or soft-shell gelatin capsule, or it may be compressed into tablets. The active agent(s) (e.g., ularitide) may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, caplets, elixirs, suspensions, syrups, wafers and the like. The orally ingestible formulation may contain high-molecular weight polymers or gel-forming agents that allow sustained release of the natriuretic peptide over an extended period of time, for example, at least 8 hours, at least 12 hours or at least 24 hours. This sustained release system achieves the slow release of the active agent over a period of time.

Examples

[0055] The following prophetic examples and/or studies are set forth to assist in understanding the invention and should not be construed as specifically limiting the invention described and claimed herein. Such variations of the invention, including the substitution of any or all equivalents now known or later developed, which would be within the purview of those skilled in the art, and changes in formulation or minor changes in therapeutic design, are to be considered to fall within the scope of the invention incorporated herein.

[0056] The following is a protocol synopsis for a clinical study according to an embodiment:

Clinical Study Title:

[0057] Single-center, randomized, double-blind, placebo-controlled clinical trial for the safety, tolerability and efficacy of ularitide in patients with refractory ascites.

Clinical Study Design:

[0058] Randomized, placebo-controlled, double-blind, single-center dose escalation clinical trial in cirrhosis patients with refractory ascites.

Number of Study Sites:

[0059] One study site in Aarhus/Denmark.

Type And Number Of Patients:

[0060] 38 patients with liver cirrhosis and refractory ascites with or without resistance against diuretics. Only patients matching all in- and exclusion criteria will be randomized to either receive ularitide or matching placebo on top of standard-of-care, i.e. furosemide (up to 160 mg/day) and spironolactone (up to 400 mg/day).

Objective:

[0061] To investigate the safety, tolerability and efficacy of ularitide on the renal response in patients with liver cirrhosis and refractory ascites for a maximum exposure duration of 48 hours Primary Efficacy Endpoints:

[0062] There are three primary endpoints:

[0063] Primary Efficacy Endpoint 1 evaluates changes (absolute and relative) in urine sodium excretion rate at 24 hours post infusion start and at the end of treatment versus baseline.

[0064] Primary Efficacy Endpoint 2 evaluates changes (absolute and relative) in urine volume at 24 hours post infusion start and at the end of treatment versus baseline.

[0065] Primary Efficacy Endpoint 3 evaluates change of absolute body weight at the end of treatment versus baseline.

[0066] Patients will be classified as “improved” if the patients experience an increase in urine sodium excretion rate, in urine volume, and/or reduction in body weight.

Secondary Endpoints:

[0067] Number of responders in the ularitide group versus the placebo group at the end of treatment as defined by

• urine volume increase of >100 % versus baseline at 2 hours post treatment start or

• urine volume increase of >50 % versus baseline at 24 hours post treatment start or

• natriuresis increase at the end of treatment by 100 % versus baseline

• body weight reduction by >2 kg at the end of treatment versus baseline

[0068] Change (absolute and relative) in sodium excretion rate at 2 and 4 hours post infusion start and at 6 hours after the end of treatment.

[0069] Change (absolute and relative) in urine volume at 2 and 4 hours post infusion start and at 6 hours after the end of treatment, versus baseline.

[0070] Change (absolute and relative) in serum creatinine at 24 hours post infusion start and at the end of treatment versus baseline. [0071] Change (absolute and relative) in waist circumference at 24 hours post infusion start and at the end of treatment versus baseline.

[0072] Change (absolute and relative) in urine and plasma osmolality at 24 hours post infusion start, at the end of treatment and at 6 hours after the end of treatment versus baseline.

[0073] Change (absolute and relative) in plasma copeptin concentration at 2, 24 hours post infusion start, at the end of treatment and at 6 hours after the end of treatment versus baseline. [0074] Change (absolute and relative) in hematocrit at 24 hours post infusion start, at the end of treatment and at 6 hours after the end of treatment versus baseline.

[0075] Change (absolute and relative) in plasma cGMP concentration at 2, 24 hours post infusion start, at the end of treatment and at 6 hours after the end of treatment versus baseline. [0076] Change (absolute and relative) in plasma aquaporin concentration at 2, 24 hours post infusion start, at the end of treatment and at 6 hours after the end of treatment versus baseline. [0077] Change (absolute and relative) in eGFR (Cockroft) and GFR-24h-Crea at 24 hours post infusion start and at the end of treatment versus baseline.

