Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/2221—Relaxins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/64—Relaxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
  • A61K38/095—Oxytocins; Vasopressins; Related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present disclosure relates to a combination therapy comprising coadministration of (a) a relaxin analogue able to activate the RXFP1 receptor, for example peptide analogues of the B-chain of human relaxin-2, and (b) an analogue of vasopressin (also termed arginine vasopressin (A VP), antidiuretic hormone (ADH), and agripressin) able to activate the VI receptor, for example terlipressin, administered to an individual in need thereof in the treatment of a renal disorder, such as renal dysfunction in cirrhosis, hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI), chronic kidney disease and acute kidney injury, and to preserve renal function in peri-operative liver transplantation.
• a relaxin analogue able to activate the RXFP1 receptor for example peptide analogues of the B-chain of human relaxin-2
• vasopressin also termed arginine vasopressin
• compositions comprising relaxin analogues and/or vasopressin analogues 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, and commercial packages thereto.
• Terlipressin is a synthetic vasopressin that is approved in many countries outside of the United States to treat the life-threatening complications of cirrhosis, including hepatorenal syndrome (HRS) and esophageal bleeding (EVB). Its use is limited to the hospital setting due to its short half-life (Nilsson, et al., (1990) Drugs Explt Clin. Res., XVI (6):307-314), and it is typically administered as an intravenous bolus usually every 4 to 6 hours.
• HRS hepatorenal syndrome
• EVB esophageal bleeding
• terlipressin may have clinical utility
• a recent study found that at 3 months, mortality in patients receiving terlipressin and standard of care (e.g., albumin) was 51%, as compared to 45% for patients receiving standard of care alone (Wong, F. et al., Terlipressin plus Albumin for the Treatment of Type 1 Hepatorenal Sydrome, NEJM (2021), 384: 818-828.
• terlipressin can cause side effects in up to 40% of patients.
• a method of preventing or treating renal failure in an individual in need thereof comprising co-administering an effective amount of a relaxin analogue and a vasopressin analogue to the individual.
• 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.
• hepatorenal syndrome is HRS-AKI (hepatorenal syndrome type 1).
• the relaxin analogue is an RXFP1 agonist.
• the relaxin analogue is a long-acting peptidyl RXFP1 agonist.
• the vasopressin analogue is a Via receptor agonist.
• the vasopressin analogue is terlipressin or a pharmaceutically acceptable salt thereof.
• the terlipressin is administered intravenously at a dosage of 0.5 to 2 mg per administration. In some embodiments, the terlipressin is administered intravenously at a dosage of 0.5 to 2 mg every 4 to 6 hours. In some embodiments, the terlipressin is administered via an intravenous infusion. In some embodiments, the terlipressin is administered at a rate of 0.5 to 2 mg per 4 to 6 hours. In some embodiments, the terlipressin is administered at a rate of 0.5 to 2 mg per 8 to 36 hours. In some embodiments, the terlipressin is administered at a rate of 0.5 to 2 mg per 10 to 30 hours.
• the terlipressin is administered at a rate of 0.5 to 2 mg per 15 to 28 hours. In some embodiments, the terlipressin is administered at a rate of 0.5 to 2 mg per 20 to 25 hours. In some embodiments, the terlipressin is administered at a rate of 0.5 to 2 mg per 24 hours.
• the relaxin analogue is administered at a dose of from about 0.01 mg/kg to about 0.5 mg/kg. In some embodiments, the relaxin analogue is administered, parenterally, intravenously, subcutaneously, rectally, transdermally, or by inhalation. In some embodiments, the relaxin analogue has an EC50 for activation RXFP1 in the in vitro OVCAR5 cAMP assay of less than 15 nM, less than 1 nM, less than 0.5 nM, or less than 0.1 nM.
• the method of preventing or treating further comprises administration of midodrine or octreotide to the individual.
• the relaxin analogue and the vasopressin analogue are administered simultaneously. In some embodiments, the relaxin analogue and the vasopressin analogue are administered in a single composition. In some embodiments, the relaxin analogue and the vasopressin analogue are administered in a separate composition. In some embodiments, the vasopressin analogue and the relaxin analogue are administered sequentially.
• the combination therapy has a synergistic therapeutic effect.
• the administration of the relaxin analogue mitigates adverse effects associated with vasopressin analogue treatment in the individual.
• the administration of the vasopressin analogue reduces risk of hypotension in said individual associated with treatment with the relaxin analogue.
• the administration of the vasopressin analogue or relaxin analogue increases renal pressure in said individual.
