COMBINATION OF RELAXIN ANALOGUE AND VASOPRESSIN ANALOGUE FOR THE TREATMENT OF RENAL DISORDERS OR CONDITIONS - Patent application

Abstract

The present disclosure relates to a combination therapy for maintaining renal function during perioperative liver transplantation, comprising co-administration of a relaxin analog, such as a peptide analog of the B chain of human relaxin2 capable of activating the RXFP1 receptor, and (b) an analog of vasopressin (also known as arginine vasopressin (AVP), antidiuretic hormone (ADH), and aglipressin), such as terlipressin, capable of activating the V1 receptor, to an individual in need thereof in the treatment of renal disorders, such as renal dysfunction in cirrhosis, hepatorenal syndrome type 1 (HRS-AKI) and hepatorenal syndrome type 2 (HRS-NAKI), chronic kidney disease, and acute kidney injury. The present disclosure also relates to compositions comprising relaxin analogs and / or vasopressin analogs for co-administration to an individual in need thereof, preparation of such compositions, and use of such compositions for co-administration to an individual in need thereof, as well as commercial packaging thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 236,090, filed August 23, 2021, and U.S. Provisional Patent Application No. 63 / 332,994, filed April 20, 2022, the entire disclosures of each of which are incorporated herein by reference for all purposes.

[0002] The present disclosure relates to a combination therapy comprising (a) a relaxin analog capable of activating the RXFP1 receptor, such as a peptide analog of the B chain of human relaxin2, and (b) an analog of vasopressin (also known as arginine vasopressin (AVP), antidiuretic hormone (ADH), and agripressin), such as terlipressin, capable of activating the V1 receptor, administered to an individual in need thereof in the treatment of renal disorders, such as renal dysfunction in cirrhosis, hepatorenal syndrome type 1 (HRS-AKI) and hepatorenal syndrome type 2 (HRS-NAKI), chronic kidney disease, and acute kidney injury, and for maintaining renal function during perioperative liver transplantation. The present disclosure also relates to compositions comprising relaxin analogs and / or vasopressin analogs for co-administration to an individual in need thereof, preparation of such compositions, and use of such compositions for co-administration to an individual in need thereof, as well as commercial packaging thereof. [Background technology]

[0003] Terlipressin is a synthetic vasopressin approved in many countries outside the United States to treat life-threatening complications of cirrhosis, including hepatorenal syndrome (HRS) and esophageal bleeding (EVB). Its short half-life limits its use to hospital settings (Nilsson, et al., (1990) Drugs Explt Clin. Res., XVI(6):307-314) and it is typically administered as an intravenous bolus, usually every 4-6 hours. However, despite the potential clinical benefit of terlipressin, a recent study found that at 3 months, the mortality rate of patients receiving terlipressin and standard treatment (e.g., albumin) was 51% compared to 45% of patients receiving standard treatment alone (Wong, F. et al., Terlipressin plus Albumin for the Treatment of Type 1 Hepatorenal Syndrome, NEJM (2021), 384: 818-828). Furthermore, terlipressin may cause side effects in up to 40% of patients. Serious side effects (including respiratory failure, myocardial infarction, angina pectoris, cardiac arrhythmias, severe hypertension, and intestinal ischemia / infarction / bleeding) have been reported to date, and up to 10% of patients may require treatment interruption (Angeli, (2011) Ascites, Hyponatremia and Hepatorenal Syndrome: Progress in Indeed, because of its rapid vasoconstrictor properties, terlipressin administered as an IV bolus should be used with caution and may not otherwise be recommended in patients with severe asthma, severe hypertension, advanced atherosclerosis, cardiac rhythm disorders, and coronary insufficiency.

[0004] Thus, there is a need for improved therapies for treating hepatorenal syndrome (HRS) and related conditions, e.g., that improve their efficacy and / or safety profile. Summary of the Invention

[0005] In some embodiments, provided herein is a method of preventing or treating renal failure in an individual in need thereof, comprising co-administering to the individual an effective amount of a relaxin analog and a vasopressin analog, in some embodiments, the renal failure is selected from the group consisting of cirrhosis-induced renal dysfunction, liver transplant-induced renal dysfunction, chronic kidney disease, and acute kidney injury.

[0006] Also provided herein, according to some embodiments, is a method of preventing or treating hepatorenal syndrome in an individual in need thereof, comprising co-administering to the individual an effective amount of a relaxin analog and a vasopressin analog. In some embodiments, the hepatorenal syndrome is HRS-AKI (type 1 hepatorenal syndrome).

[0007] In some embodiments, the relaxin analog is an RXFP1 agonist. In some embodiments, the relaxin analog is a long-acting peptidyl RXFP1 agonist. In some embodiments, the vasopressin analog is a V1a receptor agonist. In some embodiments, the vasopressin analog is terlipressin or a pharmaceutically acceptable salt thereof.

[0008] In some embodiments, terlipressin is administered intravenously at a dose of 0.5-2 mg per dose. In some embodiments, terlipressin is administered intravenously at a dose of 0.5-2 mg every 4-6 hours. In some embodiments, terlipressin is administered by intravenous infusion. In some embodiments, terlipressin is administered at a rate of 0.5-2 mg per 4-6 hours. In some embodiments, terlipressin is administered at a rate of 0.5-2 mg per 8-36 hours. In some embodiments, terlipressin is administered at a rate of 0.5-2 mg per 10-30 hours. In some embodiments, terlipressin is administered at a rate of 0.5-2 mg per 15-28 hours. In some embodiments, terlipressin is administered at a rate of 0.5-2 mg per 20-25 hours. In some embodiments, terlipressin is administered at a rate of 0.5-2 mg per 24 hours.

[0009] In some embodiments, the relaxin analog is administered at a dose of about 0.01 mg / kg to about 0.5 mg / kg. In some embodiments, the relaxin analog is administered parenterally, intravenously, subcutaneously, rectally, transdermally, or by inhalation. In some embodiments, the relaxin analog has an EC50 of less than 15 nM, less than 1 nM, less than 0.5 nM, or less than 0.1 nM for activating RXFP1 in an in vitro OVCAR5 cAMP assay.

[0010] In some embodiments, the methods of prevention or treatment further comprise administering to the individual midodrine or octreotide.

[0011] In some embodiments, the relaxin analog and the vasopressin analog are administered simultaneously. In some embodiments, the relaxin analog and the vasopressin analog are administered in a single composition. In some embodiments, the relaxin analog and the vasopressin analog are administered in separate compositions. In some embodiments, the vasopressin analog and the relaxin analog are administered sequentially.

[0012] In some embodiments, the combination therapy has a synergistic therapeutic effect. In some embodiments, administration of a relaxin analog reduces adverse effects associated with vasopressin analog treatment in an individual. In some embodiments, administration of a vasopressin analog reduces the risk of hypotension associated with relaxin analog treatment in an individual. In some embodiments, administration of a vasopressin analog or relaxin analog increases renal pressure in an individual.

[0013] Also provided herein, according to some embodiments, is a method of treating renal failure in an individual in need thereof, comprising administering a relaxin analog to the individual who has previously been administered a vasopressin analog.

[0014] Also provided herein, according to some embodiments, is a method for treating hepatorenal syndrome in an individual with cirrhosis, comprising: a) administering a relaxin analog to an individual who has previously been administered a vasopressin analog.

[0015] In some embodiments, administration of a relaxin analog reduces adverse effects associated with vasopressin analog treatment in an individual.

[0016] Also provided herein, according to some embodiments, is a method of treating renal failure in an individual in need thereof, comprising administering a vasopressin analog to the individual who has previously been administered a relaxin analog.

[0017] Also provided herein, according to some embodiments, is a method for treating hepatorenal syndrome in an individual with cirrhosis, comprising: a) administering a vasopressin analog to an individual who has previously been administered an effective amount of a relaxin analog.

[0018] In some embodiments, administration of a vasopressin analog reduces the risk of hypotension associated with treatment with a relaxin analog in an individual, hi some embodiments, administration of a vasopressin analog or relaxin analog increases renal pressure in an individual.

[0019] In some embodiments, the hepatorenal syndrome is HRS-AKI (type 1 hepatorenal syndrome).

[0020] Also provided herein, in some embodiments, are pharmaceutical compositions comprising a relaxin analog, a vasopressin analog, and one or more pharma- ceutically acceptable excipients, either separately or together.

[0021] Also provided herein, in some embodiments, is a kit comprising a relaxin analog in a pharma- ceutically acceptable composition, and a vasopressin analog in a pharma- ceutically acceptable composition.

[0022] In some embodiments, the relaxin analog is an RXFP1 agonist. In some embodiments, the relaxin analog is a long-acting peptidyl RXFP1 agonist. In some embodiments, the vasopressin analog is a V1a receptor agonist. In some embodiments, the vasopressin analog is terlipressin or a pharmaceutically acceptable salt thereof.

[0023] In some embodiments, the relaxin analog has formula (I) (SEQ ID NO: 105): N ter -Ac-X 10 -EGREX 15 -VRX 18 -X 19 -IX 21 -X 22 -EGX 25 -SX 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 -NH2-C ter1. A modified relaxin B chain peptide comprising: During the ceremony, N ter represents the N-terminus of the peptide, C ter represents the C-terminus of the peptide, Ac represents an acetyl group. X 10 represents an amino acid selected from the group consisting of leucine, 2-amino-isobutyric acid, Nε-acetyl-lysine, and α-methyl-leucine, E stands for glutamic acid, G stands for glycine, R represents arginine, X 15 represents an amino acid selected from the group consisting of lysine, arginine, homolysine, homoarginine, ornithine, glutamine, phenylalanine, and leucine; V stands for valine, X 18 represents an amino acid selected from the group consisting of alanine, 2-amino-isobutyric acid, leucine, Nε-acetyl-lysine, and glutamine, X 19 represents an amino acid selected from the group consisting of lysine, Nε-acetyl-lysine, citrulline, glutamine, alanine, and 2-amino-isobutyric acid, I stands for isoleucine, X 21 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and alanine, X 22 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and isoleucine, X 25 represents the following structure: [ka] During the ceremony, * is X in formula (I). 25 represents a covalent bond to the preceding glycine, [ka] is X in formula (I). 25 represents a covalent bond to serine followed by Z represents a group of formula (II), -[(PEG xx ) b (gE) c C d ], b and c independently represent 1, 2, 3, 4, or 5; PEG xx represents independently a polyethylene glycol derivative selected from the group consisting of PEG2, PEG2DGA, and TTDS; gE stands for gamma-glutamic acid; C d is a linear saturated C 12 ~C 22 represents an acyl group, S stands for serine, X 27 represents an amino acid selected from the group consisting of threonine, lysine, arginine, and glutamine; X 28 represents an amino acid selected from the group consisting of tryptophan, phenylalanine, 5-fluoro-tryptophan, 5-chloro-tryptophan, 5-methoxy-tryptophan, tyrosine, 4-fluoro-phenylalanine, 1-naphthylalanine, 2-naphthylalanine, α-methyl-tryptophan, α-methyl-phenylalanine, and 5-hydroxy-tryptophan, X 29 represents an amino acid selected from the group consisting of serine, D-serine, 2-amino-isobutyric acid, threonine, α-methyl-serine, Nε-acetyl-lysine, and valine, X 30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, α-methyl-lysine, D-lysine, lysine, homolysine, ornithine, arginine, and α-methyl-arginine; X 31 represents an amino acid selected from the group consisting of arginine, Nω-methyl-arginine, alanine, Nω,Nω′-dimethyl-arginine, and citrulline, X32 represents an amino acid selected from the group consisting of lysine, alanine, arginine, Nε-acetyl-lysine, and Nε,Nε,Nε-tri-methyl-lysine, X 33 represents an amino acid selected from lysine, Nε-acetyl-lysine, leucine, arginine, and alanine, or a salt or solvate thereof.

[0024] In some embodiments, b represents 2, 3, 4, or 5 and c represents 2, 3, or 4, or a salt or solvate thereof.

[0025] In some embodiments, C d is a linear saturated C 12 ~C 22 Acyl groups, e.g., C 12 (Lau), C 14 (Myr), C 15 (Penta), C 16 (Palm), C 17 (Hepta), C 18 (Stea), C 20 (Eico), and C 22 (Doco) acyl groups. In one embodiment, C d is C 12 (Lau), C 14 (Myr), C 15 (Penta), C 16 (Palm), C 17 (Hepta), or C 18 (Stea) a linear saturated acyl group selected from the group consisting of acyl groups, such as a linear saturated C 14 , C 16 , or C 18 Acyl groups, or linear C 16 Or C 18 It represents an acyl group, or a salt or solvate thereof.

[0026] In some embodiments, C d is C 12(Lau), C 14 (Myr), C 15 (Penta), C 16 (Palm), C 17 (Hepta), and C 18 (Stea) represents a linear saturated acyl group selected from the group consisting of acyl groups, or a salt or solvate thereof.

[0027] In some embodiments, C d is a linear C 16 ~C 18 It represents an acyl group, or a salt or solvate thereof.

[0028] In various embodiments, the relaxin analog has formula (Ib): N ter -Ac-X 10 -EGREX 15 -VRX 18 -X 19 -IX 21 -X- 22 -EGX 25 -SX 27 -X 28 -X 29 -X 30 -RX 32 -X 33 -NH2-C ter , During the ceremony, N ter represents the N-terminus of the peptide, C ter represents the C-terminus of the peptide, Ac represents an acetyl group. X 10 represents an amino acid selected from the group consisting of leucine, Nε-acetyl-lysine, and 2-amino-isobutyric acid, E stands for glutamic acid, G stands for glycine, R represents arginine, X 15 represents an amino acid selected from the group consisting of lysine, arginine, homolysine, glutamine, phenylalanine, and leucine; V stands for valine, X 18 represents an amino acid selected from the group consisting of alanine, 2-amino-isobutyric acid, and Nε-acetyl-lysine, X 19 represents an amino acid selected from the group consisting of lysine, Nε-acetyl-lysine, glutamine, and citrulline, I represents isoleucine, X 21 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and alanine, X 22 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and isoleucine, X 25 represents the following structure: [ka] During the ceremony, * is X in formula (Ia) 25 represents a covalent bond to the preceding glycine, [ka] is X in formula (Ia) 25followed by a covalent bond to serine, and Z is -(TTDS)2-(gE)3-Palm, -(TTDS)3-(gE)3-Palm, -(PEG2DGA)3-(gE)3-Palm, -(PEG2)4-(gE)3-Palm, -(TTDS)2-(gE)2-Palm, -(TTDS)2-(gE)3-Stea, -(TTDS)3-(gE)3-Stea, -(PEG2DGA)3-(gE)3-Stea, -(PEG2DGA)3-(gE)4-Stea, -(PEG2)3-(gE)3-Palm, -(PEG2)4-(gE)3-Stea, -(PEG2)5-(gE)3- Palm, -(PEG2)5-(gE)4-Palm, -(TTDS)3-(gE)4-Stea, -(TTDS)2-(gE)4-Palm, -(TTDS)3-(gE)2-Stea, -(TTDS)2-(gE)4-Stea, -(TTDS)4-(gE)3-Stea, -(TTDS)3-(gE)4-Palm, -(TTDS)4-(gE)3-Palm, -(TTDS)3-(gE)3-Myr, and -(TTDS)3-(gE)4-Myr, wherein gE represents γ-glutamic acid, Palm represents palmitoyl, and Stea represents stearoyl; S stands for serine, X 27 represents an amino acid selected from the group consisting of threonine, glutamine, arginine, and lysine; X 28 represents an amino acid selected from the group consisting of tryptophan, phenylalanine, 5-chlorotryptophan, α-methyl-phenylalanine, 4-fluoro-phenylalanine, and 5-fluorotryptophan, X 29 represents an amino acid selected from the group consisting of serine, D-serine, 2-amino-isobutyric acid, Nε-acetyl-lysine, threonine, and valine, X 30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, α-methyl-lysine, D-lysine, lysine, homolysine, and arginine; X 32represents an amino acid selected from the group consisting of lysine, alanine, arginine, and Nε-acetyl-lysine, X 33 represents an amino acid selected from lysine, Nε-acetyl-lysine, and arginine, or a salt or solvate thereof.

[0029] In some embodiments, the relaxin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-97.

[0030] In some embodiments, Z is -(TTDS)2-(gE)3-Palm, -(TTDS)3-(gE)3-Palm, -(PEG2DGA)3-(gE)3-Palm, -(PEG2)4-(gE)3-Palm, -(TTDS)2-(gE)2-Palm, -(TTDS)2-(gE)3-Stea, -(TTDS)3-(gE)3-Stea, -(PEG2DGA)3-(gE)3-Stea, -(PEG2)3-(gE)3-Palm, -(PEG2)4-(gE)3-Stea, -(PEG2)5-(gE)3-Palm, -(TTDS)3-(gE)4-Stea, -(TTDS)2-(gE)4-Palm, -(TTDS)2-(gE)4-Stea, -(TTDS)4-(gE)3-Stea, -(TTDS)3-(gE)4-Palm, -(TTDS)4-(gE)3-Palm, -(TTDS)3-(gE)3-Myr, and -(TTDS)3-(gE)4-Myr.

[0031] In some embodiments, the relaxin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 7, 9-12, 20-22, 26, 28, 30-34, 45, 47-49, 51, 54-62, 64, 67-69, 71-86, 91, 93, and 96.

[0032] In some embodiments, the relaxin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 7, 20, 26, 30-34, 45, 48, 49, 51, 54-61, 67, 71, 73, 75-79, 81, 83-92, and 97.

[0033] In some embodiments, the relaxin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:20.

[0034] In some embodiments, the relaxin analog comprises the amino acid sequence of SEQ ID NO:3.

[0035] Further disclosed herein is a method of preventing or treating renal failure in an individual in need thereof, comprising co-administering to the individual a dose of about 1.0 mg to about 10.0 mg of a relaxin analog and an effective amount of a vasopressin analog. In various embodiments, the renal failure is selected from the group consisting of cirrhosis-induced renal dysfunction, liver transplant-induced renal dysfunction, chronic kidney disease, and acute kidney injury.

[0036] Further disclosed herein is a method of preventing or treating hepatorenal syndrome in an individual in need thereof, comprising co-administering to the individual a relaxin analog at a dose of about 1.0 mg to about 10.0 mg and an effective amount of a vasopressin analog, hi various embodiments, the hepatorenal syndrome is HRS-AKI (type 1 hepatorenal syndrome).

[0037] In various embodiments, the relaxin analog is an RXFP1 agonist. In various embodiments, the relaxin analog is a long-acting peptidyl RXFP1 agonist.

[0038] In various embodiments, about 1.0 mg to about 3.0 mg of a relaxin analog is administered to the individual. In various embodiments, about 1.0 mg of a relaxin analog is administered to the individual. In various embodiments, about 2.0 mg of a relaxin analog is administered to the individual. In various embodiments, about 3.0 mg to about 5.0 mg of a relaxin analog is administered to the individual. In various embodiments, about 4.0 mg of a relaxin analog is administered to the individual. In various embodiments, about 5.0 mg to about 10.0 mg of a relaxin analog is administered to the individual. In various embodiments, about 5.0 mg of a relaxin analog is administered to the individual. In various embodiments, about 10.0 mg of a relaxin analog is administered to the individual. In various embodiments, the relaxin analog is administered intravenously. In various embodiments, the relaxin analog is administered intravenously over a period of about 1 hour to about 10 hours. In various embodiments, the relaxin analog is administered intravenously over a period of about 2 hours to about 8 hours. In various embodiments, the relaxin analog is administered intravenously over a period of about 3 hours to about 6 hours. In various embodiments, the relaxin analog is administered intravenously over a period of about 4 hours.

[0039] In various embodiments, the methods disclosed herein further comprise administering to the individual an additional dose of the relaxin analog. In various embodiments, the additional dose of the relaxin analog is administered 5 hours to 18 hours after administration of about 1.0 mg to about 5.0 mg of the relaxin analog. In various embodiments, the additional dose of the relaxin analog is administered 8 hours to 15 hours after administration of the dose of about 1.0 mg to about 10.0 mg of the relaxin analog. In various embodiments, the additional dose of the relaxin analog is administered 10 hours to 13 hours after administration of the dose of about 1.0 mg to about 10.0 mg of the relaxin analog. In various embodiments, the additional dose of the relaxin analog is administered about 12 hours after administration of the dose of about 1.0 mg to about 10.0 mg of the relaxin analog. In various embodiments, the additional dose of the relaxin analog is administered subcutaneously to the individual. In various embodiments, the additional dose of the relaxin analog comprises about 1 mg to about 50 mg of the relaxin analog. In various embodiments, the additional dose of the relaxin analog comprises about 5 mg to about 15 mg of the relaxin analog. In various embodiments, the additional dose of the relaxin analog comprises about 8 mg to about 12 mg of the relaxin analog. In various embodiments, the additional dose of the relaxin analog comprises about 10 mg of the relaxin analog.

[0040] In various embodiments, the methods disclosed herein further comprise administering to the individual a further additional dose of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog is administered 18 to 30 hours after administration of about 1.0 mg to about 5.0 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog is administered 20 to 26 hours after administration of the dose of about 1.0 mg to about 10.0 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog is administered about 24 hours after administration of the dose of about 1.0 mg to about 10.0 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog is administered subcutaneously to the individual. In various embodiments, the further additional dose of the relaxin analog comprises about 1 mg to about 50 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog comprises between about 2 mg and about 15 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog comprises between about 3 mg and about 8 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog comprises about 5 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog comprises between about 2 mg and about 5 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog comprises about 2.5 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog comprises between about 6 mg and about 15 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog comprises between about 8 mg and about 12 mg of the relaxin analog. In various embodiments, the further additional dose of the relaxin analog comprises about 10 mg of the relaxin analog.

[0041] In various embodiments, the methods disclosed herein further comprise administering an additional dose of the relaxin analog daily. In various embodiments, the additional dose of the relaxin analog is administered daily for about 10 days to about 20 days. In various embodiments, the additional dose of the relaxin analog is administered daily for about 12 days to about 16 days. In various embodiments, the additional dose of the relaxin analog is administered daily for about 14 days or more. In various embodiments, the additional dose of the relaxin analog is administered daily for about 3 days to about 15 days. In various embodiments, the additional dose of the relaxin analog is administered daily for about 4 days to about 13 days. In various embodiments, the additional dose of the relaxin analog is administered daily for about 5 days to about 11 days. In various embodiments, the additional dose of the relaxin analog is administered daily for about 6 days to about 9 days.

[0042] In various embodiments, the relaxin analog has an EC of activated RXFP1 of less than 15 nM, less than 1 nM, less than 0.5 nM, or less than 0.1 nM in an in vitro OVCAR5 cAMP assay. 50In various embodiments, the vasopressin analog is a V1a receptor agonist. In various embodiments, the vasopressin analog is terlipressin or a pharma- ceutically acceptable salt thereof. In various embodiments, the terlipressin is administered intravenously at a dose of about 0.5 to about 10 mg. In various embodiments, the terlipressin is administered intravenously at a dose of about 0.5 to about 2 mg every 4 to 6 hours. In various embodiments, the terlipressin is administered intravenously at a dose of about 1 mg every 6 hours. In various embodiments, the terlipressin is administered intravenously at a dose of about 2 to about 6 mg every 4 to 6 hours. In various embodiments, the terlipressin is administered intravenously at a dose of about 4 mg every 6 hours. In various embodiments, the terlipressin is administered intravenously at a dose of about 6 to about 10 mg every 4 to 6 hours. In various embodiments, the terlipressin is administered intravenously at a dose of about 6 mg every 6 hours. In various embodiments, terlipressin is administered intravenously at a dose of about 8 mg every 6 hours. In various embodiments, terlipressin is administered intravenously by bolus injection. In various embodiments, terlipressin is administered intravenously by bolus injection over about 1 minute to about 5 minutes. In various embodiments, terlipressin is administered intravenously by bolus injection over about 2 minutes to about 3 minutes. In various embodiments, terlipressin is administered intravenously by bolus injection over about 2 minutes. In various embodiments, terlipressin is administered intravenously at a dose of about 1 mg to about 10 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 1.5 mg to about 4 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 2 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 5 mg to about 8 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 6 mg. In various embodiments, terlipressin is administered intravenously over a period of about 10 hours to about 30 hours. In various embodiments, terlipressin is administered intravenously over a period of about 20 hours to about 25 hours. In various embodiments, terlipressin is administered intravenously over a period of about 24 hours.