[0078] Change (absolute and relative) in plasma renin concentration at 2, 24 hours post infusion start, at the end of treatment and at 6 hours after the end of treatment versus baseline. [0079] Change (absolute and relative) in plasma angiotensin concentration at 2, 24 hours post infusion start, at the end of treatment and at 6 hours after the end of treatment versus baseline. [0080] Change (absolute and relative) in plasma aldosterone concentration at 2, 24 hours post infusion start, at the end of treatment and at 6 hours after the end of treatment versus baseline.

Safety Endpoints:

[0081] Incidence of stopping criteria leading to a dose reduction.

[0082] Incidence of stopping criteria leading to early termination of treatment.

[0083] Incidence and severity of Adverse Events (AEs) until 6 hours post infusion end [0084] Incidence of Serious Adverse Events (SAEs) until 30 days from end of first treatment sequence.

Inclusion Criteria:

[0085] Men and women >18 years.

[0086] Patients with liver cirrhosis confirmed by FibroScan (>20 kPa), or by imaging with signs of a bulky liver surface with collaterals, or clinically by cirrhosis stigmata.

[0087] Refractory ascites defined by:

• Failure to respond to or intolerance to high dose diuretics (spironolactone up to 400 mg/d and furosemide up 160 mg/d) .

• Early ascites recurrence: reappearance of grade 2 or 3 ascites within 4 weeks of initial mobilization or >2 paracentesis within last 3 months

[0088] Urine sodium excretion <60 mmol/24 h.

[0089] Serum creatinine <150 pmol/L.

[0090] Child-Turcotte-Pugh Score of B or C (<13).

[0091] Bilirubin <150 (pmol/L).

[0092] Prothrombin time (PP) 0.20-0.60 [INR 2.5-1.3],

[0093] SBP >95 (100) mmHg.

[0094] Written informed consent to participate in the clinical trial.

Exclusion Criteria:

[0095] Gastrointestinal bleeding within 2 weeks prior to inclusion.

[0096] Proteinuria >500 mg/day.

[0097] Hemoglobin <5.5 mmol/L.

[0098] Spontaneous bacterial peritonitis within 2 weeks prior to inclusion.

[0099] Loculated ascites. [00100] Hepatic encephalopathy encephalopathy grade 2-4 (West-Haven classification).

[00101] Obstructive uropathy.

[00102] Primary kidney disease.

[00103] Known diagnosis of congestive heart failure.

[00104] Known diagnosis of acute-on-chronic liver failure.

[00105] Known diagnosis of systematic inflammatory response syndrome (SIPS).

[00106] Acute infections by known diagnosis and/or antibiotic treatment.

[00107] Known HIV infection.

[00108] Known allergy to the investigational drug or other natriuretic peptides.

[00109] Treatment with dobutamine, levosimendan, milrinone, any phosphodiesterase inhibitor, octreotide, midodrine, vasopressin, dopamine or other vasopressors within 2 weeks prior to inclusion.

[00110] Nephrotoxic drugs within 1 month prior to inclusion.

[00111] Fertile women not using contraception, either an intrauterine device or hormonal contraception (tablets, implants, transdermal patches, vaginal rings and injections).

[00112] Positive pregnancy test in pre-menopausal women or in breast-feeding women. Menopause is defined as no menses since >12 months without an alternative medical cause prior to screening.

[00113] Participation in an interventional clinical drug trial within 1 month prior to inclusion.

[00114] Legal incapacity or limited legal capacity.

[00115] Patients who are employees or relatives of the investigator.

Investigational Medicinal Product: [00116] Ularitide-2.5 mg for injection containing 2.5 mg of the active ingredient ularitide and 20 mg mannitol; sterile, white to off-white lyophilized powder for reconstitution supplied in clear 5 mL glass vials. The test product is intended for IV use after being reconstituted in 5 mL sterile saline (0.9 % sodium chloride) and dissolved in a 250 mL infusion bag containing sterile saline.

Reference Therapy:

[00117] Matching placebo, i.e. mannitol, in 5 mL glass vials that are identical to the vials containing ularitide. Reconstitution and dissolution in sterile saline is identical to ularitide.

Storage and Stability

[00118] Lyophilized ularitide and placebo has a proposed shelf-life of 6 years when stored at 2-8 °C. The product should not be frozen or shaken. The formulation contains no preservative, and study drug should therefore be used on the day of reconstitution.