• a method of treating renal failure in an individual in need thereof comprising: administering a relaxin analogue to the individual, wherein the individual previously has been administered a vasopressin analogue.
• a method of treating hepatorenal syndrome in an individual with liver cirrhosis comprising a) administering a relaxin analogue to the individual, wherein the individual previously has been administered a vasopressin analogue.
• the administration of the relaxin analogue mitigates adverse effects associated with vasopressin analogue treatment in the individual.
• a method of treating renal failure in an individual in need thereof comprising: administering a vasopressin analogue to the individual, wherein the individual previously has been administered a relaxin analogue.
• a method of treating hepatorenal syndrome in an individual with liver cirrhosis comprising a) administering a vasopressin analogue to the individual, wherein the individual previously has been administered an effective amount of a relaxin analogue.
• the administration of the vasopressin analogue reduces risk of hypotension in said individual associated with treatment with the relaxin analogue. In some embodiments, the administration of the vasopressin analogue or relaxin analogue increases renal pressure in said individual.
• the hepatorenal syndrome is HRS-AKI (hepatorenal syndrome type 1).
• a pharmaceutical composition comprising separately or together a relaxin analogue, a vasopressin analogue, and one or more pharmaceutically acceptable excipients.
• kits comprising a relaxin analogue in a pharmaceutically acceptable composition and a vasopressin analogue in a pharmaceutically acceptable composition.
• the relaxin analogue is an RXFP1 agonist. In some embodiments, the relaxin analogue is a long-acting peptidyl RXFP1 agonist. In some embodiments, the vasopressin analogue is a Via receptor agonist. In some embodiments, the vasopressin analogue is terlipressin or a pharmaceutically acceptable salt thereof.
• the relaxin analogue is a modified relaxin B chain peptide comprising formula (I) (SEQ ID NO: 105):
• Nter represents the N-terminal end of the peptide
• Cter represents the C-terminal end of the peptide
• Xio represents an amino acid selected from the group consisting of leucine, 2-amino- isobutyric acid, Ns-acetyl-lysine and a-methyl-leucine;
• E represents glutamic acid
• G represents glycine
• R represents arginine
• X15 represents an amino acid selected form the group consisting of lysine, arginine, homolysine, homoarginine, ornithine, glutamine, phenylalanine and leucine;
• V represents valine
• Xis represents an amino acid selected from the group consisting of alanine, 2-amino- isobutyric acid, leucine, Ns-acetyl-lysine and glutamine;
• X19 represents an amino acid selected from the group consisting of lysine, Ns-acetyl- lysine, citrulline, glutamine, alanine and 2-amino-isobutyric acid;
• I represents isoleucine
• X21 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and alanine;
• X22 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and isoleucine;
• X25 represents the following structure: in which:
• PEGxx independently represents a polyethylene glycol derivative selected from the group consisting of PEG2, PEG2DGA, and TTDS;
• gE represents y-glutamic acid;
• Cd represents a linear saturated C12-C22 acyl group
• X27 represents an amino acid selected from the group consisting of threonine, lysine, arginine and glutamine;
• X28 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-naphtylalanine, 2-naphtylalanine, a-methyl- tryptophan, a-methyl-phenylalanine and 5 -hydroxy -tryptophan;
• X29 represents an amino acid selected from the group consisting of serine, D-serine, 2-amino-isobutyric acid, threonine, a-methyl-serine, Ns-acetyl-lysine and valine;
• X30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, a-methyl-lysine, D-lysine, lysine, homolysine, ornithine, arginine and a-methyl- arginine;
• X31 represents an amino acid selected from the group consisting of arginine, No-methyl-arginine, alanine, No,No’-dimethyl-arginine and citrulline;
• X32 represents an amino acid selected from the group consisting of lysine, alanine, arginine, Ns-acetyl-lysine and Ns,N8,Ns-tri-methyl-lysine;
• X33 represents an amino acid selected from lysine, Ns-acetyl-lysine, leucine, arginine and alanine; or a salt or solvate thereof.
• Cd represents a linear saturated C12-C22 acyl group, for example a linear saturated acyl group selected from the group consisting of C12 (Lau), C14 (Myr), C15 (Penta), Ci6 (Palm), C17 (Hepta), Cis (Stea), C20 (Eico) and C22 (Doco) acyl group.
• Cd represents a linear saturated acyl group selected from the group consisting of C12 (Lau), C14 (Myr), C15 (Penta), Ci6 (Palm), C17 (Hepta) or Cis (Stea) acyl group, for example a linear saturated C14, Ci6 or Cis acyl group, or for example a linear Ci6 or Cis acyl group; or a salt or solvate thereof.