[0043] In various embodiments, the methods disclosed herein further comprise administering to the individual midodrine or octreotide. In various embodiments, the methods disclosed herein further comprise administering to the individual albumin. In various embodiments, the doses of relaxin analog and vasopressin analog are administered simultaneously. In various embodiments, the doses of relaxin analog and vasopressin analog are administered in a single composition. In various embodiments, the doses of relaxin analog and vasopressin analog are administered in separate compositions. In various embodiments, the doses of vasopressin analog and relaxin analog are administered sequentially. In various embodiments, the combination therapy has a synergistic therapeutic effect.

[0044] In various embodiments, the combination therapy achieves improved response rate incidence, with responders defined at least according to the International Club of Acites (ICA) criteria. In various embodiments, responders include complete or partial responders, as defined according to ICA criteria, who survive free of renal transplant therapy (RRT) for at least 30 days from the start of treatment. In various embodiments, responders include complete or partial responders, as defined according to ICA criteria, who survive free of renal transplant therapy (RRT) for at least 10 days from the start of treatment. In various embodiments, a complete responder is defined as two serum creatinine levels returning to within 0.3 mg / dL (26.5 micromoles / L) of the baseline serum creatinine value, at least two hours apart. In various embodiments, a partial responder is defined as regression of at least one stage of acute kidney injury (AKI) accompanied by a decrease in serum creatinine of 0.3 mg / dL or more above the baseline serum creatinine value. In various embodiments, the combination therapy achieves an improved incidence of response rate, where the incidence is measured based on serum creatinine returning to a value within 0.3 mg / dL (26.5 micromol / L) of the baseline value. In various embodiments, the combination therapy achieves an improved incidence of response rate, where the incidence is measured based on regression of acute kidney injury (AKI) stage with a decrease in serum creatinine of 0.3 mg / dL or more above the baseline value. In various embodiments, the combination therapy achieves an improved incidence of response rate, where the incidence is measured based on two consecutive serum creatinine values ​​at least two hours apart falling below 1.5 mg / dL. In various embodiments, administration of the relaxin analog reduces adverse effects associated with vasopressin analog treatment in an individual. In various embodiments, administration of the vasopressin analog reduces the risk of hypotension associated with relaxin analog treatment in an individual. In various embodiments, administration of the vasopressin analog increases renal pressure in the individual.

[0045] Further disclosed in the present specification is a method for preventing or treating renal failure in an individual in need thereof, the method comprising: A) intravenously administering to the individual a dose of about 4.0 mg of a relaxin analog; B) subcutaneously administering to the individual a dose of about 5.0 mg of a relaxin analog on the same day that step (A) is performed; and C) subcutaneously administering to the individual a dose of about 10 mg of a relaxin analog on a later day that is different from the days that steps (A) and (B) are performed.

[0046] Further disclosed herein is a method of preventing or treating hepatorenal syndrome in an individual in need thereof, comprising: A) administering to the individual a dose of about 4.0 mg of a relaxin analog; B) administering to the individual a dose of about 5.0 mg of a relaxin analog subcutaneously on the same day that step (A) is performed; and C) administering to the individual a dose of about 10 mg of a relaxin analog subcutaneously on a later day that is different from the days that steps (A) and (B) are performed. In various embodiments, the methods disclosed herein further comprise repeating step (c) daily for up to 13 days. In various embodiments, the methods disclosed herein further comprise administering a bolus of 1 mg of terlipressin intravenously every 6 hours on the same day that steps (A) and (b) are performed. In various embodiments, the methods disclosed herein further comprise repeating a bolus of 1 mg of terlipressin intravenously every 6 hours for up to 14 days. [Brief description of the drawings]

[0047] [Figure 1] 2 is two parts of a schema depicting the general method used to synthesize relaxin peptide analogs. [Diagram 2] 2 is two parts of a schema depicting the general method used to synthesize relaxin peptide analogs. [Figure 3A] 1 shows the change from baseline effective renal plasma flow following administration of a relaxin agonist. [Figure 3B] 1 shows the change from baseline effective renal plasma flow following administration of a relaxin agonist. [Figure 4] 1 shows the overall study design of a Phase II trial evaluating the safety, tolerability, efficacy, and pharmacokinetics of a relaxin agonist in combination with terlipressin. [Diagram 5] The design of the open-label safety run-in portion of the overall study design shown in FIG. 4 is shown. [Figure 6] The design of the single-blind, placebo-controlled, randomized treatment portion of the overall study design is shown in FIG. [Figure 7] The design of the open-label terlipressin non-responder portion of the overall study design shown in FIG. 4 is shown.

[0048] definition As used herein, "X" in the formula of a relaxin peptide analog y The term " is used with different values ​​of y to represent an amino acid as defined in the formula definition above. y indicates the position of said amino acid in the native B chain of relaxin-2. For example, X 10 represents the amino acid at position 10 of the amino acid sequence of the native B chain of relaxin-2.

[0049] As used herein, the term "pharmaceutical acceptable carrier" refers to a fluid, particularly a fluid that contains a pharmaceutical compound or combination of pharmaceutical compounds of the present invention, such that the pharmaceutical composition is physiologically tolerable, i.e., can be administered to an individual's body without toxicity or undue discomfort.

[0050] As used herein, the term "relaxin analog" refers to peptides or other compounds that are functional variants of relaxin and / or structural analogs of native relaxin-2, such as modified relaxin B chain peptides, that can activate, for example, the RXFP1 receptor. In addition to modified relaxin peptides, relaxin analogs may include small molecules that are RXFP1 receptor agonists or exhibit RXFP1 agonist properties. In some embodiments, relaxin analogs may also include other modalities, such as relaxin bound to an Fc fragment, and RXFP1 agonists, such as nanobodies or monoclonal antibodies.

[0051] As used herein, the term "vasopressin analog" refers to prodrugs of vasopressin, as well as peptides structurally similar to vasopressin and / or functional variants of vasopressin, such as terlipressin (triglycyllysine vasopressin), argipressin, desmopressin, felypressin, lypressin, or ornipressin. Vasopressin analogs can activate vasopressin receptors V1a, V2, and / or V3 (also known as V1b). Small molecule vasopressin analogs are also known to those of skill in the art and are included in the term "vasopressin analog" as used herein.

[0052] The term "agonist" in this context refers to a peptide or small molecule, as defined herein, that can bind and activate a receptor. A full agonist binds and activates a receptor with the maximum response that an agonist can induce at the receptor. A partial agonist also binds and activates a given receptor, but is only partially effective at the receptor compared to a full agonist, even at maximum receptor occupancy. A selective agonist is selective for a particular type or subtype of receptor.

[0053] A "functional variant" of a peptide is a peptide that is capable of performing essentially the same function as the peptide of which it is a functional variant. In particular, a functional variant is capable of binding to the same molecule as the peptide of which it is a functional variant, preferably with similar affinity.

[0054] As used herein, the term "native" as used herein in reference to relaxin refers to a naturally occurring or wild-type molecule.

[0055] As used herein, the term "prevent" is intended to mean reducing the risk of the phenomenon under consideration occurring. This reduction may be total or partial, i.e. resulting in a degree of risk lower than the existing risk in the use according to the invention.

[0056] As used herein, the term "treating" is intended to mean reducing or even eliminating the undesirable condition or disease under consideration. A "treatment effect" or "therapeutic effect" is manifested when there is a change in the condition being treated, as measured by the criteria constituting the definition of the terms "treating" and "treatment". There is a "change" in the condition being treated when there is at least a 5% improvement, preferably a 10% improvement, more preferably at least a 25%, even more preferably at least a 50%, at least a 75%, and most preferably a 100% improvement. This change can be based on an improvement in the severity of the treated condition in an individual, or on the difference in the frequency of improvement in a condition in a population treated and an untreated population with a bioactive agent(s). Treatment according to the invention can be prophylactic, ameliorative, and / or curative.

[0057] A "bioactive agent" (i.e., biologically active substance / drug) is any agent, drug, compound, composition or mixture of substances that provides some pharmacological, often beneficial, effect that can be demonstrated in vivo or in vitro. It refers to peptide sequences as defined herein, compounds or compositions containing them, and nucleic acid constructs encoding said peptides. As used herein, the term further includes any physiologically or pharmacologically active substance that produces a local or systemic effect in an individual. As used herein, "bioactive agent" collectively refers to peptides, nucleic acid constructs encoding said peptides, and compositions comprising peptides.

[0058] A "pharmacologically effective amount," "pharmacologically effective amount," "physiologically effective amount," or "effective amount" of a "bioactive agent" is the amount of the bioactive agent or combination of bioactive agents present in one or more pharmaceutical compositions described herein necessary to provide a desired level of the active agent(s) in the bloodstream or at the site of action (e.g., hepatic system, renal system, circulatory system, pulmonary, gastrointestinal system, colorectal system, etc.) of the individual being treated to result in the physiological response expected when such composition is administered.

[0059] As used herein, "co-administering" or "co-administration" refers to the administration of two or more bioactive agents. The two or more components can be administered separately, sequentially, or simultaneously.

[0060] The term "individual" refers to a vertebrate, a particular member of a mammalian species, preferably a primate, including a human. As used herein, "subject" and "individual" may be used interchangeably. However, treatment of animals such as mice, rats, dogs, cats, cows, horses, sheep, and pigs is also within the scope of the invention.

[0061] As used herein, "an individual in need thereof" refers to an individual who can benefit from treatment. In one embodiment, the individual in need thereof is an individual having a disease, which may be a renal disorder.

[0062] Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

[0063] As used herein, the singular articles "a," "an," and "the" include plural references unless otherwise indicated.

[0064] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting," and / or "consisting essentially of" aspects and embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Combination Therapy of Vasopressin Analogues and Relaxin Analogues According to some embodiments, disclosed herein is a method for preventing or treating renal disorders, such as hepatorenal syndrome (HRS), in an individual in need thereof, comprising co-administering a relaxin peptide analog and a vasopressin analog as bioactive agents to the individual.

[0066] Vasopressin analogs such as terlipressin act as vasoconstrictors. Terlipressin also causes vasoconstriction in the splanchnic and systemic circulation. As described in the Background section, the safety profile of terlipressin is poor. For example, as described in Snowdon, Victoria K et al. “Serelaxin as a potential treatment for renal dysfunction in cirrhosis: Preclinical evaluation and results of a randomized phase 2 trial” PLoS medicine vol.14,2 e1002248.28 Feb.2017, “splanchnic vasoconstrictors such as terlipressin...further compromise hepatic perfusion in cirrhosis and may impair organ function.” Terlipressin's selectivity for splanchnic vasoconstriction is more limited than systemic vasoconstriction, including hepatic and renal vasoconstriction, further worsening HRS. In general, the use of splanchnic vasoconstrictors such as terlipressin is suboptimal for the treatment of renal dysfunction in cirrhosis, as they can result in a variety of adverse effects that may compromise renal function and patient safety.

[0067] Relaxin analogs, such as the long-acting peptidyl RXFP1 agonists disclosed herein, function as vasodilators with a degree of functional selectivity for renal vasodilatation.

[0068] The relaxin analog and vasopressin analog combination therapy herein relies on additive and synergistic interactions between a vasoconstrictor (e.g., a vasopressin analog such as terlipressin) and a vasodilator (e.g., a relaxin analog) acting in different local vascular beds (predominantly visceral in the case of terlipressin and primarily renal in the case of relaxin analogs) to improve the safety and efficacy of treating renal conditions associated with renal vasoconstriction in an individual, as described herein.

[0069] According to some embodiments, disclosed herein are pharmaceutical compositions comprising a relaxin peptide analog and a vasopressin analog, individually or together, for use in methods to treat renal disorders, such as HRS, e.g., hepatorenal syndrome type 1 (HRS-AKI) and hepatorenal syndrome type 2 (HRS-NAKI), or to maintain renal function during peri-operative liver transplantation.

[0070] In some embodiments, pharmaceutical compositions are also disclosed herein that include a peptide analog of the B chain of relaxin-2 capable of activating the RXFP1 receptor and a vasopressin analog capable of activating the vasopressin V1 (V1a) receptor, either individually or together, for use in methods to treat renal disorders, such as HRS, e.g., hepatorenal syndrome type 1 (HRS-AKI) and hepatorenal syndrome type 2 (HRS-NAKI), or to maintain renal function during perioperative liver transplantation.

[0071] According to some embodiments, disclosed herein are pharmaceutical compositions comprising relaxin peptide analogs and terlipressin, individually or together, for use in methods to treat renal disorders, such as HRS, including hepatorenal syndrome type 1 (HRS-AKI) and hepatorenal syndrome type 2 (HRS-NAKI), or to maintain renal function during peri-operative liver transplantation.

[0072] In some embodiments, pharmaceutical compositions are also disclosed herein that include a peptide analog of the B chain of relaxin-2 capable of activating the RXFP1 receptor and terlipressin, either individually or together, for use in methods to treat renal disorders, such as HRS, including hepatorenal syndrome type 1 (HRS-AKI) and hepatorenal syndrome type 2 (HRS-NAKI), or to maintain renal function during perioperative liver transplantation.

[0073] Also disclosed herein, in some embodiments, is a method of treating renal disorders, including hepatorenal syndrome type 1 (HRS-AKI) and hepatorenal syndrome type 2 (HRS-NAKI), comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising, individually or together, a relaxin peptide analog, such as a peptide analog of the B chain of relaxin-2 capable of activating the RXFP1 receptor, and a vasopressin analog, such as terlipressin, capable of activating the V1 (V1a) receptor.

[0074] In one embodiment, the renal disorder is renal dysfunction in cirrhosis, including HRS, such as HRS type 1 hepatorenal syndrome (HRS-AKI) and HRS type 2 hepatorenal syndrome (HRS-NAKI), and / or perioperative liver transplantation, chronic kidney disease, and acute kidney injury.

[0075] Vasopressin analogues such as terlipressin As described herein, a vasopressin analog, such as terlipressin, is used in combination with a relaxin analog for the treatment of disorders associated with renal dysfunction. In some embodiments, the vasopressin analog is terlipressin, argipressin, desmopressin, felypressin, lypressin, or ornipressin.

[0076] In some embodiments, the vasopressin analog is terlipressin or a pharma- ceutically acceptable salt of terlipressin. Terlipressin (also known as triglycyllysine vasopressin) is a synthetic analog of the human neuropeptide hormone vasopressin. Terlipressin is a prohormone of lysine-vasopressin (triglycyllysine vasopressin (TGLVP)), described, for example, in Rittig et al., Movement Disorders, 1991, Vol. 6(1), p21-28. After administration and absorption into the circulation, endothelial peptidases cleave the glycyl residues from the prohormone, allowing the release of lysine-vasopressin. Thus, although terlipressin itself has weak intrinsic vasoconstrictor activity, it is converted by endothelial endopeptidases to the fully active lysine vasopressin (LVP). Terlipressin is also known by the trade names Teripress and Glypressin. The molecular weight of terlipressin is 1227.37 g / mol and is represented by the following formula: [ka] Terlipressin, administered intravenously, is used, e.g., as a vasoactive agent in the management of hypotension (low blood pressure) and to treat, e.g., bleeding esophageal varices, septic shock, HRS, and ascites.

[0077] Outside the United States, individuals with cirrhosis exhibiting hepatorenal syndrome type 1 (HRS-1) have been treated with terlipressin administered by continuous IV infusion. Doses ranged from 2.0 to 12.0 mg per 24 hours (Angeli, et al., (2009) Journal of Hepatology, 50:S73: 2.0-12.0 mg / 24 hours; Gerbes, (2009) Gastroenterology, 137:1 179-1 189: starting dose 3.0 mg / day; Robertson, et al., (2014) Hepatology, 60(6):21 25-21 26: 3.0 mg / day; Ding, et al., (2013), Gastroenterology and Hepatology, 28:1242-1246: 4.0 mg / day; Cavallin, et al., (2015), Hepatology, 62(2):567-574: 3-12 mg / day).

[0078] Currently, terlipressin is available in two forms: a lyophilized powder for reconstitution, or a liquid (0.2 milligrams / mL) in a vial. The lyophilized version is usually supplied in a vial containing 1 mg of terlipressin powder for reconstitution using a 5 mL ampoule of provided saline to deliver a 0.17 mg / mL terlipressin (0.2 mg / mL terlipressin acetate) solution for injection (e.g., Glypressin (Ferring Pharmaceuticals) is supplied as one vial containing 1 mg of terlipressin acetate for reconstitution in 5 mL of solution). Administration of this product requires two or three preliminary steps: reconstitution of the powder with diluent, withdrawal of the solution, possibly further dilution, and then injection in a slow bolus dose directly into the patient or into the patient's intravenous line or into an IV bag. Glypressin must be stored refrigerated at a temperature of 2°C to 8°C.

[0079] Liquid terlipressin acetate 0.2 milligram / mL solution for injection (Terlipressin acetate, Ever Pharma) is also not stable at room temperature (RT) and must be stored refrigerated at a temperature between 2°C and 8°C. It is supplied in vials containing 5 mL or 10 mL of solution. The solution is drawn into a syringe for administration by bolus injection. The current formulation uses acetic acid to adjust the pH of the terlipressin acetate solution.

[0080] One aspect of the present disclosure provides an aqueous composition comprising terlipressin acetate or a pharma- ceutically acceptable salt thereof, the composition comprising about 0.2 to about 10.0 mg / ml of terlipressin acetate or a pharma- ceutically acceptable salt thereof, and the composition having a pH of about 3.4 to about 5.0.

[0081] As used herein, the term "aqueous" refers to a solution in which the solvent is water. The solvent may be sterile water suitable for injection. In one embodiment, the solvent may be bacteriostatic water. In other embodiments, the solvent may be a mixture of water and other pharma- ceutically acceptable solvents, or pharma-ceutically acceptable alcohols or other bacteriostatic agents (e.g., benzyl alcohol).

[0082] The concentration of terlipressin in a liquid (e.g., an aqueous composition) can be, for example, 0.2 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 5.0 mg / ml, or 10.0 mg / ml. In some embodiments, the composition comprises about 0.2 mg / ml to about 10.0 mg / ml, about 0.5 mg / ml to about 10.0 mg / ml, about 1.0 mg / ml to about 9.0 mg / ml, about 1.5 mg / ml to about 8.5 mg / ml, about 2.0 mg / ml to about 8.0 mg / ml, about 2.5 mg / ml to about 7.5 mg / ml, about 3.0 mg / ml to about 7.0 mg / ml, about 3.5 mg / ml to about 6.5 mg / ml, about 0.5 mg / ml to about 2.0 mg / ml, or about 0.5 mg / ml to about 1.0 mg / ml of terlipressin acetate, or a pharma- ceutically acceptable salt thereof.

[0083] In some embodiments, the pH of the composition can be about 3.4 to about 5.0, about 3.5 to about 5.0, about 3.6 to about 5.0, about 3.7 to about 5.0, about 3.8 to about 5.0, about 3.9 to about 5.0, about 4.0 to about 5.0, about 4.1 to about 5.0, about 4.2 to about 5.0, about 4.3 to about 5.0, about 4.4 to about 5.0, about 4.5 to about 5.0, about 4.6 to about 5.0, about 4.7 to about 5.0, about 4.8 to about 5.0, or about 4.9 to about 5.0. In other embodiments, the pH of the composition may be about 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or 5.1.

[0084] Further information regarding the formulation and administration of terlipressin can be found, for example, in International Publication No. WO2020 / 237170, “Formulations of Terlipressin,” which is incorporated by reference in its entirety.

[0085] The compositions may be for any route of drug administration (e.g., buccal, nasal, transdermal (e.g., patch technology), parenteral, intravenous, intramuscular, or subcutaneous injection, intracisternal, intraperitoneal). In some embodiments, the compositions are for intravenous administration, e.g., by continuous infusion or a bolus IV dose.

[0086] In some embodiments, the methods described herein provide a terlipressin concentration in a subject of about 1 to about 1000 μg / mL. In some embodiments, the methods described herein provide a terlipressin concentration in a subject of about 10 to about 600 μg / mL. In some embodiments, the methods described herein provide a terlipressin concentration in a subject of about 20 to about 200 μg / mL. In some embodiments, the methods described herein provide a terlipressin concentration in a subject of about 60 μg / mL. In some embodiments, the methods described herein provide a (lysine-)vasopressin concentration in a subject of about 1 to about 200 μg / mL, where (lysine-)vasopressin is the active metabolite of terlipressin.

[0087] In some embodiments, the methods described herein provide a vasopressin analog concentration in the subject, e.g., in the subject's plasma, of about 2 to about 100 μg / mL. In some embodiments, the methods described herein provide a vasopressin analog concentration in the subject, e.g., in the subject's plasma, of about 3 to about 30 μg / mL. In some embodiments, the methods described herein provide a vasopressin analog concentration in the subject, e.g., in the subject's plasma, of about 10 μg / mL.

[0088] In some embodiments, the method includes up to two administrations per day. In some embodiments, the method includes up to three or four administrations per day. The dosage regimen utilizing the compound is selected according to various factors, including the age, weight, sex, and medical condition of the patient, the severity of the condition being treated, the route of administration, the renal or hepatic function of the patient, and the specific compound or salt thereof used. A physician of ordinary skill can easily determine and prescribe the effective amount of drug required to prevent, treat, or prevent the progression of the condition.

[0089] Another aspect of the present invention is a method of treating a subject suffering from ascites (e.g., caused by cirrhosis of the liver), comprising administering to the subject a therapeutically effective amount of a vasopressin analog, such as terlipressin and relaxin analogs. Optionally, such treatment is administered in combination with another drug used to treat ascites. One aspect of the present invention is a method of treating a subject suffering from ascites, comprising administering to the subject a therapeutically effective amount of a vasopressin analog, such as terlipressin and relaxin analogs.

[0090] Relaxin analogues Relaxin analogs or pharma- ceutically acceptable salts thereof are useful in the present invention in combination with vasopressin analogs, such as terlipressin. In some embodiments, the relaxin analogs are long-acting relaxin peptide analogs that function as RXFP1 receptor agonists. The present disclosure further relates to compositions comprising them and their use in combination with vasopressin analogs in the treatment of renal conditions, diseases or disorders.

[0091] Where applicable, each amino acid of the relaxin peptide analogs disclosed herein can be independently an L-amino acid or a D-amino acid. In certain embodiments, an amino acid is an L-amino acid. If no information is given in the text regarding the L- or D-form of a given amino acid, that amino acid is an L-amino acid. ter and C ter are conventional labels used to denote the N-terminus of a peptide and the C-terminus of a relaxin peptide analogue, respectively.

[0092] Peptide structural analogues In some embodiments, the relaxin peptide analog is a modified relaxin-2 B chain peptide.

[0093] In some embodiments, the modified relaxin B chain peptide has formula (I) (SEQ ID NO: 105): N ter -Ac-X 10 -EGREX 15 -VRX 18 -X 19 -IX 21 -X 22 -EGX 25 -SX 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 -NH2-C ter 1. A modified relaxin B chain peptide comprising: During the ceremony, N terrepresents the N-terminus of the peptide, C ter represents the C-terminus of the peptide, Ac represents an acetyl group. X 10 represents an amino acid selected from the group consisting of leucine, 2-amino-isobutyric acid, Nε-acetyl-lysine, and α-methyl-leucine, E stands for glutamic acid, G stands for glycine, R represents arginine, X 15 represents an amino acid selected from the group consisting of lysine, arginine, homolysine, homoarginine, ornithine, glutamine, phenylalanine, and leucine; V stands for valine, X 18 represents an amino acid selected from the group consisting of alanine, 2-amino-isobutyric acid, leucine, Nε-acetyl-lysine, and glutamine, X 19 represents an amino acid selected from the group consisting of lysine, Nε-acetyl-lysine, citrulline, glutamine, alanine, and 2-amino-isobutyric acid, I stands for isoleucine, X 21 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and alanine, X 22 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and isoleucine, X 25 represents the following structure: [ka] During the ceremony, * is X in formula (I). 25 represents a covalent bond to the preceding glycine, [ka] is X in formula (I). 25 represents a covalent bond to serine followed by Z represents a group of formula (II), -[(PEG xx ) b (gE) c C d ], b and c independently represent 1, 2, 3, 4, or 5; PEG xx represents independently a polyethylene glycol derivative selected from the group consisting of PEG2, PEG2DGA, and TTDS; gE stands for gamma-glutamic acid; C d is a linear saturated C 12 ~C 22 represents an acyl group, S stands for serine, X 27 represents an amino acid selected from the group consisting of threonine, lysine, arginine, and glutamine; X 28 represents an amino acid selected from the group consisting of tryptophan, phenylalanine, 5-fluoro-tryptophan, 5-chloro-tryptophan, 5-methoxy-tryptophan, tyrosine, 4-fluoro-phenylalanine, 1-naphthylalanine, 2-naphthylalanine, α-methyl-tryptophan, α-methyl-phenylalanine, and 5-hydroxy-tryptophan, X 29 represents an amino acid selected from the group consisting of serine, D-serine, 2-amino-isobutyric acid, threonine, α-methyl-serine, Nε-acetyl-lysine, and valine, X 30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, α-methyl-lysine, D-lysine, lysine, homolysine, ornithine, arginine, and α-methyl-arginine; X 31 represents an amino acid selected from the group consisting of arginine, Nω-methyl-arginine, alanine, Nω,Nω′-dimethyl-arginine, and citrulline, X 32represents an amino acid selected from the group consisting of lysine, alanine, arginine, Nε-acetyl-lysine, and Nε,Nε,Nε-tri-methyl-lysine, X 33 represents an amino acid selected from lysine, Nε-acetyl-lysine, leucine, arginine, and alanine, or a salt or solvate thereof.