Dose, Mode and Duration of treatment:

[00119] After reconstitution of the lyophilized powder in 5 mL sterile saline the Investigational Medicinal Product (IMP) is dissolved in a 250 mL infusion bag containing sterile saline (0.9% sodium chloride) and administered as continued IV infusion through a dedicated line using an automated infusion pump system (Alaris GP+ Carefusion Pump).

[00120] The dose will be calculated for each patient according to body weight and adjusted by the infusion flow rate as shown in the below table:

[00121] A vascular access will be provided by insertion of a peripheral venous catheter, most likely in a vein of the hand or arm. Patients will continuously receive ularitide or placebo infusion up to 48 hours. Patients will remain hospitalized through the 48-hour infusion period and the 6-hour post-infusion follow-up; overall 54 hours, assumed no stopping criteria have occurred. Blood and urine samples will be collected as outlined in Table 1. Investigators and study personnel will follow a checklist to ensure protocol adherence of sampling and study procedures. Table 1: Samples and measures during hospitalization.

[00122] The following parameters will be analyzed immediately after collection of blood samples:

• Hematology: hemoglobin, red blood cell count, hematocrit, platelet count, and total white blood cell count.

• Blood chemistry: sodium, potassium, urea, creatinine, eGFR, C-reactive protein, bilirubin, albumin, alanine aminotransferase, and alkaline phosphatase.

• Coagulation: international normalized ratio, coagulation factors II, VII, X, prothrombin time.

[00123] 30 mL of blood will be collected at each vein puncture (baseline, after 2 hours of treatment, after 24 hours of treatment, at end of treatment and 6 hours post end of treatment), including 10 mL of plasma and 10 mL of serum, which will be frozen and placed in a research biobank shortly after collection. These samples will eventually be batch-analyzed for concentrations of cGMP, aquaporin, copeptin, renin, angiotensin and aldosterone, as well as hematocrit and plasma osmolality.

[00124] The following parameters will be analyzed upon urinalysis: sodium, potassium, creatinine, GRF-24h-Crea, urea and osmolality.

[00125] An overview on study conduct is provided in the schedules of assessments (Table 2). Urine volume will not only be determined as one of the primary efficacy endpoints, but also as lead parameter for the treatment-response-dependent dose adjustment.

Table 2: Pre-, during-, and post-treatment period assessments.

[00126] Continuous IV infusion of ularitide or placebo at a starting dose of 30 ng/kg/min will be initiated after randomization. Dose will be maintained until end of treatment, unless no clinically meaningful effect (>100% increase in urine volume vs baseline) will be observed after 2 hours. In this case, dose will be increased to 45 ng/kg/min, and the patient will be continuously treated at this dose until end of treatment.

[00127] If patients dosed at 45 ng/kg/min will show a >50% increase in urine volume vs baseline at 24 hours post treatment start, they will be continuously treated at this dose until end of treatment. If not, treatment will be terminated early for safety reasons.

[00128] Maximum treatment duration is 48 hours.

[00129] Patients will be treated according to standard-of-care, i.e. furosemide (up to 160 mg/day) and spironolactone (up to 400 mg/day). Ularitide (or matching placebo) will be administered on top of standard-of-care.

Stopping Criteria

[00130] Systolic blood pressure (SBP) will be determined non-invasively every 15 min for the first 6 hours of treatment and continuously monitored thereafter, but no less than every six hours.

[00131] If SBP will fall <90 mmHg in two consecutive measurements 5 min apart, treatment will be transiently discontinued for approximately 2 hours and SBP will be determined non-invasively every 15 min for approximately 2 hours.

If SBP will increase to levels >90 mmHg in at least two consecutive measurements within that 2-hour-period, IMP infusion will be re-started at half the starting dose, i.e. 15 ng/kg/min. Patients will be kept at this dose until end of treatment or until stopping criteria occur.

If SBP will not increase to levels >90 mmHg in at least two consecutive measurements within that 2-hour period, treatment will be terminated early for safety reasons.

[00132] If a patient will show SBP <90 mmHg any time after restart of the infusion at a dose of 15 ng/kg/min, treatment will be permanently discontinued for safety reasons. [00133] If patients do not tolerate the treatment because of serious adverse events e.g. allergic reactions, treatment will be terminated for safety reasons.