• Cd represents a linear saturated acyl group selected from the group consisting of: C12 (Lau), C14 (Myr), C15 (Penta), Ci6 (Palm), C17 (Hepta) amd Cis (Stea) acyl group; or a salt or solvate thereof;
• Cd represents a linear Ci6 or Cis acyl group; or a salt or solvate thereof.
• the relaxin analogue comprises formula (lb): Nter-Ac-Xio-E-G-R-E-Xi 5 -V-R-Xi8-Xi9-I-X 2 i-X- 22-E-G-X25-S-X27-X28-X29-X30-R-X32- X 3 3-NH 2 -Cter, wherein:
• Nter represents the N-terminal end of the peptide
• Cter represents the C-terminal end of the peptide
• X10 represents an amino acid selected form the group consisting of leucine, Ns-acetyl- lysine and 2-amino-isobutyric acid;
• E represents glutamic acid
• G represents glycine
• R represents arginine
• X15 represents an amino acid selected form the group consisting of lysine, arginine, homolysine, glutamine, phenylalanine and leucine;
• V represents valine
• Xis represents an amino acid selected from the group consisting of alanine, 2-amino- isobutyric acid and Ns-acetyl-lysine;
• X19 represents an amino acid selected from the group consisting of lysine, Ns-acetyl- lysine, glutamine and citrulline; I represents isoleucine;
• X21 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and alanine
• X22 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and isoleucine
• X25 represents the following structure: in which:
• * represents a covalent bond with the glycine preceding X25 in formula (la); .
• + represents a covalent bond with the serine following X25 in formula (la); and Z is selected from the group consisting of a -(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
• X27 represents an amino acid selected from the group consisting of threonine, glutamine, arginine and lysine;
• X28 represents an amino acid selected from the group consisting of tryptophan, phenylalanine, 5-Chlorotryptophan, a-Methyl-phenylalanine, 4-Fluoro-phenylalanine and 5 -Fluorotryptophan;
• X29 represents an amino acid selected from the group consisting of serine, D-serine, 2- amino-isobutyric acid, Ns-acetyl-lysine, threonine and valine;
• X30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, a-methyl-lysine, D-lysine, lysine, homolysine and arginine;
• X32 represents an amino acid selected from the group consisting of lysine, alanine, arginine and Ns-acetyl-lysine; and X33 represents an amino acid selected from lysine, Ns-acetyl-lysine and arginine; or a salt or solvate thereof.
• the relaxin analogue comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 to 97.
• Z is selected from the group consisting of: -(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 )3-(gE)3-Palm, -(PEG 2 )4-(gE) 3 -Stea, -(PEG 2 ) 5 -(gE)3-Palm, -(TTDS) 3 -(gE) 4 -Stea,
• the relaxin analogue comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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.
• the relaxin analogue comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 6, 7, 20, 26, 30-34, 45, 48, 49, 51, 54- 61, 67, 71, 73, 75-79, 81, 83-92 and 97.
• the relaxin analogue 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.
• the relaxin analogue comprises the amino acid sequence of SEQ ID NO: 3.
• a method of preventing or treating renal failure in an individual in need thereof comprising co-administering a dose of a relaxin analogue from about 1.0 mg to about 10.0 mg and an effective amount of a vasopressin analogue to the individual.
• 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.
• hepatorenal syndrome is HRS-AKI (hepatorenal syndrome type 1).
• the relaxin analogue is an RXFP1 agonist. In various embodiments, the relaxin analogue is a long-acting peptidyl RXFP1 agonist. [0038] In various embodiments, from about 1.0 mg to about 3.0 mg of the relaxin analogue is administered to the individual. In various embodiments, about 1.0 mg of the relaxin analogue is administered to the individual. In various embodiments, about 2.0 mg of the relaxin analogue is administered to the individual. In various embodiments, from about 3.0 mg to about 5.0 mg of the relaxin analogue is administered to the individual. In various embodiments, about 4.0 mg of the relaxin analogue is administered to the individual.
• from about 5.0 mg to about 10.0 mg of the relaxin analogue is administered to the individual. In various embodiments, about 5.0 mg of the relaxin analogue is administered to the individual. In various embodiments, about 10.0 mg of the relaxin analogue is administered to the individual. In various embodiments, the relaxin analogue is administered intravenously. In various embodiments, the relaxin analogue is administered intravenously over from about 1 hour to about 10 hours. In various embodiments, the relaxin analogue is administered intravenously over from about 2 hours to about 8 hours. In various embodiments, the relaxin analogue is administered intravenously over from about 3 hours to about 6 hours. In various embodiments, the relaxin analogue is administered intravenously over about 4 hours.