[0094] Relaxin peptide analogs disclosed herein also include salts of the peptides of formula (I) or (Ia) as defined herein, in one embodiment pharma- ceutically acceptable salts, e.g., salts as acid additions with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid, and boric acid, or with organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and sulfanilic acid, as well as salts with metals, e.g., alkali metals such as sodium, potassium, lithium, zinc, aluminum, and the like.

[0095] In one embodiment, the salts of the peptides are pharma- ceutically acceptable salts, e.g., acid adducts with hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and sulfanilic acid, and salts with metals, e.g., alkali metals, e.g., sodium, potassium, lithium, and zinc.

[0096] The relaxin peptide analogs disclosed herein also include solvates, in one embodiment pharma- ceutically acceptable solvates, of the peptides of formula (I) or (Ia) above.

[0097] By solvate is meant a complex of a relaxin peptide analog compound or a salt thereof with solvent molecules, such as organic solvent molecules and / or water.

[0098] In accordance with standard polypeptide nomenclature (J. Biol. Chem., 243:3552-59 (1969)), the abbreviations for α-amino acid residues are as follows: [Table 1]

[0099] For unnatural or modified amino acids, the following abbreviations are used: [Table 2]

[0100] In all formulas of relaxin peptide analogs, the amino acid sequence is represented by the above abbreviations and, e.g., X 18 X etc. y When depicted using the representations, left and right orientation is the conventional direction from amino terminus to carboxy terminus.

[0101] So, for example, X 28 represents an amino acid selected from the group consisting of tryptophan, 5-fluoro-tryptophan, 5-chloro-tryptophan, 5-methoxy-tryptophan, tyrosine, phenylalanine, 4-fluoro-phenylalanine, 1-naphthylalanine, 2-naphthylalanine, α-methyl-tryptophan, α-methyl-phenylalanine and 5-hydroxy-tryptophan, the N-terminus or amine group of said amino acid may be selected from the group consisting of X 27 The C-terminus or carboxyl group of the above amino acid is bound to an amino acid represented by X 29 It is bound to an amino acid represented by

[0102] Its N-terminus (N ter ) At the tip, the relaxin peptide analog is substituted with an acetyl group (Ac): CH3C(O)--.

[0103] Its C-terminus (C ter ) At the tip, the relaxin peptide analog is substituted with an --NH2 group.

[0104] That X 25 At this position, relaxin peptide analogs include the following structures: [ka]

[0105] This structure corresponds to the lysine amino acid, where: -Alpha (α) nitrogen atom ( * ) is N represented in the formula of the relaxin peptide analog ter -C ter Based on the orientation, the 24th position (X 24 ) glycine) and is attached to the front part of the peptide (i.e., X 25 N ter (based on) -Carboxyl group ( [ka] ) carbon atom is represented by the N ter -C ter Based on the orientation, the 26-position (X 26 ) with serine, X 25 It is bound to the portion of the peptide following position X 25 C ter (based on) -The nitrogen atom of the side chain is bonded to the Z group. The Z group is defined as being of formula (II). -[(PEG xx ) b (gE) c C d ],

[0106] In formula (II), - represents X 25represents a covalent bond to the nitrogen atom of the side chain of the lysine structure in

[0107] b and c independently represent 1, 2, 3, 4 or 5, in particular 2, 3, 4 or 5.

[0108] In certain embodiments, b represents 2, 3, 4 or 5.

[0109] In certain embodiments, c represents 2, 3 or 4.

[0110] In preferred embodiments, b represents 2, 3, 4 or 5; and c independently represents 2, 3 or 4.

[0111] PEG in the formula of relaxin peptide analogs xx represents independently a polyethylene glycol derivative selected from the group consisting of PEG2, PEG2DGA, and TTDS;

[0112] The above groups are defined as follows: [Table 3] Here, (PEG xx ) b In the above, (gE) c represents a covalent bond between the group of formula (II) and X 25 represents a covalent bond linking the nitrogen atom of the side chain of the lysine structure.

[0113] In certain embodiments, (PEG xx ) b represents a polyethylene glycol derivative selected from the group consisting of (TTDS)2, (TTDS)3, (PEG2DGA)3, (PEG2)3, (PEG2)4, and (PEG2)5.

[0114] As already indicated, gE, which may also be designated γE, gGlu, or γGlu, stands for γ-glutamic acid. This amino acid has the following structure: [ka] In the formula (II), (gE) c When expressed as [ka] is C d represents a covalent bond with [ka] (PEG xx ) b Represents a covalent bond to a group.

[0115] C d is a linear saturated C 12 ~C 22 Acyl groups, e.g., C 12 (Lau), C 14 (Myr), C 15 (Penta), C 16 (Palm), C 17 (Hepta), C 18 (Stea), C 20 (Eico), and C 22 (Doco) acyl groups. In one embodiment, C d is C 12 (Lau), C 14 (Myr), C 15 (Penta), C 16 (Palm), C 17 (Hepta), or C 18 (Stea) a linear saturated acyl group selected from the group consisting of acyl groups, such as a linear saturated C 14 , C 16 , or C 18 Acyl groups, or linear C 16 Or C 18 Represents an acyl group.

[0116] In certain embodiments, C d is a linear saturated C 12 Represents an acyl group. Linear saturated C 12The acyl group is a lauroyl group (also referred to herein as "Lau").

[0117] In certain embodiments, C d is a linear saturated C 14 Represents an acyl group. Linear saturated C 14 The acyl group is a myristoyl group (also referred to herein as "Myr").

[0118] In certain embodiments, C d is a linear saturated C 15 Represents an acyl group. Linear saturated C 15 The acyl group is a pentadecanoyl group (also referred to herein as "Penta").

[0119] In certain embodiments, C d is a linear saturated C 16 Represents an acyl group. Linear saturated C 16 The acyl group is a palmitoyl group (also referred to herein as "Palm").

[0120] In certain embodiments, C d is a linear saturated C 17 Represents an acyl group. Linear saturated C 17 The acyl group is a heptadecanoyl group (also referred to herein as "Hepta").

[0121] In another particular embodiment, C d is a linear saturated C 18 Represents an acyl group. Linear saturated C 18 The acyl group is a stearoyl group (also referred to herein as "Stea").

[0122] In another particular embodiment, C d is a linear saturated C 20 Represents an acyl group. Linear saturated C 20 The acyl group is an eicosanoyl group (also referred to herein as "Eico").

[0123] In another particular embodiment, C d is a linear saturated C 22 Represents an acyl group. Linear saturated C 22 The acyl group is a docosanoyl group (also referred to herein as "Doco").

[0124] In certain embodiments, Z is -(TTDS)2-(gamma glutamic acid) 3-palmitoyl (-(TTDS)2-(gE)3-Palm), -(TTDS)3-(gamma glutamic acid) 3-palmitoyl (-(TTDS)3-(gE)3-Palm), -(PEG2DGA)3-(gamma glutamic acid) 3-palmitoyl (-(PEG2DGA)3-(gE)3-Palm), -(PEG2)4-(gamma glutamic acid) 3-palmitoyl (-(PEG2)4-(gE)3-Palm), -(TTDS)2-(gamma glutamic acid) 2-palmitoyl (-(TTDS)2-(gE)2-Palm), -(TTDS)2-(gamma glutamic acid) 3-stearoyl -(TTDS)2-(gE)3-Stea), -(TTDS)3-(γ-glutamic acid)3-stearoyl (-(TTDS)3-(gE)3-Stea), -(PEG2DGA)3-(γ-glutamic acid)3-stearoyl (-(PEG2DGA)3-(gE)3-Stea), -(PEG2DGA)3-(γ-glutamic acid)4-stearoyl (-(PEG2DGA)3-(gE)4-Stea), -(PEG2)3-(γ-glutamic acid)3-palmitoyl (-(PEG2)3-(gE)3-Palm), -(PEG2)4-(γ-glutamic acid)3-stearoyl (-(PEG2)4-(gE)3-Stea), -(PEG2) 5-(gamma glutamic acid) 3-palmitoyl (-(PEG2)5-(gE)3-Palm), -(PEG2)5-(gamma glutamic acid) 4-palmitoyl (-(PEG2)5-(gE)4-Palm), -(TTDS)3-(gamma glutamic acid) 4-stearoyl (-(TTDS)3-(gE)4-Stea), -(TTDS)2-(gamma glutamic acid) 4-palmitoyl (-(TTDS)2-(gE)4-Palm), -(TTDS)3-(gamma glutamic acid) 2-stearoyl (-(TTDS)3-(gE)2-Stea), and -(TTDS)2-(gamma glutamic acid) 4-stearoyl (-(TTDS)2-(gE) 4- Stea ) In another embodiment Z is selected from the group consisting of -(TTDS)2-(gE)3-Palm, -(TTDS)3-(gE)3-Palm, -(PEG2DGA)3-(gE)3-Palm, -(PEG2)4-(gE)3-Palm, -(TTDS)2-(gE)2-Palm, -(TTDS)2-(gE)3-Stea, -(TTDS)3-(gE)3-Stea, -(PEG2)3-(gE)3-Palm , -(PEG2)4-(gE)3-Stea, and -(TTDS)2-(gE)4-Palm, and in specific embodiments Z is selected from the group consisting of -(TTDS)3-(gE)3-Palm, -(TTDS)2-(gE)3-Stea, and -(TTDS)3-(gE)3-Stea, such as -(TTDS)3-(gE)3-Palm and -(TTDS)3-(gE)3-Stea.

[0125] In all these Z groups, the first -- symbol represents the Z group and the lysine X 25 It represents a covalent bond between a nitrogen atom of a side chain of the structure.

[0126] Thus, if the Z group is represented as, for example, -(TTDS)2-(gamma glutamic acid)3-palmitoyl (also represented as -(TTDS)2-(gE)3-Palm), the first TTDS group is X 25The covalent bond is also attached to a second TTDS group, while another covalent bond attaches this second TTDS group to a first gamma-glutamic acid (gE), which is itself attached via a covalent bond to a second gamma-glutamic acid (gE), which is attached via another covalent bond to a third gamma-glutamic acid (gE), which is further attached via a covalent bond to a palmitoyl (Palm) group.

[0127] It will further be understood from the present application that, for example, a Z group represented as -(TTDS)2-(gamma glutamic acid)3-palmitoyl (also represented as -(TTDS)2-(gE)3-Palm) could also be represented as -TTDS-TTDS-gE-gE-gE-Palm.

[0128] The same applies mutatis mutandis to the other depicted Z groups according to the invention.

[0129] According to certain embodiments, the relaxin peptide analog has formula (Ia) (SEQ ID NO: 106): N ter -Ac-LEGREX 15 -VRX 18 -X 19 -I-Aib-Aib-EGX 25 -STX 28 -X 29 -X 30 -RX 32 -X 33 -NH2-C ter During the ceremony, N ter represents the N-terminus of the peptide, C ter represents the C-terminus of the peptide, Ac represents an acetyl group. L stands for leucine, E stands for glutamic acid, G stands for glycine, R represents arginine, X 15represents an amino acid selected from the group consisting of lysine, arginine, homolysine, glutamine, phenylalanine, and leucine; V stands for valine, X 18 represents an amino acid selected from the group consisting of alanine and 2-amino-isobutyric acid, X 19 represents an amino acid selected from the group consisting of lysine, Nε-acetyl-lysine, glutamine, and citrulline, I stands for isoleucine, Aib stands for 2-amino-isobutyric acid, X 25 represents the following structure: [ka] During the ceremony, - * is X in formula (Ia) 25 represents a covalent bond to the preceding glycine, - [ka] is X in formula (Ia) 25 followed by a covalent bond to serine, and Z is -(TTDS)2-(gE)3-Palm, -(TTDS)3-(gE)3-Palm, -(PEG2DGA)3-(gE)3-Palm, -(PEG2)4-(gE)3-Palm, -(TTDS)2-(gE)2-Palm, -(TTDS)2-(gE)3-Stea, -(TTDS)3-(gE)3-Stea, -(PEG2DGA)3-(gE)3-Stea, -(PEG2DGA)3-(gE)3-Stea, -(PEG2DGA)3-(g -(PEG2)-(gE)-Palm, -(PEG2)-(gE)-Stea, -(PEG2)-(gE)-Palm, -(PEG2)-(gE)-Palm, -(PEG2)-(gE)-Palm, -(TTDS)-(gE)-Stea, -(TTDS)-(gE)-Palm, -(TTDS)-(gE)-Stea, and -(TTDS)-(gE)-Stea, wherein gE represents γ-glutamic acid; Palm stands for palmitoyl, Stea stands for stearoyl. S stands for serine, T stands for threonine, X 28 represents an amino acid selected from the group consisting of tryptophan and phenylalanine, X 29 represents an amino acid selected from the group consisting of serine, D-serine, and 2-amino-isobutyric acid; X 30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, α-methyl-lysine, D-lysine, and lysine; X 32 represents an amino acid selected from the group consisting of lysine, alanine, and arginine; X 33 is formula (Ia), or a salt or solvate thereof, which represents an amino acid selected from lysine and Nε-acetyl-lysine.

[0130] According to certain embodiments, the relaxin peptide analog has an amino acid sequence selected from the group consisting of the amino acid sequences of reference SEQ ID NOs: 1-97. In certain embodiments, the relaxin peptide analog has an amino acid sequence selected from the group consisting of the amino acid sequences of reference SEQ ID NOs: 1-32, 34-37, 39, 42, 44, 45, 47-49, 51, and 54-97.

[0131] As noted above, in certain embodiments, peptides having the formula (Ia) are X 19 represents an amino acid selected from the group consisting of Nε-acetyl-lysine and citrulline, X 25 represents the following structure: [ka] During the ceremony, - * is X in formula (Ia) 25 represents a covalent bond to the preceding glycine, - [ka] is X in formula (Ia) 25 followed by a covalent bond to serine; Z is selected from the group consisting of -(TTDS)2-(gE)3-Palm, -(TTDS)3-(gE)3-Palm, -(PEG2DGA)3-(gE)3-Palm, -(PEG2)4-(gE)3-Palm, -(TTDS)2-(gE)2-Palm, -(TTDS)2-(gE)3-Stea, -(TTDS)3-(gE)3-Stea, -(PEG2)3-(gE)3-Palm, -(PEG2)4-(gE)3-Stea, and -(TTDS)2-(gE)4-Palm, wherein gE represents γ-glutamic acid, Palm represents palmitoyl, and Stea represents stearoyl; X represents γ-glutamic acid, Palm represents palmitoyl, and Stea represents stearoyl; 32 represents an amino acid selected from the group consisting of lysine and alanine, X 33 represents Nε-acetyl-lysine, or a salt or solvate thereof.

[0132] According to another embodiment, the relaxin peptide analog has an amino acid sequence selected from the group consisting of the amino acid sequences of Reference SEQ ID NOs: 1-30.

[0133] In certain embodiments, the relaxin peptide analog of formula (Ia) is N ter represents the N-terminus of the peptide, C ter represents the C-terminus of the peptide, Ac represents an acetyl group. L stands for leucine, E stands for glutamic acid, G stands for glycine, R represents arginine, X 15 represents an amino acid selected from the group consisting of lysine and homolysine, V stands for valine, X 18 represents alanine, X 19 represents an amino acid selected from the group consisting of Nε-acetyl-lysine and citrulline, I stands for isoleucine, Aib stands for 2-amino-isobutyric acid, X 25 represents the following structure: [ka] During the ceremony, - * is X in formula (Ia) 25 represents a covalent bond to the preceding glycine, - [ka] is X in formula (Ia) 25 followed by a covalent bond to serine, Z is selected from the group consisting of -(TTDS)2-(gE)2-Palm, -(TTDS)2-(gE)3-Palm, -(TTDS)2-(gE)4-Palm, -(TTDS)3-(gE)3-Palm, -(PEG2DGA)3-(gE)3-Palm, -(TTDS)2-(gE)3-Stea, -(TTDS)3-(gE)3-Stea, and -(PEG2)4-(gE)3-Stea, wherein gE represents γ-glutamic acid, Palm represents palmitoyl, and Stea represents stearoyl; S stands for serine, T stands for threonine, X 28 represents an amino acid selected from the group consisting of tryptophan and phenylalanine, X 29 represents serine, X 30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and α-methyl-lysine, X 32 represents an amino acid selected from the group consisting of lysine, alanine, and arginine; X 33 represents an amino acid selected from lysine, and Nε-acetyl-lysine, or a salt or solvate thereof.

[0134] In certain embodiments, the relaxin peptide analog of formula (Ia) is N ter represents the N-terminus of the peptide, C ter represents the C-terminus of the peptide, Ac represents an acetyl group. L stands for leucine, E stands for glutamic acid, G stands for glycine, R represents arginine, X 15 represents lysine, V stands for valine, X 18 represents alanine, X 19 represents Nε-acetyl-lysine, I stands for isoleucine, Aib stands for 2-amino-isobutyric acid, X 25 represents the following structure: [ka] During the ceremony, - * is X in formula (Ia) 25 represents a covalent bond to the preceding glycine, - [ka] is X in formula (Ia) 25 followed by a covalent bond to serine, Z is selected from the group consisting of -(TTDS)3-(gE)3-Palm, -(TTDS)2-(gE)3-Stea, -(TTDS)3-(gE)3-Stea, and -(PEG2)4-(gE)3-Stea, wherein gE stands for gamma-glutamic acid, Palm stands for palmitoyl, and Stea stands for stearoyl. S stands for serine, T stands for threonine, X 28 represents an amino acid selected from the group consisting of tryptophan and phenylalanine, X29 represents serine, X 30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and α-methyl-lysine, X 32 represents an amino acid selected from the group consisting of lysine, alanine, and arginine; X 33 represents an amino acid selected from lysine, and Nε-acetyl-lysine, or a salt or solvate thereof.

[0135] According to certain embodiments, the relaxin peptide analog has formula (Ib): N ter -Ac-X 10 -EGREX 15 -VRX 18 -X 19 -IX 21 -X- 22 -EGX 25 -SX 27 -X 28 -X 29 -X 30 -RX 32 -X 33 -NH2-C ter During the ceremony, N ter represents the N-terminus of the peptide, C ter represents the C-terminus of the peptide, Ac represents an acetyl group. X 10 represents an amino acid selected from the group consisting of leucine, Nε-acetyl-lysine, and 2-amino-isobutyric acid, E stands for glutamic acid, G stands for glycine, R represents arginine, X 15 represents an amino acid selected from the group consisting of lysine, arginine, homolysine, glutamine, phenylalanine, and leucine; V stands for valine, X 18represents an amino acid selected from the group consisting of alanine, 2-amino-isobutyric acid, and Nε-acetyl-lysine, X 19 represents an amino acid selected from the group consisting of lysine, Nε-acetyl-lysine, glutamine, and citrulline, I represents isoleucine, X 21 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and alanine, X 22 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and isoleucine, X 25 represents the following structure: [ka] During the ceremony, - * is X in formula (Ia) 25 represents a covalent bond to the preceding glycine, - [ka] is X in formula (Ia) 25followed by a covalent bond to serine, and Z represents -(TTDS)2-(gE)3-Palm, -(TTDS)3-(gE)3-Palm, -(PEG2DGA)3-(gE)3-Palm, -(PEG2)4-(gE)3-Palm, -(TTDS)2-(gE)2-Palm, -(TTDS)2-(gE)3-Stea, -(TTDS)3-(gE)3-Stea, -(PEG2DGA)3-(gE)3-Stea, -(PEG2DGA)3-(gE)4-Stea, -(PEG2)3-(gE)3-Palm, -(PEG2)4-(gE)3-Stea, -(PEG2)5-(gE)3-Palm, -(PEG2)5-(gE)4-Palm, -(TTDS)3-(gE)4-Stea, -(TTDS)2-(gE)4-Palm, -(TTDS)3-(gE)2-Stea, -(TTDS)2-(gE)4-Stea, -(TTDS)4-(gE)3-Stea, -(TTDS)3-(gE)4-Palm, -(TTDS)4-(gE)3-Palm, -(TTDS)3-(gE)3-Myr, and -(TTDS)3-(gE)4-Myr, wherein gE represents γ-glutamic acid; Palm stands for palmitoyl, and Stea stands for stearoyl. S stands for serine, X 27 represents an amino acid selected from the group consisting of threonine, glutamine, arginine, and lysine; X 28 represents an amino acid selected from the group consisting of tryptophan, phenylalanine, 5-chlorotryptophan, α-methyl-phenylalanine, 4-fluoro-phenylalanine, and 5-fluorotryptophan, X 29 represents an amino acid selected from the group consisting of serine, D-serine, 2-amino-isobutyric acid, Nε-acetyl-lysine, threonine, and valine, X 30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, α-methyl-lysine, D-lysine, lysine, homolysine, and arginine; X 32represents an amino acid selected from the group consisting of lysine, alanine, arginine, and Nε-acetyl-lysine, X 33 represents an amino acid selected from lysine, Nε-acetyl-lysine, and arginine, or a salt or solvate thereof.

[0136] In some embodiments, Z is -(TTDS)2-(gE)3-Palm, -(TTDS)3-(gE)3-Palm, -(PEG2DGA)3-(gE)3-Palm, -(PEG2)4-(gE)3-Palm, -(TTDS)2-(gE)2-Palm, -(TTDS)2-(gE)3-Stea, -(TTDS)3-(gE)3-Stea, -(PEG2DGA)3-(gE)3-Stea, -(PEG2)3-(gE)3-Palm, -(PEG2 )4-(gE)3-Stea, -(PEG2)5-(gE)3-Palm, -(TTDS)3-(gE)4-Stea, -(TTDS)2-(gE)4-Palm, -(TTDS)2-(gE)4-Stea, -(TTDS)4-(gE)3-Stea, -(TTDS)3-(gE)4-Palm, -(TTDS)4-(gE)3-Palm, -(TTDS)3-(gE)3-Myr, and -(TTDS)3-(gE)4-Myr.

[0137] In certain embodiments, the relaxin peptide analog has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 7, 9, 10, 11, 12, 13, 20, 21, 22, 26, 28 and 30.

[0138] In certain embodiments, the relaxin peptide analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 7, 9-12, 20-22, 26, 28, 30-34, 45, 47-49, 51, 54-62, 64, 67-69, 71-93, 96, and 97. As illustrated above and in the enclosed examples, these peptides have an EC 50 has.

[0139] In particular, the relaxin peptide analog has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 7, 13, 20, 26 and 30.

[0140] In particular, the relaxin peptide analogs have an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 7, 20, 26, 30-34, 45, 48, 49, 51, 54-61, 67, 71, 73, 75-79, 81, 83-92, and 97. As illustrated in the included examples, all peptides according to this embodiment have an EC of ≦0.5 nM in an in vitro OVCAR5 cell line cyclic adenosine monophosphate (cAMP) assay, in which OVCAR5 cells express endogenous human RXFP1. 50 (See Example 3).

[0141] Furthermore, in some embodiments, relaxin peptide analogs exhibit improved solubility at pH 4.5 or pH 7.5, improved rat and human plasma or blood stability, and in vivo pharmacokinetic half-life compared to prior art RXFP1 agonist peptides.