[00134] An adverse event (AE) is any untoward medical occurrence in a patient or clinical investigation patient administered a medicinal product and which does not necessarily have to have a causal relationship with the treatment. An AE can therefore be any unfavorable and unintended sign (including e.g. an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product.

[00135] Any worsening in severity or frequency of a concomitant disease or any new disease diagnosed in the study must be documented as an AE.

[00136] A serious adverse event (SAE) is any AE occurring at any dose that: results in death, is life-threatening, requires prolongation of existing hospitalization, results in persistent or significant disability/incapacity , or is an important medical event.

[00137] Other important medical events may also be considered an SAE when, based on appropriate medical judgment, they jeopardize the patient or require intervention to prevent one of the outcomes listed.

Study Design:

[00138] Before treatment start urine will be collected from patients over a period of approximately 2 hours. This urine volume serves as baseline.

[00139] All patients will be asked to drink the same pre-defined volume of water (2.5 L/24 h) over the 48-hour treatment period and 6-hour follow-up period with 200 ml at start of baseline and 200 ml before infusion start.

[00140] Eligible patients will be randomly assigned in a 2:1 ratio to ularitide or placebo by means of a computer-generated randomization list. Block randomization will be performed with blocks of six patients ensuring four patients treated with ularitide and two with placebo in each block.

[00141] Patients will be treated with a ularitide starting dose of 30 ng/kg/min (or matching placebo at the same infusion rate) for 2 hours. After 2 hours urine volume since start of treatment will be determined.

• If urine volume increase will be >100% increase vs baseline, patients will be continuously treated at the starting dose until end of treatment or until stopping criteria occur. Patients will be counted as responders (Table 3, scenario 1).

• If urine volume increase will be <100% vs baseline, dose will be increased to 45 ng/kg/min (Table 3, scenario 2-4).

[00142] After 4 hours since start of treatment urine volume from 2 to 4 hours post infusion start will be determined in all patients (2-4 hours).

[00143] In case of a dose increase to 45 ng/kg/min, the following criteria for dose adjustment will apply:

• If urine volume increase at 4 hours will be >100% versus baseline, treatment will be continued at the same dose until the end of treatment, or until stopping criteria will apply. Patients will be counted as responders (Table 3, scenario 2)

• If urine volume increase at 4 hours is <100% vs baseline, treatment will be continued at the same dose until 24 hours post treatment start, or until stopping criteria will apply (Table 3, scenario 3-4).

[00144] After 24 hours since start of treatment urine volume from 4 to 24 hours post infusion start will be determined in all patients (4-24 hours).

[00145] In patients dosed at 45 ng/kg/min and with a urine volume increase of <100 % versus baseline at the previous quantification at 4 hours post infusion start, the following criteria for dose adjustment will apply: • If urine volume increase is >50% vs baseline, treatment will be continued at the same dose until the end of treatment start, or until stopping criteria will apply. Patients will be counted as responders (Table 3, scenario 3).

• If urine volume increase is <50% versus individual baseline level, treatment will be terminated early for safety reasons. Patients will be counted as non-responders (Table 3, scenario 4).

[00146] Thus, during the trial, urine volume will be determined after 2, 4 and 24 hours of treatment, and at end of treatment (after 48 hours unless stopping criteria have applied), as well as after a 6-hour post treatment follow-up period.

[00147] After end of treatment patients will be followed-up for SAEs at day 30. Additionally, information concerning paracentesis in the meantime will be collected as well as body weight and waist circumference are measured.

Table 3: Treatment response to ularitide/placebo.

[00148] In the foregoing description, numerous specific details are set forth, such as specific materials, dimensions, processes parameters, etc., to provide a thorough understanding of the present invention. The particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. The words “example” or “exemplary” are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “example” or “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the words “example” or “exemplary” is simply intended to present concepts in a concrete fashion. As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or”. That is, unless specified otherwise, or clear from context, “X includes A or B” is intended to mean any of the natural inclusive permutations. That is, if X includes A; X includes B; or X includes both A and B, then “X includes A or B” is satisfied under any of the foregoing instances. Reference throughout this specification to “an embodiment”, “certain embodiments”, or “one embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase “an embodiment”, “certain embodiments”, or “one embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.

[00149] The present invention has been described with reference to specific exemplary embodiments thereof. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.