[0142] These properties enable relaxin peptide analog formulations to be used as agents that maintain in vivo effectiveness over a wide concentration range for extended periods (i.e., long-acting) and can be administered once daily via intravenous or subcutaneous routes.

[0143] In certain embodiments, the relaxin peptide analog has an amino acid sequence selected from the group consisting of the amino acid sequences of reference SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:20, and specifically has the amino acid sequence of reference SEQ ID NO:3.

[0144] In some embodiments, relaxin analogs further described herein and related embodiments can be used in combination with vasopressin analogs, as described herein. Such disclosures of relaxin analogs can be found in WO 2019 / 149782, entitled "Modified Lipidated Relaxin B Chain Peptides and Their Therapeutic Use," published on August 8, 2019 as the publication of International Application No. PCT / EP2019 / 052298, WO 2019 / 149780, entitled "Modified Relaxin B Chain Peptides and Their Therapeutic Use," published on August 8, 2019 as the publication of International Application No. PCT / EP2019 / 052296, and WO 2019 / 149781, entitled "Modified Lipidated Relaxin B Chain Peptides and Their Therapeutic Use," published on August 8, 2019 as the publication of International Application No. PCT / EP2019 / 052297, which are incorporated by reference in their entireties. In some embodiments, other relaxin peptide analogs known in the art can be used in combination with vasopressin analogs, such as terlipressin, to treat an individual in need of treatment as described herein. For example, RXFP1 agonist relaxin peptide analogs are disclosed in International Publication No. WO 2015 / 157829, "Modified relaxin B chain peptides," published on October 22, 2015 as the publication of International Application No. PCT / AU2015 / 050184, which is incorporated herein by reference in its entirety.

[0145] Relaxin peptide analogs can be produced by any technique known per se in the art, such as, but not limited to, any chemical, biological, genetic, or enzymatic technique, alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can easily produce said polypeptide by standard techniques for polypeptide production, or according to the methods described herein, even when unnatural amino acids are used.

[0146] For example, relaxin peptide analogs can be synthesized using well-known solid-phase methods, e.g., using a commercially available peptide synthesizer (such as those manufactured by Applied Biosystems, Foster City, Calif., Gyros Protein technologies, Tucson, Ariz., or CEM corporation, Matthews, NC) following the manufacturer's instructions.

[0147] Examples of suitable methods are illustrated in the included Examples.

[0148] Relaxin Analogs - Small Molecule RXFP1 Agonists In some embodiments, the relaxin analog is a small molecule with orthosteric or allosteric agonist activity at RXFP1. Such small molecule agonists are known to those skilled in the art, such as ML290 and ML290 analogs (see, for example, McBride, A., Hoy, AM, Bamford, MJ et al. In search of a small molecule agonist of the relaxin receptor RXFP1 for the treatment of liver fibrosis. Sci Rep 7, 10806 (2017)) and Kocan, M., Sarwar, M., Ang, SY et al. ML290 is a biased allosteric agonist at the relaxin receptor RXFP1. Sci Rep 7, 2968 (2017)).

[0149] Dosage regimen for relaxin analogs and / or vasopressin analogs Disclosed herein is a method for preventing or treating renal failure in an individual in need thereof, comprising co-administering to the individual a dose of about 1.0 mg to about 5.0 mg of a relaxin analog and an effective amount of a vasopressin analog. In various embodiments, the renal failure is selected from the group consisting of cirrhosis-induced renal dysfunction, liver transplant-induced renal dysfunction, chronic kidney disease, and acute kidney injury. Disclosed herein further is a method for preventing or treating hepatorenal syndrome in an individual in need thereof, comprising co-administering to the individual a dose of about 1.0 mg to about 5.0 mg of a relaxin analog and an effective amount of a vasopressin analog. In various embodiments, the hepatorenal syndrome is HRS-AKI (type 1 hepatorenal syndrome). In certain embodiments, the relaxin analog is an RXFP1 agonist, such as a long-acting peptidyl RXFP1 agonist.

[0150] In various embodiments, one or more doses of the relaxin analog are administered to the individual. In various embodiments, two or more doses, three or more doses, four or more doses, five or more doses, six or more doses, seven or more doses, eight or more doses, nine or more doses, ten or more doses, eleven or more doses, twelve or more doses, thirteen or more doses, fourteen or more doses, fifteen or more doses, sixteen or more doses, seventeen or more doses, eighteen or more doses, nineteen or more doses, or twenty or more doses of the relaxin analog are administered to the individual. In various embodiments, a single dose of the relaxin analog is administered to the individual. In various embodiments, two or more doses of the relaxin analog are administered on the same day. In various embodiments, two doses of the relaxin analog are administered to the individual on the same day, followed by one or more doses of the relaxin analog on a subsequent day.

[0151] In the following description, reference is made to doses of a relaxin analog. In embodiments in which multiple doses of a relaxin analog are administered to an individual, the following description is equally applicable to each individual dose of the relaxin analog administered to the individual.

[0152] In certain embodiments, the relaxin analog is administered to the individual in a fixed dose. In various embodiments, the relaxin analog is administered to the individual in a dose of about 0.1 mg to about 100 mg. In various embodiments, the relaxin analog is administered to the individual in a dose of about 0.5 mg to about 50 mg. In various embodiments, the relaxin analog is administered to the individual in a dose of about 1.0 mg to about 50 mg. In various embodiments, the relaxin analog is administered to the individual in a dose of about 1 mg to about 25 mg, about 2 mg to about 20 mg, about 3 mg to about 19 mg, about 4 mg to about 18 mg, about 5 mg to about 15 mg, about 6 mg to about 14 mg, about 7 mg to about 13 mg, about 8 mg to about 12 mg, or about 9 mg to about 11 mg.

[0153] In various embodiments, the relaxin analog is administered to the individual at a dose of about 1 mg to about 10 mg, about 1 mg to about 9 mg, about 1 mg to about 8 mg, about 1 mg to about 7 mg, about 1 mg to about 6 mg, about 1 mg to about 5 mg, about 1 mg to about 4 mg, about 1 mg to about 6 mg, or about 1 mg to about 2 mg. In various embodiments, the relaxin analog is administered to the individual at a dose of about 1 mg to about 10 mg, about 2 mg to about 8 mg, about 3 mg to about 6 mg, about 3 mg to about 5 mg, or about 4 mg to about 5 mg. In various embodiments, the relaxin analog is administered to the individual at a dose of about 1 mg to about 9 mg, about 2 mg to about 6 mg, about 3 mg to about 5 mg, or about 3.5 mg to about 4.5 mg.

[0154] In various embodiments, the relaxin analog is administered to the individual at a dose of about 10 mg to about 20 mg, about 10.5 mg to about 18 mg, about 11 mg to about 16 mg, about 11.5 mg to about 14 mg, or about 12 mg to about 13 mg. In various embodiments, the relaxin analog is administered to the individual at a dose of about 20 mg to about 30 mg, about 21 mg to about 29 mg, about 22 mg to about 28 mg, about 23 mg to about 26 mg, or about 24 mg to about 25 mg. In various embodiments, the relaxin analog is administered to the individual at a dose of about 40 mg to about 50 mg, about 42 mg to about 49.5 mg, about 44 mg to about 49 mg, about 46 mg to about 48.5 mg, or about 47 mg to about 48 mg.

[0155] In certain embodiments, the relaxin analog is administered to the individual at a dose of about 2.0 mg. In certain embodiments, the relaxin analog is administered to the individual at a dose of about 4.0 mg. In certain embodiments, the relaxin analog is administered to the individual at a dose of about 5.0 mg. In certain embodiments, the relaxin analog is administered to the individual at a dose of about 10.0 mg. In certain embodiments, the relaxin analog is administered to the individual at a dose of about 12.0 mg. In certain embodiments, the relaxin analog is administered to the individual at a dose of about 15.0 mg. In certain embodiments, the relaxin analog is administered to the individual at a dose of about 24.0 mg. In certain embodiments, the relaxin analog is administered to the individual at a dose of about 48.0 mg.

[0156] In various embodiments, the relaxin analog is administered subcutaneously. In various embodiments, the relaxin analog is administered intravenously. In various embodiments, the relaxin analog is administered intravenously as a bolus injection. In various embodiments, the relaxin analog is administered intravenously over a period of about 10 minutes to about 24 hours, about 30 minutes to about 16 hours, about 45 minutes to about 12 hours, about 1 hour to about 10 hours, about 1.5 hours to about 9 hours, about 2 hours to about 8 hours, about 2.5 hours to about 7 hours, about 3 hours to about 6 hours, about 3.5 hours to about 5 hours, or about 4 hours to about 4.5 hours. In certain embodiments, the relaxin analog is administered intravenously over a period of about 4 hours.

[0157] In various embodiments, at least two doses of the relaxin analog are administered to the individual. In various embodiments, a second dose (e.g., a booster dose) of the relaxin analog is administered to the individual about 1 hour to about 24 hours after administration of the first dose of the relaxin analog. In various embodiments, the second dose of the relaxin analog is administered to the individual about 2 hours to about 22 hours, about 3 hours to about 20 hours, about 4 hours to about 19 hours, about 5 hours to about 18 hours, about 6 hours to about 17 hours, about 7 hours to about 16 hours, about 8 hours to about 15 hours, about 9 hours to about 14 hours, about 10 hours to about 13 hours, or about 11 hours to about 12.5 hours after administration of the first dose of the relaxin analog.

[0158] In various embodiments, the timing of the second dose of the relaxin analog is measured relative to the completion of administration of the first dose of the relaxin analog. For example, assuming that the first dose of the relaxin analog is a four-hour continuous intravenous administration, the timing of the second dose of the relaxin analog is measured relative to the completion of the four-hour continuous intravenous administration. In various embodiments, the timing of the second dose of the relaxin analog is measured relative to the start of administration of the first dose of the relaxin analog. For example, assuming that the first dose of the relaxin analog is a four-hour continuous intravenous administration, the timing of the second dose of the relaxin analog is measured relative to the start of the four-hour continuous intravenous administration.

[0159] In various embodiments, the first and second doses of the relaxin analog are administered to the individual through different routes of administration. For example, the first dose of the relaxin analog may be administered to the individual intravenously and the second dose of the relaxin analog may be administered to the individual subcutaneously. As another example, the first dose of the relaxin analog may be administered to the individual subcutaneously and the second dose of the relaxin analog may be administered to the individual intravenously. In various embodiments, the first and second doses of the relaxin analog are administered to the individual through the same route of administration. For example, the first and second doses of the relaxin analog are both administered to the individual intravenously. As another example, the first and second doses of the relaxin analog are both administered to the individual subcutaneously.

[0160] In various embodiments, the second dose of the relaxin analog is higher than the first dose of the relaxin analog. In various embodiments, the second dose of the relaxin analog is at least 100% higher than the first dose of the relaxin analog. For example, the first dose may be about 2.0 mg and the second dose may be about 5.0 mg. As another example, the first dose may be about 4.0 mg and the second dose may be about 10.0 mg. In various embodiments, the second dose of the relaxin analog is less than 30% higher than the first dose of the relaxin analog. In one embodiment, the second dose of the relaxin analog may be 25% higher than the first dose of the relaxin analog. For example, the first dose may be about 4.0 mg and the second dose may be about 5.0 mg.

[0161] In various embodiments, at least a third dose (e.g., a further additional dose) of the relaxin analog is administered to the individual. In various embodiments, a third dose of the relaxin analog is administered to the individual. In various embodiments, the third dose of the relaxin analog is administered to the individual about 12 hours to about 48 hours after administration of the first dose of the relaxin analog. In various embodiments, the third dose of the relaxin analog is administered to the individual about 13 hours to about 45 hours, about 14 hours to about 42 hours, about 15 hours to about 39 hours, about 16 hours to about 36 hours, about 17 hours to about 33 hours, about 18 hours to about 30 hours, about 19 hours to about 28 hours, about 20 hours to about 26 hours, about 22 hours to about 25 hours, or about 23 hours to about 24.5 hours after administration of the first dose of the relaxin analog.

[0162] In various embodiments, the timing of the third dose of the relaxin analog is measured relative to the completion of administration of the first dose of the relaxin analog. For example, assuming that the first dose of the relaxin analog is a four-hour continuous intravenous administration, the timing of the third dose of the relaxin analog is measured relative to the completion of the four-hour continuous intravenous administration. In various embodiments, the timing of the third dose of the relaxin analog is measured relative to the start of administration of the first dose of the relaxin analog. For example, assuming that the first dose of the relaxin analog is a four-hour continuous intravenous administration, the timing of the third dose of the relaxin analog is measured relative to the start of the four-hour continuous intravenous administration.

[0163] In various embodiments, the first dose and the third dose of the relaxin analog are administered to the individual through different routes of administration. For example, the first dose of the relaxin analog may be administered to the individual intravenously and the third dose of the relaxin analog may be administered to the individual subcutaneously. As another example, the first dose of the relaxin analog may be administered to the individual subcutaneously and the third dose of the relaxin analog may be administered to the individual intravenously. In various embodiments, the first dose and the third dose of the relaxin analog are administered to the individual through the same route of administration. For example, the first dose and the third dose of the relaxin analog are both administered to the individual intravenously. As another example, the first dose and the third dose of the relaxin analog are both administered to the individual subcutaneously.

[0164] In various embodiments, the third dose of the relaxin analog is higher than the first dose of the relaxin analog. In various embodiments, the third dose of the relaxin analog is at least 100% higher than the first dose of the relaxin analog. For example, the first dose may be about 2.0 mg and the third dose may be about 5.0 mg. As another example, the first dose may be about 4.0 mg and the third dose may be about 10.0 mg. In various embodiments, the third dose of the relaxin analog is less than 30% higher than the first dose of the relaxin analog. In one embodiment, the third dose of the relaxin analog may be 25% higher than the first dose of the relaxin analog. For example, the first dose may be about 4.0 mg and the third dose may be about 5.0 mg.

[0165] In various embodiments, doses greater than the third dose are administered to the individual. In various embodiments, the doses greater than the third dose replicate the amount and route of administration of the third dose. For example, assuming the third dose comprises 10 mg of a relaxin analog administered subcutaneously, the doses greater than the third dose also comprise 10 mg of a relaxin analog administered subcutaneously.

[0166] In various embodiments, doses greater than the third dose are administered daily. In various embodiments, doses greater than the third dose are administered daily for about 5 days to about 25 days. In various embodiments, doses greater than the third dose are administered daily for about 6 days to about 24 days, about 7 days to about 23 days, about 8 days to about 22 days, about 9 days to about 21 days, about 10 days to about 20 days, about 11 days to about 18 days, about 12 days to about 16 days, or about 13 days to about 15 days. In certain embodiments, doses greater than the third dose are administered daily for 13 days. In certain embodiments, doses greater than the third dose are administered daily for 14 days. In certain embodiments, doses greater than the third dose are administered daily for 15 days.

[0167] In various embodiments, doses greater than the third dose are administered daily. In various embodiments, doses greater than the third dose are administered daily for about 3 days to about 15 days. In various embodiments, doses greater than the third dose are administered daily for about 4 days to about 13 days, about 5 days to about 11 days, or about 6 days to about 9 days. In certain embodiments, doses greater than the third dose are administered daily for 6 days. In certain embodiments, doses greater than the third dose are administered daily for 7 days. In certain embodiments, doses greater than the third dose are administered daily for 8 days. In certain embodiments, doses greater than the third dose are administered daily for 9 days.

[0168] In various embodiments, a method for preventing or treating renal failure in an individual in need thereof and / or a method for preventing or treating hepatorenal syndrome in an individual includes intravenous administration of a first dose of 2.0 mg of relaxin analog over approximately 4 hours, subcutaneous administration of 5.0 mg of relaxin analog 12 hours after initiation of the first dose, subcutaneous administration of 5.0 mg of relaxin analog 24 hours after initiation of the first dose, and daily subcutaneous administration of 5.0 mg of relaxin analog thereafter for up to 12-15 days.

[0169] In various embodiments, a method for preventing or treating renal failure in an individual in need thereof and / or a method for preventing or treating hepatorenal syndrome in an individual comprises intravenous administration of a first dose of 4.0 mg of relaxin analog over approximately 4 hours, subcutaneous administration of 10.0 mg of relaxin analog 12 hours after the start of the first dose, subcutaneous administration of 10.0 mg of relaxin analog 24 hours after the start of the first dose, and subcutaneous administration of 5.0 mg of relaxin analog daily thereafter for up to 12-15 days.

[0170] In various embodiments, a method for preventing or treating renal failure in an individual in need thereof and / or a method for preventing or treating hepatorenal syndrome in an individual includes intravenous administration of a first dose of 4.0 mg of relaxin analog over approximately 4 hours, subcutaneous administration of 5.0 mg of relaxin analog 12 hours after initiation of the first dose, subcutaneous administration of 10.0 mg of relaxin analog 24 hours after initiation of the first dose, and daily subcutaneous administration of 5.0 mg of relaxin analog thereafter for up to 12-15 days.

[0171] In various embodiments, a method for preventing or treating renal failure in an individual in need thereof and / or a method for preventing or treating hepatorenal syndrome in an individual comprises co-administering to the individual effective amounts of a relaxin analog and a vasopressin analog, hi certain embodiments, the vasopressin analog is terlipressin or a pharma- ceutical acceptable salt thereof.

[0172] In various embodiments, terlipressin is administered intravenously at a dose of about 0.5 to about 10 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 0.6 mg to about 5 mg, about 0.7 mg to about 3 mg, about 0.8 mg to about 2 mg, or about 0.9 mg to about 1.5 mg. In certain embodiments, terlipressin is administered intravenously at a dose of about 1 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 0.8 mg to about 6 mg, about 1 mg to about 5 mg, about 1.2 mg to about 4 mg, about 1.4 mg to about 3 mg, about 1.6 mg to about 2.5 mg, about 1.8 mg to about 2.2 mg, or about 1.9 mg to about 2.1 mg. In certain embodiments, terlipressin is administered intravenously at a dose of about 2 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 1 mg to about 8 mg, about 2 mg to about 6 mg, about 3 mg to about 5 mg, about 3.2 mg to about 4.8 mg, about 3.4 mg to about 4.6 mg, about 3.6 mg to about 4.4 mg, or about 3.8 mg to about 4.2 mg. In certain embodiments, terlipressin is administered intravenously at a dose of about 4 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 2 mg to about 9 mg, about 4 mg to about 8 mg, about 5 mg to about 7 mg, about 5.2 mg to about 6.8 mg, about 5.4 mg to about 6.6 mg, about 5.6 mg to about 6.4 mg, or about 5.8 mg to about 6.2 mg. In certain embodiments, terlipressin is administered intravenously at a dose of about 6 mg.

[0173] In various embodiments, terlipressin is administered intravenously every 2-10 hours. In various embodiments, terlipressin is administered intravenously every 3-8 hours, or every 4-6 hours. In various embodiments, terlipressin is administered intravenously every 6 hours. In various embodiments, terlipressin is administered intravenously by bolus injection. In various embodiments, terlipressin is administered intravenously by bolus injection over about 30 seconds to about 10 minutes, about 1 minute to about 5 minutes, or about 2 minutes to about 3 minutes. In various embodiments, terlipressin is administered intravenously by bolus injection over about 2 minutes.

[0174] In various embodiments, about 0.5 mg to about 2 mg of terlipressin is administered intravenously every 4 to 6 hours. In certain embodiments, about 1.0 mg of terlipressin is administered intravenously every 6 hours. In various embodiments, about 2 mg to about 6 mg of terlipressin is administered intravenously every 4 to 6 hours. In certain embodiments, about 4.0 mg of terlipressin is administered intravenously every 6 hours. In various embodiments, about 6 mg to about 10 mg of terlipressin is administered intravenously every 4 to 6 hours. In certain embodiments, about 6.0 mg of terlipressin is administered intravenously every 6 hours. In various embodiments, about 8.0 mg of terlipressin is administered every 8 hours.

[0175] In various embodiments, terlipressin is administered intravenously for about 8 hours to about 36 hours. In various embodiments, terlipressin is administered intravenously for about 10 hours to about 30 hours, about 15 hours to about 28 hours, or about 20 hours to about 25 hours. In certain embodiments, terlipressin is administered intravenously for about 24 hours.

[0176] In various embodiments, terlipressin is administered intravenously at a dose of about 1 mg to about 10 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 1.5 mg to about 4.0 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 2.0 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 5.0 mg to about 8.0 mg. In various embodiments, terlipressin is administered intravenously at a dose of about 6.0 mg.

[0177] In certain embodiments, a method for preventing or treating renal failure in an individual in need thereof, and / or a method for preventing or treating hepatorenal syndrome in an individual, comprises a combination of one of the following (1A), (1B), or (1C) with (2): (1A) Terlipressin 1.0 mg bolus intravenous infusion every 6 hours; (1B) terlipressin 2.0 mg intravenous infusion over 24 hours; (1C) Terlipressin 6.0 mg intravenous infusion over 24 hours; (2) a first dose of 2.0 mg of relaxin analog administered intravenously over approximately 4 hours, followed by 5.0 mg of relaxin analog administered subcutaneously 12 hours after the start of the first dose, followed by 5.0 mg of relaxin analog administered subcutaneously 24 hours after the start of the first dose, and then 5.0 mg of relaxin analog administered subcutaneously daily for up to 12-15 days.

[0178] In certain embodiments, a method for preventing or treating renal failure in an individual in need thereof, and / or a method for preventing or treating hepatorenal syndrome in an individual, comprises a combination of one of the following (1A), (1B), or (1C) with (2): (1A) Terlipressin 1.0 mg bolus intravenous infusion every 6 hours; (1B) terlipressin 2.0 mg intravenous infusion over 24 hours; (1C) Terlipressin 6.0 mg intravenous infusion over 24 hours; (2) a first dose of 4.0 mg of relaxin analog administered intravenously over approximately 4 hours, followed by 10.0 mg of relaxin analog administered subcutaneously 12 hours after the start of the first dose, followed by 10.0 mg of relaxin analog administered subcutaneously 24 hours after the start of the first dose, and then 5.0 mg of relaxin analog administered subcutaneously daily for up to 12-15 days.

[0179] In certain embodiments, a method for preventing or treating renal failure in an individual in need thereof, and / or a method for preventing or treating hepatorenal syndrome in an individual, comprises a combination of one of the following (1A), (1B), or (1C) with (2): (1A) Terlipressin 1.0 mg bolus intravenous infusion every 6 hours; (1B) terlipressin 2.0 mg intravenous infusion over 24 hours; (1C) Terlipressin 6.0 mg intravenous infusion over 24 hours; (2) a first dose of 4.0 mg of relaxin analog administered intravenously over approximately 4 hours, followed by 5.0 mg of relaxin analog administered subcutaneously 12 hours after the start of the first dose, followed by 10.0 mg of relaxin analog administered subcutaneously 24 hours after the start of the first dose, and then 5.0 mg of relaxin analog administered subcutaneously daily for up to 12-15 days.

[0180] Compositions and Agents The present application also relates to medicaments or pharmaceutical compositions comprising the above vasopressin analogs and relaxin analogs, or pharma- ceutically acceptable salts or solvates thereof, individually or in combination, and at least one pharma- ceutically acceptable carrier.

[0181] In some embodiments, a relaxin analog and / or a vasopressin analog is present as an active ingredient in a medicament or pharmaceutical composition of the present invention.

[0182] The composition or medicament of the present invention is in a form suitable for administration to an individual in need thereof.

[0183] The compositions or medicaments of the present invention can be administered, for example, parenterally, intravenously, subcutaneously, rectally, transdermally, topically, or by inhalation. In particular, the compositions of the present invention are administered by the intravenous or subcutaneous route.

[0184] According to certain embodiments, the pharma- ceutically acceptable carrier of the composition of the present invention is suitably selected from the group consisting of injectable carrier liquids, such as sterile water for injection, and aqueous solutions, such as physiological saline.

[0185] The composition or medicament of the present invention may contain the peptide of the present invention at a content of 0.01 mg / mL to 30 mg / mL, particularly 0.3 mg / mL to 3 mg / mL.

[0186] The medicament or pharmaceutical composition of the present invention may comprise at least one peptide of the present invention as the sole active ingredient, or may further comprise at least one other active ingredient (so long as said other active ingredient does not interfere with the biological activity of the peptide of the present invention).

[0187] The pharmaceutical composition or medicament of the present invention may further comprise at least one antioxidant, dispersing agent, emulsifier, antifoaming agent, flavoring agent, preservative, solubilizer, and / or dye, provided that this / these additional substances do not interfere with the biological properties of the peptide of the present invention.

[0188] The sterile compositions of the present invention for parenteral administration may be, in certain embodiments, aqueous or non-aqueous solutions, suspensions, or emulsions. Solvents or vehicles that can be used include water, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate, or other suitable organic solvents. These compositions may further include auxiliary agents such as wetting agents, isotonicity agents, emulsifiers, dispersants, and stabilizers. Sterilization can be carried out in several ways, for example, by sterile filtration, by incorporating sterilizing agents into the composition, by irradiation, or by heating. They can also be prepared in the form of sterile solid compositions that can be dissolved in sterile water or any other injectable sterile medium at the time of use.

[0189] Compositions for topical administration may be, for example, nasal drops or aerosols.

[0190] For subcutaneous, intramuscular or intravenous administration, the peptides of the invention used are converted into solutions, suspensions or emulsions, if necessary, using substances customary for this purpose, such as solubilizers, emulsifiers or other excipients. Examples of suitable solvents are water, physiological saline or alcohols, such as ethanol, propanol, glycerol and sugar solutions, such as glucose or mannitol solutions, or mixtures of the various solvents mentioned above.

[0191] In a particular embodiment, the composition of the invention, the agent of the invention, or the peptide of the invention, or one of its pharma- ceutically acceptable salts or solvates, is administered to an individual by a parenteral route, in particular transdermally, intravenously, subcutaneously, or intramuscularly, in particular intravenously or subcutaneously.

[0192] Methods for preparing parenterally administrable compositions will be apparent to those skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Sciences, 15th ed., Mack Publishing Company, Easton, Pa.

[0193] Administration of the compositions of the invention or the peptides of the invention to an individual may be systemic or localized to tissues, organs and / or sites of an individual organism.

[0194] Uses of relaxin analog and vasopressin analog peptides and compositions The present invention relates to combination therapies of vasopressin analogs and relaxin analogs, as well as related compositions comprising these bioactive agents, as well as pharma- ceutically acceptable salts or solvates thereof, for treating individuals in need thereof.

[0195] The present invention further relates to one or more pharmaceutical compositions of the present invention comprising a vasopressin analog and a relaxin analog bioactive agent for use as a combination therapy.

[0196] Furthermore, the present invention relates to the peptide of the present invention, its pharma- ceutical acceptable salt or solvate, or the pharmaceutical composition of the present invention, for use in combination therapy for the treatment and / or prevention of various diseases or conditions involving the RXFP1 receptor and / or the V1a vasopressin receptor, more particularly for the treatment and / or prevention of diseases or conditions associated with renal failure, including renal dysfunction induced by liver cirrhosis, renal dysfunction induced by liver transplantation, chronic kidney disease, and acute kidney injury. Such combination therapy can be used in particular for the treatment and / or prevention of HRS, in particular HRS-AKI (type 1 hepatorenal syndrome) or HRS-NAKI (type 2 hepatorenal syndrome).

[0197] In one embodiment, the combination therapy of relaxin analog and vasopressin analog peptides, pharma- ceutically acceptable salts or solvates thereof, or pharmaceutical compositions comprising one or more of these bioactive agents, is administered once daily, particularly by intravenous or subcutaneous routes.

[0198] The dose and frequency of administration of relaxin analog and vasopressin analog peptides, or pharma- ceutically acceptable salts or solvates thereof, will vary according to the desired effect, potency, and duration of action of the compound used, as well as the nature and severity of the disease or condition being treated, and the sex, age, weight, and individual responsiveness of the individual being treated. In general, the physician will determine the appropriate dosage as a function of age, weight, and all other factors specific to the individual being treated.

[0199] Further provided herein is a method for preventing and / or treating a disease or condition associated with renal failure, including cirrhosis-induced renal dysfunction, liver transplant-induced renal dysfunction, chronic kidney disease, and acute kidney injury, comprising co-administering to an individual in need of said prevention and / or treatment the pharmaceutical composition(s) of the invention comprising a combination of relaxin analogs and vasopressin analog peptides of the invention, pharma- ceutical acceptable salts or solvates thereof, or a vasopressin analog and / or relaxin analog bioactive agent of the invention, or a therapeutically effective amount of a combination of vasopressin analogs and relaxin analog peptides, pharma-ceutical acceptable salts or solvates thereof, or a vasopressin analog and / or relaxin analog bioactive agent according to the invention.

[0200] Further described is the use of the relaxin analog and vasopressin analog bioactive agents described herein, or combinations of pharma- ceutical acceptable salts or solvates thereof, or pharmaceutical compositions of the invention comprising relaxin analog and vasopressin analog peptides according to the invention, individually or in combination, or a therapeutically effective amount of the combinations of these, or combinations of pharma- ceutical acceptable salts or solvates thereof, or relaxin analog and vasopressin analog peptides of the invention, individually or in combination, for the manufacture of a medicament for the prevention and / or treatment in an individual of a disease or condition associated with renal failure, including renal dysfunction induced by liver cirrhosis, renal dysfunction induced by liver transplantation, chronic kidney disease, and acute kidney injury.

[0201] Further described is the use of the relaxin analog and vasopressin analog bioactive agents described herein, or combinations of pharma- ceutical acceptable salts or solvates thereof, or the pharmaceutical compositions of the invention comprising relaxin analog and vasopressin analog peptides according to the invention, individually or in combination, or a therapeutically effective amount of the combinations of these, or combinations of pharma- ceutical acceptable salts or solvates thereof, or the relaxin analog and vasopressin analog peptides of the invention, individually or in combination, for the manufacture of a medicament for the prevention and / or treatment in an individual of HRS, e.g., HRS-AKI (Hepatorenal Syndrome Type 1) and HRS-NAKI (Hepatorenal Syndrome Type 2).

[0202] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above Description, but is as set forth in the appended claims.

[0203] In the claims, articles such as "a," "an," and "the" can mean one or more, unless the contrary is indicated or otherwise clear from the context. A claim or description containing "or" between one or more elements of a group is considered to be satisfied if one, more than one, or all of the group elements are present in, employed in, or otherwise relevant to a given product or process, unless the contrary is indicated or otherwise clear from the context. The invention includes embodiments in which exactly one element of a group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the group elements are present in, employed in, or otherwise relevant to a given product or process.

[0204] It should also be noted that the term "comprising" is intended to be open-ended, allowing, but not requiring, the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0205] Where ranges are given, endpoints are included. Moreover, unless otherwise indicated or otherwise evident from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges are to be understood as contemplating any specific value or subrange within the ranges set forth in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

[0206] All cited sources, e.g., references, publications, databases, database entries, and techniques cited herein, are incorporated herein by reference, even if not explicitly stated in the citation. In the event of a conflict between the statements in the cited sources and this application, the statements in this application take precedence.

[0207] The section and table headings are not intended to be limiting. EXAMPLES

[0208] Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0209] The practice of the present invention employs, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); ALLehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

[0210] Example 1: Synthesis of relaxin peptide analogs. Materials used For the synthesis of C-terminal amides, various rink amide resins were used (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin sold by Chem-Impex or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidomethyl resin sold by Millipore Merck).

[0211] These were loaded in the range of 0.2 to 0.4 mmol / g.

[0212] Fmoc (fluorenylmethyloxycarbonyl)-protected natural amino acids were purchased from various sources, i.e., Protein Technologies Inc., Merck Biosciences, Novabiochem, Iris Biotech, Bachem, Chem-Impex International, or MATRIX Innovation.

[0213] The following standard amino acids were used throughout the synthesis: Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Glu-OtBu, Fmoc-Gly-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, and Fmoc-L-Val-OH.

[0214] In addition, the following special amino acids were purchased from the same supplier: Fmoc-L-hArg(Pbf)-OH, Fmoc-L-Cit-OH, Fmoc-L-Arg(Me,Pbf)-OH, Fmoc-L-hLys(Boc)-OH, Fmoc-L-Orn(Boc)-OH, Fmoc-L-Lys(Dde)-OH, Fmoc-L-Lys(ivDde)-OH, Fmoc-L-Lys(Mtt)-OH, Fmoc-L-Lys(aloc)-OH, Fmoc-L-Lys(Ac)-OH, Fmoc-Aib-OH, Fmoc-L-α-Me-Ser(tBu)-OH, Fmoc-L-α Me-Lys(Boc)-OH, Fmoc-L-α-Me-Arg(Pbf)-OH, Fmoc-L-α-Me-Leu-OH, Fmoc-L-Nle-OH and Fmoc-L-1-Nal-OH, Fmoc-L-2-Nal-OH Fmoc-5-Wox-OH, Fmoc-Pfp-OH, Fmoc-L-α-Me-Trp-OH, Fmoc-L-α-Me-Phe-OH. [Table 4-1] [Table 4-2]

[0215] To synthesize the C-terminally lipidated relaxin peptide analogs, an orthogonally protected lysine was used, as shown below.

[0216] 1.A. General Methods Used for the Synthesis of Selected Relaxin Peptide Analogs Relaxin peptide analogs of sequences SEQ ID NOs: 1-97 were synthesized based on the methods depicted in FIGS.

[0217] 0.2 mmol of Rink amide AM resin was placed into a CEM Liberty blue microwave peptide synthesizer and the complete peptide sequence was assembled (Figure 1).

[0218] The entire synthesis was carried out in DMF as the solvent. The peptides were synthesized using standard heating protocols on a 0.1-0.2 mmol scale.

[0219] Standard heating protocol: 170 watts, 75°C, 15 seconds, followed by 30 watts, 90°C, 120 seconds.

[0220] Deprotection was carried out using 20 v / v % piperidine in DMF, followed by three DMF washing steps.

[0221] Heating protocol for deprotection: 170 watts, 75° C., irradiation for 15 seconds, followed by 30 watts, 90° C., irradiation for 50 seconds.

[0222] Amino acid couplings were carried out using 5 equivalents of Fmoc-AA as a 0.2 M solution in DMF using 5 equivalents of N,N'-diisopropylcarbodiimide (DIC) 0.5 M and 5 equivalents of Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma Pure®) 1 M as coupling reagents.

[0223] To increase the final yield, each amino acid required double coupling at 90° C. for 120 seconds. 21 and X 22 2-aminoisobutyric acid at position X, 29 For serine at position 1, triple coupling at 90° C. for 2 min was used.

[0224] For expensive amino acid derivatives such as Fmoc-α-methyllysine (Boc)-OH, manual coupling using 3 equivalents of amino acid and 3 equivalents of coupling agent such as HATU or HCTU at room temperature for 1-18 hours or microwave heating for 120 seconds using DIC and Oxyma Pure™ may be advantageous.

[0225] X 25 At this position, a derivative of Fmoc-lysine-OH was used that bears an orthogonal protecting group on the side chain nitrogen. Selective deprotection allowed modification of this amino acid side chain.

[0226] At the end of the synthesis, Fmoc deprotection was performed manually twice with 20 v / v % piperidine in DMF for 30 min at room temperature.

[0227] Acetylation at the N-terminus was carried out by treatment with 5-10 equivalents of acetic anhydride and 5-10 equivalents of DIEA in DMF for 15 min. The resin containing the fully protected peptide was then washed three times with DCM / DMF / DCM respectively and dried under vacuum.

[0228] Next, X 25The N-protecting group of the lysine at position 1 (Dde, ivDde, Aloc, or Mtt) was removed using dilute hydrazine in DMF (Dde, ivDde), phenylsilane or dimethylaminoborane in the presence of a palladium(0) catalyst (aloc) in DCM, or 1% TFA in DCM (Mtt).

[0229] The Z group is then attached to the solid support by the following steps: 25 Attachment was made to the side chain nitrogen of lysine.

[0230] X 25 When the lysine at position 1 was protected on its side chain by Dde or iv-Dde, the resin peptide was transferred to a 50 ml polypropylene syringe. The resin was infiltrated with 80 ml of a 5% solution of hydrazine in DMF, followed by washing with DMF (3 times). The reaction was monitored by Kaiser test.

[0231] X 25 When the lysine at position 1 was protected on its side chain by an allyl-oxy-carbonyl group (aloc), the resin peptide was transferred to a 50 ml polypropylene syringe, swollen in dichloromethane, and treated with 20 equivalents of phenylsilane (PhSiH3) (or borane dimethylamine complex ((CH3)2NH.BH3) and piperidine) and 10% (mol / mol) tetrakis-(triphenylphosphine)palladium (Pd(PPh3)4) under argon for 2 hours. This treatment was repeated until no starting aloc-protected peptide was detected by UPLC / MS analysis after cleavage of an aliquot of the resin.

[0232] Once the reaction was complete, the resin was washed with dichloromethane, 1% DIEA in DMF, 5% diethyl-dithio-carbamate in DMF, DMF, 10% DIEA in DMF, DMF and dichloromethane (three times each).

[0233] X 25If the lysine at position 1 was protected on its side chain with a methyl-trityl group (Mtt), the resin-peptide was transferred to a 50 ml polypropylene syringe, swollen in dichloromethane and treated with 10 ml of a DCM / TIS / TFA (88 / 5 / 2) mixture. After shaking for 1 h, the solvent was drained and the resin was washed with DCM, shaken with 10 ml of a DCM / DIEA (90 / 10) mixture and washed several times with DCM.

[0234] Z-group PEG xx The group(s) may be substituted with 3 equivalents of Fmoc-PEG along with 3 equivalents of DIC and 3 equivalents of HOAt, if applicable for a given relaxin peptide analog, while monitoring the reaction by the ninhydrin (Kaiser) test. xx The Fmoc-PEG was introduced by single acylation with -OH for 18 h. The resin was then treated with 20% v / v piperidine in DMF to remove the Fmoc protecting group (2 x 30 min) and washed three times with DMF. xx The -OH coupling, Fmoc removal, and washing steps were repeated x times (x=0-5).

[0235] Next, if gE was present in the Z group of the relaxin peptide analog, Fmoc-Glu-OtBu ((4S)-5-tert-butoxy-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid) was introduced by a single coupling using 3 equivalents of amino acid with 3 equivalents of DIC and 3 equivalents of HOAt for 18 h, with the reaction monitored by Kaiser test.

[0236] The resin was then treated with 20% v / v piperidine in DMF to remove the Fmoc protecting group (2 × 30 min) and washed with DMF three times. The Fmoc removal and Fmoc-Glu-OtBu coupling steps were repeated y times (y = 1-5).

[0237] The side chain was then modified with 3 equivalents of Ck lauric acid (C 12 ), myristic acid (C 14 ), pentadecanoic acid (C 15 ), palmitic acid (C 16), heptadecanoic acid (C 17 ), stearic acid (C 18 ), eicosanoic acid (C 20 ), or docosanoic acid (C 22 ), with 3 equivalents of DIC and 3 equivalents of HOAt in NMP, with the corresponding acyl chloride and DIEA as base in dichloromethane, or with the corresponding N-succinimidyl ester and DIEA as base in DMF.

[0238] The reaction was monitored by Kaiser test and left overnight.

[0239] The resin containing the fully protected peptide was then washed three times with DCM / DMF / DCM respectively and dried under vacuum.

[0240] 1.B. Peptide-Resin Cleavage Upon completion of the solid-phase synthesis, the peptide was cleaved from the solid support by treatment with cleavage reagent B: TFA / phenol / H2O / TIPS (87.5% / 5% / 5% / 2.5% / 25 ml) for 3 h (TIPS stands for triisopropylsilane).

[0241] In certain cases, the addition of a dithiol such as 1,2-ethanedithiol or DODT (2,2'-(ethylenedioxy)diethanethiol) may be advantageous (e.g. cleavage reagent K). The TFA solution containing the peptide was filtered and concentrated under reduced pressure at T<30°C.

[0242] The desired product was precipitated with ice-cold MTBE (methyl tert-butyl ether) or diethyl ether and centrifuged at 3000 rpm for 30 minutes. The centrifuged pellet was then washed with ice-cold diethyl ether and centrifuged. This process was repeated three times.

[0243] In certain cases, it may be necessary to treat the crude peptide to remove undesired by-products such as TFA esters, CO2 adducts (carbamic acid) on the indole nitrogen of tryptophan, and 2-t-butyl-sulfanylethyl adducts on methionine residues.

[0244] To remove the CO2 adduct on the indole nitrogen of tryptophan, the crude peptide was dissolved in water containing 10-20% CH3CN, 5 mg / ml, and lyophilized.

[0245] To remove the 2-t-butyl-sulfanylethyl adduct on Met, the crude peptide was dissolved (2 mg / ml) in a solution of H2O / CH3CN (50:50 v / v) containing 0.1% formic acid.

[0246] The mixture was gently shaken overnight at 37°C.

[0247] To remove the TFA esters, the crude peptide was dissolved (2 mg / ml) in a solution of HO / CHCN (50:50 v / v) containing 0.1% formic acid. The mixture was gently shaken at 37° C. for 1-4 h.

[0248] In both cases the crude peptide solutions thus obtained were partially concentrated under reduced pressure at T<30° C. and lyophilized.

[0249] 1.C. Purification process Following any of the methods described above for synthesizing relaxin peptide analogs, the peptides were purified prior to use.

[0250] 80 mg of peptide was dissolved in 1.5 mL of DMSO and purified by reverse phase high pressure liquid chromatography (RP-HPLC), as described below.

[0251] A GX271 Liquid Handler, 333 / 334 pumps, and a UV / VIS 151 Gilson system were used.

[0252] Two different systems were used to purify the above peptides. System A -Column: Waters Delta-Pack C4 15μm 300Å. 250×20mm - Solution A: 0.1% trifluoroacetic acid (TFA) in HO - Solution B: 0.1% TFA in acetonitrile - Gradient: 15% B for 5 min, 15% B to 50% B in 20 min -Flow rate: 80ml / min System B -Column: Waters CSH C18 5μm 250×50mm, or Waters Sunfire C18 10μM 250×50mm - Solution A = 0.1% TFA in water - Solution B = 0.1% TFA in acetonitrile - Gradient: 1%B to 18%B in 5 min, 18%B to 28%B in 10 min, 28%B for 15 min, 28%B to 48%B in 10 min - Next, column wash from 48% B to 90% B for 10 min. -Flow rate: 150ml / min.

[0253] The peptide of interest eluted in the 35-40 min time frame.

[0254] The gradient was slightly adjusted according to the polarity of each peptide, which was characterized by its retention time on the analytical UHPLC system.

[0255] Fractions containing the pure peptide were then partially concentrated under reduced pressure at T<35° C. and lyophilized to constant weight.

[0256] For certain applications (e.g., in vivo testing), it was advantageous to exchange the TFA salt for an acetate salt. Three corresponding methods were used and are described in the following section 1.D.

[0257] 1.D. Acetate Exchange (i) Acetate exchange with TOYOPEARL® DEAE 650C (Tosoh Corporation) Ion exchange was carried out using TOYOPEARL® DEAE650 C grade resin (a weak anion exchange resin).

[0258] 120 ml of resin was washed successively with 15 volumes of NaOH 1M, 5 volumes of H2O, 5 volumes of acetic acid 1.6M, 5 volumes of acetic acid 0.16M, and finally with 5 volumes of H2O.

[0259] Next, 41.8 mg of peptide was dissolved in 4 ml of distilled water and downloaded onto the resin and mixed gently for 2 hours.

[0260] Finally, the peptide was recovered by elution, washed with water and lyophilized.

[0261] Peptide recovery amount: 35 mg ( 19 F NMR (400 MHz) ns 1028 as acetate).

[0262] (ii) Acetate exchange using a Sepharose HiTrap Q HP column (strong anion exchange column) In this second method, ion exchange was carried out using a HiTrap Q HP.

[0263] The column (5 ml total volume) was connected to a peristaltic pump set at 48 (4.5 ml / min) and washed with 50 ml (10 column volumes) of HO, 100 ml (20 column volumes) of a 1 M solution of sodium acetate, 150 ml (30 column volumes) of HO, and 50 ml (10 column volumes) of a 0.16 M solution of acetic acid before loading the peptide.

[0264] The pure peptide was dissolved at 2 mg / ml in 0.16 M acetic acid, slowly loaded onto the column and eluted at 4.5 ml / min.

[0265] The recovered solution was freeze-dried.

[0266] The effectiveness of ion exchange is 19 This was verified by F NMR (400 MHz) ns 1028.

[0267] (iii) Acetate exchange with BIO RAD AG1X4® anion exchange resin

[0268] A 125 ml reaction vessel with sintered glass bottom was charged with 6.2 g of BIO RAD AG1X4® anion exchange resin, 100-200 dry mesh size (OH-- form).

[0269] The resin was shaken on a stir plate with 3×100 ml of 1.6 M aqueous acetic acid (10% v / v) and 3×50 ml of 0.16 M aqueous acetic acid (1% v / v), respectively, for 20 min.

[0270] The purified peptide as the TFA salt (100 mg) was dissolved in 50 ml of distilled water, poured onto the exchange resin and shaken on a stir plate for 120 min.

[0271] The aqueous solution was drained into a 100 ml round bottom flask and the resin was washed with 2×15 ml of 0.16 M aqueous acetic acid (1% v / v).

[0272] The combined solutions containing the peptide as the acetate salt were lyophilized to constant weight. Yield = 80 mg of peptide as the acetate salt.

[0273] The effectiveness of ion exchange is 19 This was verified by F NMR (400 MHz, ns 1028) and / or ion chromatography.

[0274] Example 2: Exemplary synthesis of relaxin peptide analogs 2.A Loading of Fmoc-Lys(Ac)-OH onto Rink Amide Resin In a 100 ml reaction vessel with sintered glass at the bottom, 6 g of Novabiochem or CheMImpex Rink Amide AM resin (low loading 0.47 mmol / g) was swollen in 40 ml of DMF. The solvent was drained and 30 ml of a 20% piperidine solution in DMF was added. After shaking for 15 minutes, the solvent was drained. This was repeated twice to ensure complete removal of the Fmoc protecting group. The resin was washed with 5 x 30 ml of DMF.

[0275] In a separate flask, a solution containing Fmoc-Lys(Ac)-OH (3.5 g, 8 mmol, 3 equiv.) HOBT·HO (1.3 g, 8.5 mmol) in 30 ml of DMF was prepared. Diisopropylcarbodiimide (DIC) (1 g, 8.5 mmol) was added to this solution and after 5 min, the resulting mixture was added to the resin. The suspension was shaken on a stir plate for 4 h or until completion of the reaction as judged by Kaiser test (ninhydrin test) on an aliquot portion of the resin.

[0276] The solvent was then drained and the resin was washed three times with 30 ml of DMF. The Fmoc-Lys(Ac)-NH2 loaded resin was either used immediately for the next step or stored moist at 4° C. until required.

[0277] 2.B. Synthesis of a peptide having SEQ ID NO:3 The following syntheses were carried out using five times the amount of resin obtained in step 2.A., corresponding to 0.2 mmol of Fmoc-Lys(Ac)-NH2 each. The syntheses were carried out separately on individual batches using a CEM Liberty Blue microwave peptide synthesizer to build the second and third residues of the peptide sequence (starting from the C-terminus).

[0278] Peptide synthesis was carried out by using DIC 0.5M / Oxyma 1M in DMF.

[0279] All amino acids were introduced in double couplings using standard heating protocols.

[0280] The resin was removed from the synthesizer and Fmoc-α-methyl-lysine(Boc)-OH (3 eq.) was manually coupled using 3 eq. Oxyma™ and 3 eq. DIC with microwave heating (75° C., 15 s and 90° C., 110 s). The completion of the reaction was controlled by Kaiser test. In case of positive, 3 eq. DIC was added followed by microwave heating as above.

[0281] Once coupling of Fmoc-α-methyl-lysine(Boc)-OH was completed, the remainder of the peptide sequence was constructed using a CEM Liberty Blue microwave peptide synthesizer.

[0282] All amino acids were introduced by double coupling at 90° C. as above, except aminoisobutyric acid at position 21, which was triple coupled for 2 min at 90° C., and serine at position 29. Fmoc-Lys(Dde)-OH was used at position 25.

[0283] At the end of the five syntheses, the five batches of resin were combined and transferred to a 50 mL polypropylene syringe and the peptide was acetylated at the N-terminus with acetic anhydride (944 μL, 10 mmol) in DMF (30 mL) for 20 min, and this cycle was repeated twice.

[0284] The Dde protecting group on the lysine 25 side chain was then removed by filtering 50 mL of a 5% w / v solution of hydrazine in DMF, followed by DMF washes (5 x 20 ml). The reaction was monitored by Kaiser test and an aliquot of the resin was cleaved and subjected to UPLC / MS analysis.

[0285] The three TTDS spacer units were introduced by single coupling by carrying out the following procedure three times. To the resin was added a solution of Fmoc-TTDS-OH (1.62 g, 3 mmol) in 30 mL of DMF, followed by HOAt (5 ml of a 0.6 ml solution in DMF, 3 mmol) and DIC (1 ml, 6 mmol). The syringe was agitated on an orbital table for 18 h. The reaction was monitored by Kaiser test. The resin was washed with DMF (2 x 30 mL). To the resin was then added 30 mL of 20 v / v % piperidine in DMF. The syringe was agitated for 20 min on an orbital table. This deprotection procedure was repeated once more and the resin was washed with DMF (2 x 30 mL) and dichloromethane (3 x 30 mL).

[0286] Three gamma glutamic acid spacers were introduced by performing double couplings of each Fmoc-Glu-OtBu, therefore the following procedure was applied three times.

[0287] To the resin was added a solution of (4S)-5-tert-butoxy-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid (Fmoc-Glu-OtBu) (1.275 g, 3 mmol) in 30 mL of DMF, followed by HOAt (0.6 ml solution in 5 ml of DMF, 3 mmol) and DIC (1 ml, 6 mmol). The syringe was agitated on an orbital table for 4 h. The resin was washed with DMF (2 x 20 mL) and the coupling was repeated once more. The reaction was monitored by Kaiser test. The resin was washed with DMF (2 x 30 mL). To the resin was then added 30 mL of 20 v / v % piperidine in DMF. The syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated once more and the resin was washed with DMF (3×30 mL) and dichloromethane (3×30 mL).

[0288] Finally, the peptide was acylated by activation with palmitic acid (768 mg, 3 mmol), HOAt (5 ml of a 0.6 M solution in DMF, 3 mmol) and DIC (1 ml, 6 mmol) in DMF (30 mL) for 2.5 h. The resin was washed with DMF (2×30 mL) and dichloromethane (3×30 mL) and dried under vacuum.

[0289] Cleavage of the peptide from the resin was carried out using a solution of phenol (6.25 g), water (6.25 mL) and TIPS (3 mL) in TFA (QSP 125 mL) for 2.5 h at room temperature. The resin was filtered off and washed with 2×30 mL TFA. The combined filtrates were transferred to a 250 mL round-bottom flask, partially concentrated under vacuum at T<30° C., and the peptide was precipitated by adding 100 mL ice-cold MTBE and centrifuged at 3600 rpm for 30 min.

[0290] The centrifuged pellet was then washed with ice-cold diethyl ether and centrifuged. This process was repeated three times. 4.3 g of crude peptide was obtained. The crude peptide was dissolved (10 mg / mL) in a solution of H2O / CH3CN (50:50 v / v) containing 0.1% formic acid, and the mixture was gently shaken at 37°C for 1 h, partially concentrated, and lyophilized.

[0291] Purification was performed using Purification System A with 10 injections of 350 mg each, and fractions containing the pure desired peptide were lyophilized. The peptide as the trifluoroacetate salt was obtained as a white solid. m=428mg(17.5%) UPLC / MS: RT: 4.98 minutes (Analysis conditions A), purity 99% (UV) Observed mass m / z (ion type): 1454.5 (M+3H); 1091.1 (M+4H); 873.1 (M+5H).

[0292] 2.C. Synthesis of a peptide having SEQ ID NO:7 Two batches of resin obtained in 2.A., each corresponding to 0.2 mmol of Fmoc-Lys(Ac)-NH2 (total 0.4 mmol), were treated in parallel following the same procedure as in Example 2.B. After coupling of the third (4S)-5-tert-butoxy-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid (Fmoc-Glu-OtBu), the combined resin batches weighing 2.6 g were transferred into a 50 mL polypropylene syringe.

[0293] The resin was then added with 30 mL of 20% v / v piperidine in DMF. The syringe was agitated on the orbital table for 20 min. This deprotection procedure was repeated once more and the resin was washed with DMF (3×30 mL) and dichloromethane (3×30 mL).

[0294] The peptide was acylated by addition of a solution of stearoyl chloride (362 mg, 1.2 mmol) and DIPEA (0.255 ml, 1.5 mmol) in 20 ml DCM over 2.5 h. The resin was washed with DMF (2×30 mL) and dichloromethane (3×30 mL) and dried under vacuum.

[0295] Cleavage of the peptide from the resin was carried out using a solution of phenol (1.5 g), water (1.5 mL) and TIPS (0.6 mL) in TFA (QSP 30 mL) for 2.5 h at room temperature. The resin was filtered off and washed with 2 x 10 mL TFA. The combined filtrates were transferred to a 250 mL round bottom flask, partially concentrated under vacuum at T < 30 °C, and the peptide was precipitated by adding 100 mL ice-cold MTBE and centrifuged at 3600 rpm for 30 min.

[0296] The centrifuged pellet was then washed with ice-cold diethyl ether and centrifuged. This process was repeated three times. 720 mg of crude peptide was obtained. Purification was performed using purification system B with three injections of 240 mg each. Fractions containing the pure desired peptide were lyophilized. The peptide as the trifluoroacetate salt was obtained as a white solid. m = 72 mg (3.6%) UPLC / MS: RT: 5.86 minutes (Analysis conditions A), purity 98% (UV) Observed mass m / z (ion type): 1463.9 (M+3H); 1098.2 (M+4H); 878.7 (M+5H).

[0297] 2.D. Synthesis of peptide having SEQ ID NO:6 A batch of the resin obtained in 2.A. corresponding to 0.1 mmol of Fmoc-Lys(Ac)-NH2 was placed in the reaction vessel of a CEM Liberty Blue microwave peptide synthesizer. Peptide synthesis was carried out using DIC 0.5M / Oxyma 1M in DMF.

[0298] All amino acids were introduced by double coupling at 90° C. as above, except aminoisobutyric acid at position 21, which was triple coupled for 2 min at 90° C., and serine at position 29. Fmoc-Lys(ivDde)-OH was used at position 25.

[0299] At the end of peptide assembly, the resin was transferred to a 20 ml polypropylene syringe and the peptide was acetylated at the N-terminus with acetic anhydride (95 μL, 1 mmol) and DIPEA (174 μL, 1 mmol) in DMF (10 mL) for 20 min, and this cycle was repeated twice.

[0300] The ivDde protecting group on the lysine 25 side chain was then removed by stirring with 10 mL of a 5% w / v solution of hydrazine in DMF for 20 min as many times as necessary until no starting material was detectable after cleavage and UPLC / MS analysis of an aliquot of the resin. Once deprotection was deemed complete, the resin was washed with DMF (5 x 10 ml).

[0301] The two TTDS spacer units were introduced by single coupling by carrying out the following procedure twice: To the resin was added a solution of Fmoc-TTDS-OH (163 mg, 0.3 mmol), HOAt (42 mg, 0.3 mmol) and DIC (77 μL, 0.5 mmol) in 7 mL DMF. The syringe was agitated on an orbital table for 18 h. The reaction was monitored by Kaiser test. If necessary, double coupling was performed. The resin was washed with DMF (2×10 mL). Then, 10 mL of 20% v / v piperidine in DMF was added to the resin. The syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated once more and the resin was washed with DMF (2×10 mL) and dichloromethane (3×10 mL).

[0302] Three gamma glutamic acid spacers were introduced by performing double couplings of each Fmoc-Glu-OtBu, therefore the following procedure was applied three times.

[0303] The resin was treated with a solution of (4S)-5-tert-butoxy-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid (Fmoc-Glu-OtBu) (127 mg, 0.3 mmol), HOAt (42 mg, 0.3 mmol) and DIC (77 μL, 0.5 mmol) in 7 mL of DMF. The syringe was agitated on an orbital table for 18 h. The reaction was monitored by Kaiser test. The resin was washed with DMF (2×10 mL). The resin was then added with 10 mL of 20 v / v % piperidine in DMF. The syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated once more and the resin was washed with DMF (3×10 mL) and dichloromethane (3×30 mL).

[0304] Finally, the peptide was acylated with stearoyl chloride (62 mg, 0.2 mmol) and DIPEA (54 μL, 0.3 mmol) in 5 ml DCM for 2.5 h. The resin was washed with DMF (2×30 mL) and dichloromethane (3×30 mL) and dried under vacuum.

[0305] Cleavage of the peptide from the resin was carried out using a solution of phenol (0.5 g), water (0.5 mL) and TIPS (0.2 mL) in TFA (QSP 10 mL) for 2.5 h at room temperature. The resin was filtered off and washed with 2 x 4 mL of TFA. The combined filtrates were transferred to a 100 mL round bottom flask, partially concentrated under vacuum at T < 30 °C, and the peptide was precipitated by adding 50 mL of ice-cold MTBE and centrifuged at 3600 rpm for 30 min.

[0306] The centrifuged pellet was then washed with ice-cold diethyl ether and centrifuged. This process was repeated three times. 240 g of crude peptide was obtained. Purification was performed using purification system B and the fractions containing the pure desired peptide were lyophilized. The peptide as the trifluoroacetate salt was obtained as a white solid. m=38 mg (8%) UPLC / MS: RT: 5.40 min. (analysis condition A), purity 97% (UV) Observed mass m / z (ion type): 1376.1 (M+3H); 1032.0 (M+4H); 826.0 (M+5H).

[0307] 2.E. Synthesis of peptide having SEQ ID NO:20 This compound was obtained following the same procedure as used in Example 2.D, except that three TTDS were introduced into the lysine 25 side chain.

[0308] Thus, a white solid was obtained from 250 mg of resin obtained in 2.A., corresponding to 0.1 mmol of Fmoc-Lys(Ac)-NH2. m = 20 mg (4.5%). UPLC / MS: RT: 5.44 min. (analysis condition A), purity 99% (UV). Observed mass m / z (ion type): 2215.0 (M+2H); 1476.8 (M+3H); 1107.9 (M+4H); 886.7 (M+5H).

[0309] 2.F. Synthesis of peptide having SEQ ID NO:22 This compound has Fmoc-Lys(Aloc)-OH at the 25th position (X) instead of Fmoc-Lys(Dde)-OH. 25This was obtained following the same procedure as used in Example 2.B., except that 2.

[0310] Therefore, 250 mg of resin obtained in 2.A., corresponding to 0.1 mmol of Fmoc-Lys(Ac)-NH2, was treated as in 2.B. up to the N-terminal acetylation step.

[0311] The Aloc group on the Lys25 side chain was removed by adding a solution of 1 ml (8.33 mmol) of phenylsilane in 2 ml of degassed DCM and a solution of 10 mg (25.96 μmol) of tetrakis-(triphenylphosphine)palladium in 4 ml of DCM to the resin under an argon atmosphere. The resin was shaken on an orbital table for 60 min and the reaction medium was replaced twice with fresh reagents, shaking for 60 min each time.

[0312] Once the reaction was complete, the resin was washed with dichloromethane, 1% DIEA in DMF, 5% diethyl-dithio-carbamate in DMF, DMF, 10% DIEA in DMF, DMF and dichloromethane (three times each).

[0313] Three PEG2DGA spacer units were introduced by single coupling by carrying out the following procedure three times. To the resin was added a solution of Fmoc-PEG2DGA-OH (170 mg, 0.3 mmol) in 8 mL of DCM, followed by HOAt (0.5 mL of a 0.6 mL solution in DMF, 0.3 mmol) and DIC (100 μl, 0.642 mmol). The syringe was agitated on an orbital table for 18 h. The reaction was monitored by Kaiser test. The resin was washed with DMF (2×10 mL). To the resin was then added 10 mL of 20 v / v % piperidine in DMF. The syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated once more and the resin was washed with DMF (2×30 mL) and dichloromethane (3×30 mL).

[0314] Three gamma glutamic acid spacers were introduced by performing double couplings of each Fmoc-Glu-OtBu, therefore the following procedure was applied three times.

[0315] To the resin was added a solution of (4S)-5-tert-butoxy-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid (Fmoc-Glu-OtBu) (0.13 g, 0.3 mmol) in 8 mL of DMF, followed by HOAt (0.6 ml solution in 0.5 ml of DMF, 0.3 mmol) and DIC (0.1 ml, 0.6 mmol). The syringe was agitated on an orbital table for 4 h. The resin was washed with DMF (2×20 mL) and the coupling was repeated once more. The reaction was monitored by Kaiser test. The resin was washed with DMF (2×10 mL). To the resin was then added 10 mL of 20% v / v piperidine in DMF. The syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated once more and the resin was washed with DMF (3×30 mL) and dichloromethane (3×30 mL).

[0316] Finally, the peptide was acylated by activation with palmitic acid (80 mg, 0.3 mmol), HOAt (0.5 ml of a 0.6 M solution in DMF, 3 mmol) and DIC (0.1 ml, 6 mmol) in DMF (10 ml) for 2.5 h. The resin was washed with DMF (2×30 ml) and dichloromethane (3×30 ml) and dried under vacuum.

[0317] Cleavage of the peptide from the resin was carried out using a solution of phenol (0.5 g), water (0.5 mL) and TIPS (0.25 mL) in TFA (QSP 10 mL) for 2.5 h at room temperature. The resin was filtered off and washed with 2 x 5 mL of TFA. The combined filtrates were transferred to a 250 mL round bottom flask, partially concentrated under vacuum at T<30°C and the peptide was precipitated by adding 100 mL of ice-cold MTBE and centrifuged at 3600 rpm for 30 min.

[0318] The centrifuged pellet was then washed with ice-cold diethyl ether and centrifuged. This process was repeated three times. 220 mg of crude peptide was obtained.

[0319] Purification was carried out using purification system B. Fractions containing the pure desired peptide were lyophilized. The peptide as the trifluoroacetate salt was obtained as a white solid. m = 37 mg (8%) UPLC / MS: RT: 4.99 minutes. (Analysis conditions A), purity 99% (UV) Observed mass m / z (ion type): 2205.2 (M+2H); 1470.5 (M+3H); 1103.1 (M+4H); 882.7 (M+5H).

[0320] 2.G.Results The results obtained using SEQ ID NOs: 1 to 32, 34 to 37, 39, 44, 45, 47 to 49, 51, and 54 to 97 are shown in Table 1 below. [Table 5-1] [Table 5-2] [Table 5-3] Condition A is as follows. -Column: Acquity Peptide CSH, C18, 130Å, 2.1×100mm, 1.7um; - Column temperature: 50°C; flow rate = 0.6 ml / min; - Solvent A: 0.1% TFA in HO; - Solvent B: 0.1% TFA in CH3CN; - Gradient: 0-1 min B=2%, 1-7 min B=2%-70%, 7-8 min B=70%-100%; -UV detector: Wavelength: 220nm; MS acquisition: ESI+200~3000uma. Condition B is as follows: - Column: Acquity BEH C18, 130Å, 2.1×50mm, 1.7μm - column temperature 60 °C; - Flow rate 0.6 ml / min Solvent A: 0.05% TFA in H2O Solvent B: 0.05% TFA in CH3CN - Gradient: 0-1 min B=2%, 1-16 min B=2%-100%, 16-17 min B=100% -UV detector: Wavelength: 220nm; MS acquisition: ESI+200~3000uma Condition C is as follows: - Column: ACQUITY BEH C4, 130Å, 2.1×50mm, 1.7μm; - column temperature 45 °C; -Flow rate: 0.4ml / min; - Solvent A: 0.1% TFA in H2O; Solvent B: 0.05% TFA in CH3CN - Gradient: 0-1 min B=30%, 1-5 min B=30%-50%, 5-6 min B=80% -UV detector: Wavelength: 220nm; MS acquisition: ESI+200~3000uma Condition D is as follows: -Column: Acquity Peptide CSH, C18, 130Å, 2.1×150mm, 1.7um; - column temperature 60 °C; -Flow rate=0.4ml / min; - Solvent A: 0.1% TFA in H2O; Solvent B: 0.1% TFA in CH3CN; - Gradient: 0-1 min B=2%, 1-14 min B=2%-70%, 14-16 min B=70%-100%; -UV detector: Wavelength: 220nm; MS acquisition: ESI+200~3000uma.

[0321] The following relaxin peptide analogs can each be prepared as described above (SEQ ID NOs: 33, 38, 40, 41, 43, 46, 50, 52, and 53).

[0322] Example 3: In vitro analysis of relaxin peptide analogs on the RXFP1 receptor (OVCAR5 cAMP assay) A. Method OVCAR5 cells expressing endogenous human RXFP1 were used to test the RXFP1 agonist properties of relaxin peptide analogs, particularly peptides of sequences SEQ ID NOs: 1-97.

[0323] Because RXFP1 is a Gs-coupled GPCR, the increase in cAMP was used as a readout for RXFP1 activation.

[0324] Isobutylmethylxanthine (IBMX) was used to inhibit phosphodiesterase activity and facilitate cAMP measurement. HTRF (homogeneous time-resolved fluorescence) technique was used for cAMP detection due to its high sensitivity.

[0325] Briefly, OVCAR5 was grown in regular medium (RPMI) containing 10% fetal calf serum (FCS) and 1% antibiotics (penicillin / streptomycin).

[0326] Before the experiment, cells were detached with Accutase and incubated with 1 mM (3-isobutyl-1-methylxanthine) IBMX for 40 min at 37°C.

[0327] The cells were then distributed into 384 black well plates containing increasing concentrations of the various peptides in a fixed volume of medium (without FCS).

[0328] After 30 min of incubation at 37°C in a humidified incubator with 5% CO2, the reaction was stopped by adding a volume of a solution containing lysis buffer and cAMP-D2 (cAMP labeled with the dye d2), as well as an anti-cAMP antibody linked to europium and used for cAMP detection.

[0329] The experimental readout was carried out on a fluorometer enabling HTRF measurements. Activation curves were generated by plotting the intracellular values ​​of cAMP against the log10 of compound concentration.

[0330] 50% activation concentration (EC 50 ) were calculated by nonlinear regression with a sigmoidal dose-response (variable slope) equation using Prism 5 software.

[0331] Emax% was determined by dividing the maximum intracellular value of cAMP for the test compound (upper limit of cAMP vs. concentration curve) by the maximum intracellular value of cAMP for human relaxin 2 (H2-R1x) determined on the same test occasion and multiplying by 100.

[0332] Emax% = 100 × [cAMP test cpd ] / [cAMP H2-R1x ]

[0333] B. Results The results obtained with the relaxin peptide analogs are shown in Table 2 below. [Table 6-1] [Table 6-2] [Table 6-3]

[0334] The relaxin peptide analogs described herein are potent agonists of human RXFP1, with consistently high E values ​​comparable to native human relaxin 2 in the in vitro OVCAR5 cAMP assay. max It has a value.

[0335] The same experiment was performed using peptides from the prior art (Bathgate et al., International Publication No. WO 2015 / 157829, incorporated by reference in its entirety) known under the designations B7-33 C11.23S, AcB7-33 C11.23S, and KKKK (AcB7-29 C11.23S).

[0336] EC in the following OVCAR5 cAMP assays 50 results were obtained for these three peptides. [Table 7] As demonstrated above, the relaxin peptide analogs in Table 2 have very interesting RXFP1 agonist properties and are highly effective at activating RXFP1. Moreover, all of them are significantly and unexpectedly superior to prior art peptides.

[0337] Example 4: A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single and Multiple Ascending Dose-Ranging Study in Healthy Volunteers to Evaluate the Safety, Tolerability, and Measure the Pharmacokinetics and Pharmacodynamics of a Relaxin Agonist This example describes a phase 1, randomized, double-blind, pacebo-controlled study to evaluate the safety, tolerability, PK, and PD of a relaxin agonist (i.e., a relaxin peptide analog of SEQ ID NO:3).

[0338] Study design and safety results of the study Part A of the SAD was a SAD study for a total of 40 subjects (n=8 per cohort, 6 to relaxin agonist, 2 to placebo) enrolled sequentially in up to five cohorts (Cohorts A1-A5). Four cohorts received doses by SC injection only, and one cohort (Cohort A2, 4.0 mg) received doses by both SC and IV injections with at least a 2-week washout period between SC and IV injections. The 4.0 mg dosed cohort (Cohort A2) received the IV dose after the 12.0 mg SC cohort (Cohort A3) was completed. Subjects in each cohort received one of five dose levels (1.0 mg, 4.0 mg, 12.0 mg, 24.0 mg, and 48.0 mg) or a matching placebo as follows: Cohort A1: A single SC dose of 1.0 mg relaxin agonist (n=6) or matching placebo (n=2). Cohort A2: Period 1 Single SC dose of 4.0 mg relaxin agonist (n=6) or matching placebo (n=2) Cohort A2: Period 2: A single IV dose of 4.0 mg relaxin agonist (n=6) or matching placebo (n=2) over 15 minutes Cohort A3: A single SC dose of 12.0 mg relaxin agonist (n=6) or matching placebo (n=2). Cohort A4: A single SC dose of 24.0 mg relaxin agonist (n=6) or matching placebo (n=2). Cohort A5: A single SC dose of 48.0 mg relaxin agonist (n=6) or matching placebo (n=2).

[0339] In this first-in-human study, all SAD cohort subjects were dosed according to a sentinel dosing design to ensure optimal safety. This meant that two subjects were dosed initially (one subject with relaxin agonist and one with placebo). If the safety and tolerability results after the first 48 hours of dosing for the first subject at the lowest dose level (cohort A1) were acceptable to the investigator, six other subjects (five active, one placebo) were also to be dosed. In cohorts A2-A5, safety and tolerability results after the first 24 hours of dose administration were evaluated in the first two subjects before dosing the rest of the cohorts.

[0340] In part B of the MAD of the study, a total of 24 subjects (n=8 per cohort, 6 to relaxin agonist, 2 to placebo) were enrolled sequentially (cohorts B1–B3), with subjects within a cohort enrolled in parallel. Cohorts B1–B3 will receive one of the following three SC doses: 5.0 mg, 15.0 mg, or 30.0 mg once daily (QD). Cohort B1: 5.0 mg SC relaxin agonist (n=6) or matching placebo (n=2) QD for 14 days Cohort B2: 15.0 mg SC relaxin agonist (n=6) or matching placebo (n=2) QD for 14 days Cohort B3: 30.0 mg SC relaxin agonist (n=6) or matching placebo (n=2) QD for 14 days

[0341] In summary, in Part A, single subcutaneous (sc) doses of 1.0 mg, 4.0 mg, 12.0 mg, 24 mg, and 48 mg and a single intravenous (iv) dose of 4.0 mg were administered. All doses were well tolerated and safe. All reported adverse events were mild (grade 1) and the majority resolved spontaneously. The most frequently reported adverse events included injection site erythema, injection site pruritus, and injection site burning sensation, all reported to be related to the study drug.

[0342] In Part B, single and multiple once-daily subcutaneous doses of 5.0 mg, 15.0 mg, and 30 mg were administered for 14 days. All doses were well tolerated and safe. All reported adverse events were mild (grade 1) and most resolved spontaneously. The most frequently reported adverse events included injection site erythema, injection site pruritus, and injection site burning, all reported to be related to the study drug and all resolved rapidly (mostly within the same day).

[0343] Clinical Pharmacokinetics of Research Preliminary PK results following subcutaneous (sc) administration of single ascending doses of relaxin agonist are summarized in Table 4. Exposure increased dose-proportional up to a dose of 12 mg relaxin agonist. Greater than dose-proportional exposure was observed following SC administration of 24 mg and 48 mg (approximately 15% higher than expected). [Table 8]

[0344] Preliminary PK results after a single intravenous (iv) dose of 4 mg of the relaxin agonist are summarized in Table 5. The Cmax and AUCinf observed after iv administration were approximately 3.5-fold and 1.3-fold higher compared to sc administration. Thus, after sc administration, the absolute bioavailability (F) is approximately 0.76. [Table 9]

[0345] Preliminary PK results after subcutaneous (sc) administration of single and multiple ascending doses of relaxin agonist are summarized in Table 6. Approximately 2.6-fold accumulation was observed on day 14 at all dose regimens (5 mg, 15 mg, and 30 mg). Exposure increased dose-proportionally up to the 30 mg dose regimen of relaxin agonist. Terminal half-lives were observed around 23-24 hours. [Table 10] [Table 11]

[0346] Renal plasma flow (PAH) Renal plasma flow (RPF) measurements were estimated based on para-aminohippuric acid (PAH) clearance. PAH measurements were performed only in the MAD (Part B) study. Baseline measurements were performed on day 1, and measurements on day 13 were performed at -2.5, -1, and -0.5 hours pre-dose and 4, 5.5, and 6 hours post-dose, which was the predicted Tmax.

[0347] Reference is made to Figures 3A and 3B, which show the change from baseline effective renal plasma flow following administration of a relaxin agonist. After multiple doses of relaxin agonist or placebo, RPF assessed using PAH biomarkers tended to increase in the active dose groups compared to placebo, with the effect being more pronounced at day 13 post-dose compared to day 13 pre-dose in all treatment groups.

[0348] The change from baseline in PAH clearance after multiple doses of 5 mg QD to 30 mg QD varied from 118 mL / min to 187 mL / min on day 13 pre-dose and from 212 mL / min to 223 mL / min on day 13 post-dose. There was no clear trend in the change from baseline in PAH clearance related to dose. The change from baseline in PAH clearance in the placebo group was lower compared with the active group, with the change from baseline PAH clearance being 40 mL / min on day 13 pre-dose and 13 mL / min on day 13 post-dose. Consistent with PAH clearance, the effective RPF (eRPF) was higher in the active dose group compared with the placebo group, with no clear relationship to dose.

[0349] Study design and safety results of the study This study is an open-label, Phase 1, multicenter, single-dose study in subjects with severe renal impairment and matched subjects with normal renal function. The preliminary evaluation included five subjects with severe renal impairment and two subjects with normal renal function.

[0350] Two adverse events were reported in one subject (palpitations and headache). One subject reported grade 1 (mild) palpitations possibly related to the study drug. An ECG was performed with no clinically significant abnormalities. No action was taken with study drug and the event resolved spontaneously. The same subject also reported a grade 1 headache, which was not related and no action was taken with study drug. This event also resolved spontaneously. In summary, five subjects with severe renal impairment and two subjects with normal renal function received a single subcutaneous dose of 4.0 mg of the relaxin agonist. All doses were well tolerated and safe. Two AEs were reported in subjects, both grade 1 (mild) and resolved spontaneously.

[0351] Clinical Pharmacokinetics of the Study This is a phase 1, multicenter, single-dose study in subjects with severe renal impairment and matched subjects with normal renal function. Five subjects with severe renal impairment and two subjects with normal renal function were included. PK sampling was performed pre-dose and 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 hours post-dose.

[0352] The mean Cmax in the severe renal impairment group is 391.8 ng / mL. The mean Cmax in the normal group is 302.0 and 355.7 in the SAD study after 4 mg. The mean AUCinf shows similar increases in the severe renal impairment group compared to the normal group and compared to the SAD 4 mg group. The mean Cmax increases by 10-30%. The mean AUCinf increases by 14-31%. [Table 12]

[0353] Example 5: Phase II study evaluating the safety, tolerability, efficacy, and pharmacokinetics of a relaxin agonist in combination with terlipressin This example describes a randomized, single-blind, controlled, two-arm, multicenter study in patients with hepatorenal syndrome (HRS)-acute kidney injury (AKI) after a safety run-in. The purpose of this randomized, single-blind, active-controlled study is to evaluate the safety and efficacy of a relaxin agonist (i.e., relaxin peptide analog of SEQ ID NO: 3) in combination with terlipressin compared to terlipressin alone in the treatment of patients with HRS-AKI. Efficacy will be assessed through the primary endpoint of reversal of established hepatorenal syndrome (HRS). Other key efficacy parameters will also be evaluated. Safety data will be collected and incorporated into the overall safety evaluation of the relaxin agonist.

[0354] In this study, responders are defined according to the International Club of Ascites (ICA) criteria. A complete responder is defined as a return of two serum creatinine levels within 0.3 mg / dL (26.5 micromol / L) of the baseline serum creatinine value, at least 2 hours apart. A partial responder is defined as a regression of at least one acute kidney injury (AKI) stage accompanied by a serum creatinine decrease of 0.3 mg / dL or more above the baseline serum creatinine value. An established reversal of HRS (clinical responder) is defined as a patient with a complete or partial response based on serum creatinine level and AKI stage, and who survives without renal transplant therapy (RRT) for at least 30 days after the start of initial treatment.

[0355] The main objectives of the study include: • To evaluate the tolerability and safety of relaxin agonists in combination with terlipressin versus terlipressin alone. • To evaluate the efficacy of combining a relaxin agonist with terlipressin versus terlipressin alone based on the number of responding patients (responder rates for established HRS reversal, considering complete and partial responses as separate outcome measures and combining these in a single arm). • To evaluate the efficacy of adding a relaxin agonist to terlipressin in terlipressin non-responders.

[0356] Secondary objectives of the study include: • To evaluate the efficacy of a relaxin agonist plus terlipressin versus terlipressin alone on mortality at 30, 60, and 90 days. • To evaluate the effectiveness of a relaxin agonist plus terlipressin versus terlipressin alone on liver transplantation rates at 30, 60, and 90 days. • To evaluate the effectiveness of a relaxin agonist in combination with terlipressin versus terlipressin alone on the incidence of RRT at 30, 60, and 90 days. • To evaluate the efficacy of a relaxin agonist in combination with terlipressin versus terlipressin alone on HRS response based on SCr / AKI stage (total response = complete + partial response, complete response, or partial response). • To evaluate the effectiveness of a relaxin agonist in combination with terlipressin versus terlipressin alone on the durability of established HRS reversal (number of patients responding and free of RRT by days 45, 60, and 90). • To evaluate the efficacy of a combination of relaxin agonist and terlipressin versus terlipressin alone on recurrence of HRS. • To evaluate the efficacy of a combination of a relaxin agonist and terlipressin versus terlipressin alone in preventing progression to stage 3 acute or chronic liver failure (ACLF) or reducing ACLF stage. • To evaluate the efficacy of a relaxin agonist in combination with terlipressin versus terlipressin alone on change in Model for End-Stage Liver Disease (MELD) score at 30, 60, and 90 days. • To evaluate the efficacy of a relaxin agonist in combination with terlipressin versus terlipressin alone on SCr / AKI stage and the number of patients who achieved complete and partial HRS response based on patients being alive and RRT-free 10 days after achieving HRS response (instead of 30 days from the start of treatment).

[0357] The exploratory objectives of the study include: • To assess changes from baseline in serum and urinary biomarkers (cystatin C, endothelin-1, von Willebrand factor (vWF), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM1)). • To evaluate the population pharmacokinetics (PK) of relaxin agonist in combination with terlipressin.

[0358] research design This is a randomized, single-blind, controlled, two-arm, multicenter study in patients with hepatorenal syndrome-acute kidney injury (AKI) followed by a safety run-in. Referring now to Figure 4, an exemplary study design for a Phase II study evaluating the safety, tolerability, efficacy, and pharmacokinetics of a relaxin agonist in combination with terlipressin is shown.

[0359] As shown in Figure 4, the study consists of the following: A. An open-label safety run-in section with three patient cohorts, followed by: B. A single-blind, placebo-controlled, randomized part with two patient cohorts treated in parallel; and C. Open-label terlipressin non-responder cohort.

[0360] All patients in all cohorts will be treated with terlipressin, administered as a bolus IV infusion of 1 mg every 6 hours, increasing to a maximum of 2.0 mg infusion every 6 hours if clinically appropriate. Terlipressin administration should be continued until 24 hours after achievement of HRS response (either partial or complete response) based on serum creatinine (SCr) / AKI stage or until day 14.

[0361] Referring now to Figure 5, there is shown the design of the open-label safety run-in portion of the overall study design shown in Figure 4, in which three initial cohorts of three patients each (labeled Cohorts 1, 2, and 3 in Figure 4) are treated open-label with a combination of terlipressin and a relaxin agonist to confirm its safety.

[0362] Cohort 1 (N=3) will receive terlipressin and 1.0 mg relaxin agonist iv over 4 hours, then 2.5 mg relaxin agonist sc 12 hours after IV initiation, 2.5 mg relaxin agonist sc at 24 hours, and then once daily thereafter until 24 hours after achievement of HRS response (either partial or complete) based on SCr / AKI stage or day 14. Cohort 1 will finish dosing before cohort 2 begins.

[0363] Cohort 2 (N=3) will receive terlipressin and 2.0 mg relaxin agonist IV over 4 hours, then 5.0 mg relaxin agonist SC 12 hours after IV initiation, 5.0 mg relaxin agonist SC at 24 hours, and the same doses once daily thereafter until 24 hours after achievement of HRS response (either partial or complete) based on SCr / AKI stage or day 14. Cohort 2 will finish dosing before cohort 3 begins.

[0364] Cohort 3 (N=3) received terlipressin and 4.0 mg of relaxin agonist IV over 4 hours, then 5.0 mg of relaxin agonist SC 12 hours after IV initiation, 10.0 mg of relaxin agonist SC at 24 hours, and then the same dose (10.0 mg) once daily thereafter until 24 hours after achievement of HRS response (either partial or complete) based on SCr / AKI stage or until day 14 (i.e., the same dose and schedule used for Cohort 4 in the randomization portion of the study).

[0365] Cohort 3 will complete dosing prior to the start of Part B (single-blind, placebo-controlled, randomized part). Based on the safety and tolerability of the various relaxin agonist dose schedules in Cohorts 1, 2, and 3, the SRC will determine the appropriate relaxin agonist dose schedule to proceed to Cohorts 4 and 5. This relaxin agonist schedule will be one of the three treatment schedules from Cohorts 1, 2, or 3.

[0366] Referring now to Figure 6, there is shown the design of the single-blind, placebo-controlled, randomized treatment portion of the overall study design depicted in Figure 4. After completion of the open-label safety run-in portion (shown in Figure 5) and after the appropriate relaxin agonist dose schedule has been determined, approximately 80 patients will be randomized 1:1 to relaxin agonist and terlipressin (Cohort 4), or terlipressin and placebo (Cohort 5). At randomization, patients will be stratified according to whether they have systemic inflammatory response syndrome (SIRS), because patients with SIRS respond better to terlipressin than patients without SIRS.

[0367] Provided that the highest dose schedule of the relaxin agonist (studied in cohort 3) is safe and well tolerated, the relaxin agonist will be administered to patients in cohort 4 as a 4.0 mg infusion over 4 hours on the first day, followed by 5.0 mg SC 12 hours after the start of the infusion, followed by 10.0 mg SC at the 24 hour time point, and then once daily thereafter until 24 hours after achievement of HRS response (either partial or complete) based on SCr / AKI stage or until day 14.

[0368] Patients in Cohort 4 may be treated with a lower dose schedule. In Cohort 5, patients will receive IV terlipressin and IV / SC placebo on the same schedule as Cohort 4.

[0369] Referring now to FIG. 7, the design of the open-label terlipressin non-responder portion of the overall study design shown in FIG. 4 is shown. Patients in cohort 5 who do not respond to terlipressin are discontinued. After discontinuation, patients can enter cohort 6 (the terlipressin non-responder portion) and receive relaxin agonist at the same dose and schedule as cohort 4. Patients in all cohorts other than cohort 5 are not eligible to enter cohort 6. Terlipressin non-responders are defined as follows: On day 4, if serum creatinine improves less than 10% or is at baseline level or above, the patient is considered a non-responder and drops out of cohort 5. These patients are eligible to enter cohort 6. All patients receive standard of care with albumin.

[0370] Study Inclusion Criteria Patients will be assessed for eligibility to participate in the study according to the following inclusion criteria: 1. AKI stage 2 or 3 (see Table 11; AKI is defined by either 1) an increase in SCr (SCr) ≥ 0.3 mg / dl (or ≥ 26.5 micromol / L) within 48 hours or 2) an increase of ≥ 50% above baseline SCr, known or presumed to have occurred within the past 7 days. 2. QLY SCr is ≥ 1.5 mg / dl. 3. No sustained improvement in renal function after 48 hours of discontinuing diuretics and initiating plasma volume expansion with albumin (<20% decrease in SCr and SCr=>1.5mg / dL). 4. Female patients and female partners of male patients must avoid pregnancy for the duration of the study (>90 days).

[0371] Exclusion criteria Patients who met any of the following exclusion criteria were ineligible to participate in the study: 1. Significant comorbidity determined by the investigator to prevent participation in the study. 2. QLY SCr level >5mg / dL. 3. AKI Stage 1 4. ACLF stage 3. 5. Model for End-Stage Liver Disease (MELD) score >35. 6. At least one large volume paracentesis (LVP) of >4 liters occurred during the 4 days prior to randomization. 7. Current or recent (within 4 weeks) treatment with nephrotoxic drugs (e.g., aminoglycosides, amphotericin, cyclosporine, NSAIDS (e.g., ibuprofen, naproxen, celecoxib), significant exposure to radiographic contrast agents (large or multiple injections of iodized contrast material). 8. Shock (hypovolemic, cardiogenic, or vasodilatory / distributive shock) and hypoperfusion with mean arterial blood pressure (MAP) ≤ 70mmHg or systolic blood pressure ≤ 90mmHg. 9. Sepsis or uncontrolled bacterial infection (e.g., persistent bacteremia, persistent ascites leukocytosis, fever, leukocytosis with vasomotor instability) as measured by the quick sepsis-related organ dysfunction assessment (qSOFA) score. 10. Anti-infective treatment for confirmed or suspected infections for less than 2 days. 11. Superimposed acute liver disease caused by drugs, herbal preparations, or dietary supplements, excluding alcoholic hepatitis. 12. Estimated life expectancy is less than 5 days. 13. Proteinuria >500mg / day. 14. Renal tubular epithelial casts, heme granule casts. 15. Hematuria or microhematuria (>50 red blood cells per high-power field). 16. Abnormal renal ultrasound unless there is no known chronic structural disease (e.g., diabetic or hypertensive nephropathy). 17. Current or recent (within 4 weeks) renal transplant therapy (RRT). 18. Severe cardiovascular and pulmonary disease, including but not limited to unstable angina, pulmonary edema, congestive heart failure requiring increased doses of medication, persistent symptomatic peripheral vascular disease, or any other cardiovascular disease deemed severe by the investigator. 19. Transjugular intrahepatic systemic shunt (TIPS) unless known to be non-functioning or obstructed. 20. Continuous use of vasopressors, including midodrine, unless used for only the 48 hours prior to screening, in which case an 8 hour washout period will be used prior to randomization. 21. Known allergy or hypersensitivity to terlipressin or any other component of the study treatment. 22. Subject is, in the opinion of the Investigator, unsuitable to participate in the study for any reason (including but not limited to comorbid conditions, history of non-compliance with study visits, procedures, or medications). 23. Women of childbearing potential (those who are not surgically sterilized or have not been postmenopausal for at least one year) will be excluded from participation in the study unless they agree to use an appropriate method of contraception as described in Section 11.3. 24. Unsterilized men whose female partners are of childbearing potential must agree to use highly effective methods of contraception from the time of signing the Informed Consent Form (ICF) until 90 days after the last dose of study drug. Male patients must agree to notify the investigator immediately if their partner becomes pregnant during the study.

[0372] research treatment Treatment details for the various cohorts in the study design are shown in Table 9 below. [Table 13]

[0373] Regarding terlipressin dosing across all cohorts, all patients in all cohorts (1-6) will be treated with terlipressin administered as a 1.0 mg bolus IV infusion (1 mg over 2 minutes) every 6 hours. Terlipressin administration should continue until 24 hours after achievement of HRS response (either partial or complete) based on SCr / AKI stage or until day 14.

[0374] Regarding terlipressin dose modifications across all cohorts, if the SCr decreases to <25% of the QLY SCr (i.e., <25% of the SCr value at the time the patient was randomized) on day 3 (after 2 days of terlipressin treatment) and terlipressin demonstrates an acceptable safety profile, terlipressin may be increased to 8.0 mg per day (i.e., 2.0 mg every 6 hours). The dose should not be increased in patients with coronary artery disease or if there is circulatory overload, pulmonary edema, or bronchospasm. If dosing is interrupted due to an adverse event (AE), terlipressin may be resumed at the same or lower dose per protocol at the investigator's discretion. Dosing should not be resumed if the AE consists of cardiac or mesenteric ischemia.

[0375] Regarding the definition of terlipressin non-responders (applicable only to cohort 5), if SCr is reduced to <10% of QLY SCr or at a level equal to or greater than QLY SCr on day 4, patients will be considered non-responders and will be dropped from cohort 5. These patients will be eligible to enter cohort 6.

[0376] For combination therapy with terlipressin and a relaxin agonist, in cohorts where terlipressin is administered in combination with a relaxin agonist (cohorts 1, 2, 3, 4, and 6), the first relaxin agonist administration will begin immediately after the first terlipressin administration. The relaxin agonist should be administered up to 24 hours or until day 14 after achievement of HRS response (either partial or complete) based on SCr / AKI stage.

[0377] Prior Treatment Period (Cohorts 1, 2, 3, 4, and 5) The pretreatment period occurs before administration of study drug and includes performing baseline assessments and collecting past medication information. A qualifying SCr value (a SCr value at least 48 hours after both discontinuation of diuretics and initiation of albumin loading) is considered the QLY SCr value and is derived within 8 hours of initiating study drug. The QLY SCr value should be ≥ 1.5 mg / dL. A subject should not be randomized unless their QLY SCr is obtained within 8 hours prior to randomization and study drug initiation. If there is a delay in randomizing a subject, the QLY SCr value is rederived such that the value is collected within 8 hours prior to randomization and study drug initiation to verify that the subject still meets the inclusion criteria for QLY SCr. Other baseline assessments are performed within 24 hours prior to initiating study drug.

[0378] Active Study Period (All Cohorts) The active study period will extend from the start of study treatment through day 14 or until discharge for any reason, whichever occurs first. Investigational drug will be administered as described in the study design above.

[0379] Terlipressin administration should continue until 24 hours after achievement of HRS response based on SCr / AKI stage or until day 14. A second SCr value will be obtained a minimum of 2 hours after the first SCr value after the first SCr level has returned to within 0.3 mg / dl (26.5 micromol / L) of the BL SCr value or after the SCr has decreased by ≥ 0.3 mg / dl below the BL SCr value and the AKI stage has regressed. Efforts will be made to collect these SCr values. Any information regarding RRT, TIPS, liver transplant, or open-label vasopressor use will be collected.

[0380] The follow-up period begins after completion of study treatment and ends 90 days after treatment initiation. All subjects return for follow-up on day 30 (± 2) and are contacted by telephone for follow-up on days 60 (± 7) and 90 (± 7) to assess survival, RRT, TIPS, and liver transplant status. Study days are counted from the first day of study drug administration (or from randomization for subjects not receiving study drug). In addition, during the day 30 follow-up, a physical examination is performed and updated data on medical history, vital signs, concomitant medications, and evaluation of SAEs are collected.

[0381] Efficacy assessments will be assessed as follows: Serum creatinine will be collected once daily at baseline during treatment, then once daily (regardless of treatment status) until day 14 or discharge, whichever occurs first. If SCr assessments are performed multiple times per day as part of the subject's medical care, all values ​​obtained each day will be recorded in the eCRF. SCr values ​​obtained after RRT, TIPS, liver transplant, or use of open-label vasopressors will be excluded from efficacy assessments.

[0382] Main efficacy variables included: ● Safety and tolerability will be assessed by the occurrence of AEs, changes in physical examination, vital signs, ECG, and clinical laboratory parameters. • Incidence of responders assessed separately and combined as two distinct outcome groups (established HRS reversal defined as patients with complete or partial HRS response (based on SCr / AKI stage) and surviving without renal transplant therapy (RRT) for at least 30 days after the first dose of study drug). ● Patients undergoing liver transplantation within the first 30 days after initiating treatment will be evaluated for pre-transplant SCr and AKI stage and will be considered responders if they meet the SCr / AKI stage criteria for HRS response (complete or partial) before liver transplantation, are alive at 30 days after initiating treatment, and do not have RRT. If there is relapse and retreatment within the first 30 days, the response during the second treatment period will be evaluated.

[0383] Secondary efficacy variables included: - The number of patients who died at 30, 60, and 90 days (mortality). • Number of patients with kidney and / or liver transplant rates at 30, 60, and 90 days. • Number of patients receiving RRT at 30, 60, and 90 days. • Number of patients with sustained HRS reversal (number of patients who responded by days 45, 60, and 90 and were free of RRT). • Number of patients with recurrent HRS. • Number of patients whose ACLF1 or 2 progressed to stage 3. Mean change from baseline in MELD score at 30, 60, and 90 days • Number of responding patients with established HRS reversal (i.e., via the CONFIRM study) (clinical definition of which is patients alive and RRT-free 10 days after achieving HRS response).

[0384] Exploratory evaluation criteria include: - Mean change from baseline in serum and urinary biomarkers (including cystatin C, endothelin-1, vWF, NGAL, and KIM1) Population pharmacokinetic analysis can be used to evaluate primary PK parameters (e.g., absorption rate (ka), apparent clearance (Cl / F), and apparent volume of distribution (V / F)) and secondary PK parameters (maximum concentration (C max ), C max Time (t max ), minimum plasma concentration (C min ), the area under the concentration-time curve over the dosing interval (AUC 0-24h ), elimination half-life (t 1 / 2 ), and accumulation rate (Racc).

[0385] Safety assessment Physical Examination: A physical examination will be performed by the Investigator or designee, including evaluation of the following: head, ears, nose, and throat, neck / thyroid, extremities, and the cardiovascular, integumentary, lymphatic, nervous, musculoskeletal, and respiratory systems will be evaluated at specific time points. Any clinically significant changes in physical examination findings during the study will be considered an AE and recorded on the eCRF.

[0386] Vital Signs: Assessment of vital signs (heart rate, blood pressure, respiratory rate, temperature, height, and weight will be recorded.

[0387] Safety / 12-Lead ECG: 12-lead ECGs will be performed three times at specific time points, 1 minute apart. ECG results will be recorded in the eCRF as normal or abnormal. All abnormal results will be assessed by the Investigator as either clinically significant or clinically insignificant. New abnormal clinically significant ECG results will be recorded as an AE. A copy of each ECG tracing will remain with the original documentation.

[0388] Clinical Safety Laboratory Evaluation: All protocol specified laboratory tests will be performed at the central laboratory, except for urine pregnancy test, serum creatinine, CBC, INR, and electrolytes. Detailed instructions for collection, handling, and reporting of clinical test samples will be provided to the site in a laboratory manual provided by the central laboratory prior to the start of the site.

[0389] If an abnormal clinically significant laboratory result occurs, the abnormal test may be repeated, if necessary, to confirm the validity of the abnormal result. If an abnormal clinically significant abnormal result is valid, the laboratory test(s) will be repeated every 2 weeks until the result is within normal range or is no longer deemed clinically significant by the investigator. Any new abnormal clinically significant laboratory result will be recorded as an AE.

[0390] Laboratory evaluation included: Hemoglobin, Hematocrit, RBC, MCV, MCH, MCHC, Reticulocytes, Haptoglobin, WBC with differential, Platelet count and platelet aggregation studies, BUN, Serum creatinine, Cystatin C, AST, ALT, Alkaline phosphatase, LDH, Total bilirubin, Indirect and direct bilirubin, Sodium, Potassium, Chloride, Calcium, Phosphate, Glucose, Total protein, Albumin, Total cholesterol, LDL, HDL, Triglycerides, C-reactive protein, Uric acid, Cortisol ACTH, Lactate, Ammonia, Urinalysis: Specific gravity (females only), Protein, Blood, Ke ton, glucose, assessment of multiple organ dysfunction (e.g. based on CLIF-SOFA score), encephalopathy score, systemic inflammatory response syndrome (SIRS) assessment, rapid sepsis-related organ dysfunction assessment (qSOFA) (calculated using three criteria: low systolic blood pressure (≦100 mmHg), high respiratory rate (≧22 breaths per minute), and altered mental status (<15 on the Glasgow Coma Scale), each assigned one point), and model for end-stage liver disease score (based on SCr, bilirubin, and INR values).

[0391] Sequence Listing SEQ ID NO:1 Ac-LEGREKVRAK(Ac)-I-Aib-Aib-EGK(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- WS-Aib-RKK(Ac)-NH2 SEQ ID NO:2 Ac-LEGREKVRAK(Ac)-I-Aib-Aib-EGK(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- FS-Aib-RAK(Ac)-NH2 SEQ ID NO:3 Ac-LEGREKVRAK(Ac)-I-Aib-Aib-EGK(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- FS-Mly-RAK(Ac)-NH2 SEQ ID NO:4 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-Palm)-S-T-W-- S- Aib-R-K-K(Ac)-NH2 Sequence number 5 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2-PEG2-PEG2-P- EG2-gE-gE-gE-Palm)-S- T-W-S-Aib-R-K-K(Ac)-NH2 Sequence number 6 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-Stea)-S-T-W-- S- Aib-R-K-K(Ac)-NH2 Sequence number 7 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Stea)-S- -T-F- S-Mly-R-A-K(Ac)-NH2 Sequence number 8 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2-PEG2-PEG2-P- EG2-gE-gE-gE-Palm)-S- T-F-S-Mly-R-A-K(Ac)-NH2 Sequence number 9 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-Palm)-S-T-F-S-M- ly-R- A-K(Ac)-NH2 Sequence number 10 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-gE-Palm)-S-T- -F-S- Mly-R-A-K(Ac)-NH2 Sequence number 11 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Mly-R-A-K(Ac)-NH2 Sequence number 12 Ac-L-E-G-R-E-K-V-R-A-Cit-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S-T- -F-S- Mly-R-A-K(Ac)-NH2 Sequence number 13 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-S-K-R-A-K(Ac)-NH2 Sequence number 14 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-Dser-K-R-A-K(Ac)-NH2 Sequence number 15 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-Aib-K-R-A-K(Ac)-NH2 Sequence number 16 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-Palm)-S-T-F-- S- Dlys-R-A-K(Ac)-NH2 Sequence number 17 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-Palm)-S-T-W-- S- Dlys-R-A-K(Ac)-NH2 Sequence number 18 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-Palm)-S-T-W-- S- Dlys-R-K-K(Ac)-NH2 Sequence number 19 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2-PEG2-PEG2-g- E-gE-gE-Palm)-S-T-W- S-Aib-R-K-K(Ac)-NH2 Sequence number 20 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Stea)-S- -T- W-S-Aib-R-K-K(Ac)-NH2 Sequence number 21 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2-PEG2-PEG2-P- EG2-gE-gE-gE-Stea)-S- T-W-S-Aib-R-K-K(Ac)-NH2 Sequence number 22 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2DGA-PEG2DGA-PEG.s- ub.2DGA-gE-gE- gE-Palm)-S-T-F-S-Mly-R-A-K(Ac)-NH2 Sequence number 23 Ac-L-E-G-R-E-L-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T-F- S-Mly-R-A-K(Ac)-NH2 Sequence number 24 Ac-L-E-G-R-E-F-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T-F- S-Mly-R-A-K(Ac)-NH2 Sequence number 25 Ac-L-E-G-R-E-Q-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-S-Mly-R-A-K(Ac)-NH2 Sequence number 26 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2-PEG2-PEG2-PEG2-gE-gE-gE-St- ea)-S- T-F-S-Mly-R-A-K(Ac)-NH2 Sequence number 27 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-S-Mly-R-A-K(Ac)-NH2 Sequence number 28 Ac-L-E-G-R-E-Hly-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)- -S-T- F-S-Mly-R-A-K(Ac)-NH2 Sequence number 29 Ac-L-E-G-R-E-K-V-R-Aib-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)- -S-T- F-S-Mly-R-A-K(Ac)-NH2 Sequence number 30 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Stea)-S- -T- W-S-Mly-R-A-K(Ac)-NH2 Sequence number 31 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Aib-R-R-K(Ac)-NH2 Sequence number 32 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2DGA-PEG2DGA-PEG.s- ub.2DGA-gE-gE- gE-Stea)-S-T-F-S-Mly-R-A-K(Ac)-NH2 Sequence number 33 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-Palm)-S-T-F-- S- Mly-R-A-K(Ac)-NH2 Sequence number 34 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-Stea)-S-T-F-- S-Mly- R-A-K(Ac)-NH2 Sequence number 35 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-Aib-K-R-A-K(Ac)-NH2 Sequence number 36 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-S-Dlys-R-A-K(Ac)-NH2 Sequence number 37 Ac-L-E-G-R-E-L-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Aib-R-R-K(Ac)-NH2 Sequence number 38 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2-PEG2-PEG2-g- E-gE-gE-Palm)-S-T-F- S-Mly-R-A-K(Ac)-NH2 Sequence number 39 Ac-L-E-G-R-E-F-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Aib-R-R-K(Ac)-NH2 Sequence number 40 Ac-L-E-G-R-E-Q-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Aib-R-R-K(Ac)-NH2 Sequence number 41 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2DGA-PEG2DGA-PEG.s- ub.2DGA-gE-gE- gE-Palm)-S-T-W-S-Mly-R-A-K(Ac)-NH2 Sequence number 42 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2-PEG2-PEG2-P- EG2-gE-gE-gE-Palm)-S- T-W-S-Mly-R-A-K(Ac)-NH2 Sequence number 43 Ac-L-E-G-R-E-Hly-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Stea)- -S-T- W-S-Mly-R-A-K(Ac)-NH2 Sequence number 44 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-Dser-K-R-A-K(Ac)-NH2 Sequence number 45 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2-PEG2-PEG2-P- EG2-PEG2-gE-gE-gE- Palm)-S-T-F-S-Mly-R-A-K(Ac)-NH2 Sequence number 46 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2-PEG2-PEG2-P-EG2-PEG2-gE-gE-gE-gE-Palm)-S-T-F-S-Mly-R-A-K(Ac)-NH2 Sequence number 47 Ac-L-E-G-R-E-K-V-R-A-K-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S-T-F--S-Mly-R-A-K(Ac)-NH2 Sequence number 48 Ac-L-E-G-R-E-K-V-R-A-Q-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S-T-F--S-Mly-R-A-K(Ac)-NH2 Sequence number 49 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S--T-F-S-Mly-R-A-K-NH2 Sequence number 50 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2DGA-PEG2DGA-PEG.s-ub.2DGA-gE-gE-gE-Stea)-S-T-W-S-Aib-R-K-K(Ac)-NH2 Sequence number 51 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-gE-Stea-)-S-T-W-S-Aib-R-K-K(Ac)-NH2 Sequence number 52 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-Stea)-S-T--W-S-Aib-R-K-K(Ac)-NH2 Sequence number 53 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(PEG2DGA-PEG2DGA-PEG.sub.2DGA-gE-gE-gE-gE-Stea)-S-T-W-S-Aib-R-K-K(Ac)-NH2 Sequence number 54 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-gE-Stea)-S-T-W-S-Aib-R-K-K(Ac)-NH2SEQ-ID-NO: 55 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S--T-W-S-Aib-R-R-K(Ac)-NH2 Sequence number 55 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S-T-W-S-Aib-R-K-K(Ac)-NH2 Sequence number 56 Ac-L-E-G-R-E-K-V-R-A-Cit-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Stea)-S-T--F-S-Mly-R-A-K(Ac)-NH2 Sequence number 57 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S--T-W-S-Hly-R-A-K(Ac)-NH2 Sequence number 58 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-TTDS-gE-gE-gE-St-ea)-S-T-W-S-Aib-R-K-K(Ac)-NH2 Sequence number 59 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-TTDS-gE-gE-gE-St- ea)- S-T-F-S-Aib-R-K-K(Ac)-NH2 Sequence number 60 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-gE-gE-gE-Stea)-S-T-F-- S-Aib- R-K-K(Ac)-NH2 Sequence number 61 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Stea)-S- -T-F- S-Aib-R-K-K(Ac)-NH2 Sequence number 62 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-gE-Palm- )-S- T-W-S-Aib-R-K-K(Ac)-NH2 Sequence number 63 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -Q- W-S-Aib-R-K-K(Ac)-NH2 Sequence number 64 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -R- W-S-Aib-R-K-K(Ac)-NH2 Sequence number 65 Ac-L-E-G-R-E-K-V-R-K(Ac)-Q-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Aib-R-K-K(Ac)-NH2 Sequence number 66 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-K(Ac)-Aib-R-A-K(Ac)-NH2 Sequence number 67 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Aib-R-K-K(Ac)-NH2 Sequence number 68 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Aib-R-K(Ac)-K-NH2 Sequence number 69 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Aib-R-K(Ac)-R-NH2 Sequence number 70 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-I-E-G-K(TTDS-TTDS-TTD S-gE-gE-gE-Palm)-S-T-W- S-Aib-R-K-K(Ac)-NH2 Sequence number 71 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- W-S-Aib-R-A-K-NH2 Sequence number 72 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-Aib-R-R-A-K(Ac)-NH2 Sequence number 73 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -K- W-S-Aib-R-A-K-NH2 Sequence number 74 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-I-E-G-K(TTDS-TTDS-TTD S-TTDS-gE-gE-gE-Palm)-S- T-W-S-Aib-R-K-K(Ac)-NH2 Sequence number 75 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-TTD S-gE-gE-gE- Palm)-S-T-W-S-Aib-R-R-K(Ac)-NH2 Sequence number 76 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-S-Aib-R-R-K(Ac)-NH2 Sequence number 77 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-TTDS-gE-gE-gE- Palm)-S-T-F-S-Aib-R-R-K(Ac)-NH2 Sequence number 78 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-TTDS-gE-gE-gE- Palm)-S-T-F-S-Mly-R-A-K(Ac)-NH2 Sequence number 79 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -K- F-S-Aib-R-K-K(Ac)-NH2 Sequence number 80 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- Trp(5-C1)-S-Aib-R-R-K(Ac)-NH2 Sequence number 81 Ac-K(Ac)-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Pal- m)- S-T-F-S-Mly-R-A-K(Ac)-NH2 Sequence number 82 Ac-L-E-G-R-E-K-V-R-Aib-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)- -S-T- W-S-Aib-R-A-K(Ac)-NH2 Sequence number 83 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -K- W-S-Aib-R-K-K(Ac)-NH2 Sequence number 84 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-I-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S-T- -F-S- Mly-R-A-K(Ac)-NH2 Sequence number 85 Ac-L-E-G-R-E-R-V-R-Aib-K(Ac) I Aib Aib E G K(TTDS-TTDS-TTDS-gE-gE-gE-Palm) S T W S Aib R R K(Ac)-NH, Sequence number 86 Ac-Aib-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-I-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T-F- S-Mly-R-A-K(Ac)-NH2 Sequence number 87 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-S-R-R-A-K(Ac)-NH2 Sequence number 88 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-S-Hly-R-A-K(Ac)-NH2 Sequence number 89 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- Mph-S-R-R-A-K(Ac)-NH2 Sequence number 90 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-A-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S-T- -F- S-Mly-R-A-K(Ac)-NH2 Sequence number 91 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- Pfp-S-Aib-R-R-K(Ac)-NH2 Sequence number 92 Ac-L-E-G-R-E-R-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- Trp(5-F)-S-Aib-R-R-K(Ac)-NH2 Sequence number 93 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-T-R-R-A-K(Ac)-NH2 Sequence number 94 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Palm)-S- -T- F-V-R-R-A-K(Ac)-NH2 Sequence number 95 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Myr)-S-- T-F- S-Mly-R-A-K(Ac)-NH2 Sequence number 96 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-Myr)-S-- T-F- S-Mly-R-A-K-NH2 Sequence number 97 Ac-L-E-G-R-E-K-V-R-A-K(Ac)-I-Aib-Aib-E-G-K(TTDS-TTDS-TTDS-gE-gE-gE-gE-Myr)- -S- T-F-S-Mly-R-A-K-NH2 Sequence number 100 = H2-relaxin chain A H-Gln-Leu-Tyr-Ser-Ala-Leu-Ala-Asn-Lys-Cys-Cys-His-Val-Gly-Cys-Thr-Lys-Arg-- Ser-Leu- Ala-Arg-Phe-Cys-OH Sequence number 101 = H2-relaxin chain B H-Asp-Ser-Trp-Met-Glu-Glu-Val-Ile-Lys-Leu-Cys-Gly-Arg-Glu-Leu-Val-Arg-Ala-- Gln-Ile-Ala- Ile-Cys-Gly-Met-Ser-Thr-Trp-Ser-OH Sequence number 102 = Sequence number B7-33 C11.235 * H-Val-Ile-Lys-Leu-Ser-Gly-Arg-Glu-Leu-Val-Arg-Ala-Gln-Ile-Ala-Ile-Ser-Gly-- Met-Ser-Thr- Trp-Ser-Lys-Arg-Ser-Leu-NH2 SEQ ID NO:103 = SEQ ID NO:AcB7-33 C11.235 * Ac-Val-Ile-Lys-Leu-Ser-Gly-Arg-Glu-Leu-Val-Arg-Ala-Gln-Ile-Ala-Ile-Ser-Gly- -Met-Ser-Thr- Trp-Ser-Lys-Arg-Ser-Leu-NH2 SEQ ID NO:104 = SEQ ID NO:KKKK (AcB7-29 C11.235) * Ac-Val-Ile-Lys-Leu-Ser-Gly-Arg-Glu-Leu-Val-Arg-Ala-Gln-Ile-Ala-Ile-Ser-Gly- -Met-Ser-Thr- Trp-Ser-Lys-Lys-Lys-Lys-NH2

[0392] Other embodiments It is understood that the words which have been used are words of description rather than of limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

[0393] While the present invention has been described in some detail with respect to certain illustrated embodiments, it is not intended that the invention be limited to such details or embodiments, or to specific embodiments, but should be interpreted by reference thereto. Reference should be made to the appended claims, which should be interpreted as broadly as possible in view of the prior art, so as to effectively encompass the intended scope of the invention.

[0394] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. A relaxin analog and a vasopressin analog for use in a method for preventing or treating renal failure or hepatorenal syndrome in an individual in need thereof, comprising co-administering effective amounts of a relaxin analog and a vasopressin analog to the individual, wherein optionally the relaxin analog is an RXFP1 agonist, or preferably a long-acting peptidyl RXFP1 agonist, and optionally the vasopressin analog is a V1a receptor agonist.

2. The relaxin analog and vasopressin analog for use according to claim 1, wherein the renal failure is selected from the group consisting of renal dysfunction induced by liver cirrhosis, renal dysfunction induced by liver transplantation, chronic kidney disease, and acute kidney injury, or the hepatorenal syndrome is HRS-AKI (type 1 hepatorenal syndrome).

3. 2. The relaxin analog and vasopressin analog for use according to claim 1, wherein the vasopressin analog is terlipressin or a pharmaceutically acceptable salt thereof, and optionally the terlipressin is administered intravenously at a dose of 0.5 to 2 mg per dose, optionally every 4 to 6 hours, and / or via intravenous infusion.

4. 2. The relaxin analog and vasopressin analog for use according to claim 1, wherein the individual is further administered midodrine or octreotide.

5. the relaxin analog has an EC50 of activated RXFP1 of less than 15 nM, less than 1 nM, less than 0.5 nM, or less than 0.1 nM in an in vitro OVCAR5 cAMP assay. 50 2. The relaxin analogue and vasopressin analogue for use according to claim 1, wherein

6. The relaxin analog and vasopressin analog for use according to claim 1, wherein the relaxin analog is administered at a dose of about 0.01 mg / kg to about 0.5 mg / kg and / or the relaxin analog is administered parenterally, intravenously, subcutaneously, rectally, transdermally, or by inhalation.

7. The relaxin analog and vasopressin analog for use according to claim 1, wherein the relaxin analog and the vasopressin analog are administered simultaneously, sequentially, in a single composition, or in separate compositions.

8. Relaxin analogs and vasopressin analogs for use in a method for treating renal failure in an individual in need thereof or for treating hepatorenal syndrome in an individual with cirrhosis, the method comprising administering the relaxin analog to the individual who has previously been administered a vasopressin analog.

9. A relaxin analogue and a vasopressin analogue for use in a method for treating renal failure in an individual in need thereof or for treating hepatorenal syndrome in an individual with cirrhosis, the method comprising administering a vasopressin analogue to the individual who has previously been administered a relaxin analogue.

10. The relaxin analog has formula (I) (SEQ ID NO: 105): N ter -Ac-X 10 -E-G-R-E-X 15 -V-R-X 18 -X 19 -I-X 21 -X 22 -E-G-X 25 -S-X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 -NH 2 -C ter 1. A modified relaxin B chain peptide comprising: During the ceremony, N ter represents the N-terminus of the peptide, C ter represents the C-terminus of the peptide, Ac represents an acetyl group; X 10 represents an amino acid selected from the group consisting of leucine, 2-amino-isobutyric acid, Nε-acetyl-lysine, and α-methyl-leucine; E represents glutamic acid, G represents glycine, R represents arginine, X 15 represents an amino acid selected from the group consisting of lysine, arginine, homolysine, homoarginine, ornithine, glutamine, phenylalanine, and leucine; V represents valine, X 18 represents an amino acid selected from the group consisting of alanine, 2-amino-isobutyric acid, leucine, Nε-acetyl-lysine, and glutamine; X 19 represents an amino acid selected from the group consisting of lysine, Nε-acetyl-lysine, citrulline, glutamine, alanine, and 2-amino-isobutyric acid; I represents isoleucine, X 21 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and alanine, and X 22 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and isoleucine; X 25 represents the following structure: 【Chemical 1】 During the ceremony, * is X in formula (I). 25 represents a covalent bond to the preceding glycine, 【Chemistry 2】 is X in formula (I). 25 represents a covalent bond to serine followed by Z represents a group of formula (II), -[(PEG xx ) b (gE) c C d ]、 b and c independently represent 1, 2, 3, 4, or 5, optionally b represents 2, 3, 4, or 5 and c represents 2, 3, or 4, or a salt or solvate thereof; PEG xx are independently PEG 2 , PEG 2 DGA, and TTDS; gE stands for gamma-glutamic acid; C d is a linear saturated C 12 ~C 22 represents an acyl group, S represents serine, X 27 represents an amino acid selected from the group consisting of threonine, lysine, arginine, and glutamine; X 28 represents an amino acid selected from the group consisting of tryptophan, phenylalanine, 5-fluoro-tryptophan, 5-chloro-tryptophan, 5-methoxy-tryptophan, tyrosine, 4-fluoro-phenylalanine, 1-naphthylalanine, 2-naphthylalanine, α-methyl-tryptophan, α-methyl-phenylalanine, and 5-hydroxy-tryptophan; X 29 represents an amino acid selected from the group consisting of serine, D-serine, 2-amino-isobutyric acid, threonine, α-methyl-serine, Nε-acetyl-lysine, and valine; X 30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, α-methyl-lysine, D-lysine, lysine, homolysine, ornithine, arginine, and α-methyl-arginine; X 31 represents an amino acid selected from the group consisting of arginine, Nω-methyl-arginine, alanine, Nω,Nω′-dimethyl-arginine, and citrulline; X 32 is lysine, alanine, arginine, Nε-acetyl-lysine, and Nε,Nε,N represents an amino acid selected from the group consisting of ε-tri-methyl-lysine, X 33 represents an amino acid selected from lysine, Nε-acetyl-lysine, leucine, arginine, and alanine; 10. The relaxin analogue and vasopressin analogue for use according to any one of claims 1 to 9, which is a compound selected from the group consisting of benzodiazepine, benzodiazepine, benzophenone, benzodiazepine ...

11. In the formula, C d But C 12 (Lau), C 14 (Myr), C 15 (Penta), C 16 (Palm), C 17 (Hepta), and C 18 (Stea) represents a linear saturated acyl group selected from the group consisting of acyl groups, or C d But linear C 16 or C 18 Relaxin analogues and vasopressin analogues for use according to claim 10, which represent an acyl group, or a salt or solvate thereof.

12. The relaxin analog has the formula (Ib): N ter -Ac-X 10 -E-G-R-E-X 15 -V-R-X 18 -X 19 -I-X 21 -X- 22 -E-G-X 25 -S-X 27 -X 28 -X 29 -X 30 -R-X 32 -X 33 -NH 2 -C ter (SEQ ID NO: 107), During the ceremony, N ter represents the N-terminus of the peptide, C ter represents the C-terminus of the peptide, Ac represents an acetyl group; X 10 represents an amino acid selected from the group consisting of leucine, Nε-acetyl-lysine, and 2-amino-isobutyric acid; E represents glutamic acid, G represents glycine, R represents arginine, X 15 represents an amino acid selected from the group consisting of lysine, arginine, homolysine, glutamine, phenylalanine, and leucine; V represents valine, X 18 represents an amino acid selected from the group consisting of alanine, 2-amino-isobutyric acid, and Nε-acetyl-lysine; X 19 represents an amino acid selected from the group consisting of lysine, Nε-acetyl-lysine, glutamine, and citrulline, and I represents isoleucine; X 21 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and alanine, and X 22 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and isoleucine; X 25 represents the following structure: 【Chemistry 3】 During the ceremony, * is X in formula (Ia) 25 represents a covalent bond to the preceding glycine, 【Chemistry 4】 is X in formula (Ia) 25 represents a covalent bond to serine followed by Zは、-(TTDS) 2 -(gE) 3 -Palm、-(TTDS) 3 -(gE) 3 -Palm、-(PEG) 2 DGA) 3 -(gE) 3 -Palm、-(PEG) 2 , 4 -(gE) 3 -Palm、-(TTDS) 2 -(gE) 2 -Palm、-(TTDS) 2 -(gE) 3 -Stea、-(TTDS) 3 -(gE) 3 -Stea、-(PEG 2 DGA) 3 -(gE) 3 -Stea、-(PEG 2 DGA) 3 -(gE) 4 -Stea、-(PEG 2 , 3 -(gE) 3 -Palm、-(PEG) 2 , 4 -(gE) 3 -Stea、-(PEG 2 , 5 -(gE) 3 -Palm、-(PEG) 2 , 5 -(gE) 4 -Palm、-(TTDS) 3 -(gE) 4 -Stea、-(TTDS) 2 -(gE) 4 -Palm、-(TTDS) 3 -(gE) 2 -Stea、-(TTDS) 2 -(gE) 4 -Stea、-(TTDS) 4 -(gE) 3 -Stea、-(TTDS) 3 -(gE) 4 -Palm、-(TTDS) 4 -(gE) 3 -Palm、-(TTDS) 3 -(gE) 3 -Myr, and -(TTDS) 3 -(gE) 4 -Myr, wherein gE represents γ-glutamic acid, Palm represents palmitoyl, and Stea represents stearoyl; S represents serine, X 27 represents an amino acid selected from the group consisting of threonine, glutamine, arginine, and lysine; X 28 represents an amino acid selected from the group consisting of tryptophan, phenylalanine, 5-chlorotryptophan, α-methyl-phenylalanine, 4-fluoro-phenylalanine, and 5-fluorotryptophan; X 29 represents an amino acid selected from the group consisting of serine, D-serine, 2-amino-isobutyric acid, Nε-acetyl-lysine, threonine, and valine; X 30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, α-methyl-lysine, D-lysine, lysine, homolysine, and arginine; X 32 represents an amino acid selected from the group consisting of lysine, alanine, arginine, and Nε-acetyl-lysine; X 33 represents an amino acid selected from lysine, Nε-acetyl-lysine, and arginine, or a salt or solvate thereof.

13. the relaxin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-97; or the relaxin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 7, 9-12, 20-22, 26, 28, 30-34, 45, 47-49, 51, 54-62, 64, 67-69, 71-86, 91, 93, and 96; or the relaxin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 7, 20, 26, 30-34, 45, 48, 49, 51, 54-61, 67, 71, 73, 75-79, 81, 83-92, and 97; or the relaxin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:20; or The relaxin analog comprises the amino acid sequence of SEQ ID NO:

3. Relaxin analogues and vasopressin analogues for use according to claim 10.

14. A pharmaceutical composition comprising, separately or together, a relaxin analog, a vasopressin analog, and one or more pharmaceutically acceptable excipients, optionally wherein the vasopressin analog is A pharmaceutical composition wherein said relaxin analog is a V1a agonist or terlipressin, and / or said relaxin analog is an RXFP1 peptidyl agonist.

15. A kit comprising a relaxin analog in a pharmaceutically acceptable composition and a vasopressin analog in a pharmaceutically acceptable composition.

16. 10. The relaxin analog and vasopressin analog for use in the method of claim 9, wherein the individual has previously been considered a non-responder to the vasopressin analog.

17. A relaxin analog for use in a method for preventing or treating renal failure or treating hepatorenal syndrome in an individual in need thereof, wherein the relaxin analog is administered to the individual intravenously at a dose of about 4.0 mg and subcutaneously at a dose of about 5.0 mg on the same day, and on a different, later day, the relaxin analog is administered subcutaneously at a dose of about 10 mg.

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