• methods disclosed herein further comprise administering an additional dose of the relaxin analogue to the individual.
• the additional dose of the relaxin analogue is administered between 5 hours and 18 hours after administration of the about 1.0 mg to about 5.0 mg of the relaxin analogue.
• the additional dose of the relaxin analogue is administered between 8 hours and 15 hours after administration of the dose from about 1.0 mg to about 10.0 mg of the relaxin analogue.
• the additional dose of the relaxin analogue is administered between 10 hours and 13 hours after administration of the dose from about 1.0 mg to about 10.0 mg of the relaxin analogue.
• the additional dose of the relaxin analogue is administered about 12 hours after administration of the dose from about 1.0 mg to about 10.0 mg of the relaxin analogue. In various embodiments, the additional dose of the relaxin analogue is administered subcutaneously to the individual. In various embodiments, the additional dose of the relaxin analogue comprises from about 1 mg to about 50 mg of the relaxin analogue. In various embodiments, the additional dose of the relaxin analogue comprises from about 5 mg to about 15 mg of the relaxin analogue. In various embodiments, the additional dose of the relaxin analogue comprises from about 8 mg to about 12 mg of the relaxin analogue. In various embodiments, the additional dose of the relaxin analogue comprises about 10 mg of the relaxin analogue.
• methods disclosed herein further comprise administering a yet additional dose of the relaxin analogue to the individual.
• the yet additional dose of the relaxin analogue is administered between 18 hours and 30 hours after administration of the about 1.0 mg to about 5.0 mg of the relaxin analogue.
• the yet additional dose of the relaxin analogue is administered between 20 hours and 26 hours after administration of the dose from about 1.0 mg to about 10.0 mg of the relaxin analogue.
• the yet additional dose of the relaxin analogue is administered about 24 hours after administration of the dose from about 1.0 mg to about 10.0 mg of the relaxin analogue.
• the yet additional dose of the relaxin analogue is administered subcutaneously to the individual. In various embodiments, the yet additional dose of the relaxin analogue comprises from about 1 mg to about 50 mg of the relaxin analogue. In various embodiments, the yet additional dose of the relaxin analogue comprises from about 2 mg to about 15 mg of the relaxin analogue. In various embodiments, the yet additional dose of the relaxin analogue comprises from about 3 mg to about 8 mg of the relaxin analogue. In various embodiments, the yet additional dose of the relaxin analogue comprises about 5 mg of the relaxin analogue. In various embodiments, the yet additional dose of the relaxin analogue comprises from about 2 mg to about 5 mg of the relaxin analogue.
• the yet additional dose of the relaxin analogue comprises about 2.5 mg of the relaxin analogue. In various embodiments, the yet additional dose of the relaxin analogue comprises from about 6 mg to about 15 mg of the relaxin analogue. In various embodiments, the yet additional dose of the relaxin analogue comprises from about 8 mg to about 12 mg of the relaxin analogue. In various embodiments, the yet additional dose of the relaxin analogue comprises about 10 mg of the relaxin analogue.
• methods disclosed herein further comprise administering daily the yet additional dose of the relaxin analogue.
• the yet additional dose of the relaxin analogue is administered daily from about 10 days to about 20 days.
• the yet additional dose of the relaxin analogue is administered daily from about 12 days to about 16 days.
• the yet additional dose of the relaxin analogue is administered daily for about 14 days or more.
• the yet additional dose of the relaxin analogue is administered daily from about 3 days to about 15 days.
• the yet additional dose of the relaxin analogue is administered daily from about 4 days to about 13 days.
• the yet additional dose of the relaxin analogue is administered daily from about 5 days to about 11 days.
• the yet additional dose of the relaxin analogue is administered daily from about 6 days to about 9 days.
• the relaxin analogue has an ECso for activation RXFP1 in the in vitro OVCAR5 cAMP assay of less than 15 nM, less than 1 nM, less than 0.5 nM, or less than 0.1 nM.
• the vasopressin analogue is a Via receptor agonist.
• the vasopressin analogue is terlipressin or a pharmaceutically acceptable salt thereof.
• the terlipressin is administered intravenously at a dosage from about 0.5 to about 10 mg.
• the terlipressin is administered intravenously at a dosage from about 0.5 to about 2 mg every 4 to 6 hours.
• the terlipressin is administered intravenously at a dosage of about 1 mg every 6 hours. In various embodiments, the terlipressin is administered intravenously at a dosage from about 2 to about 6 mg every 4 to 6 hours. In various embodiments, the terlipressin is administered intravenously at a dosage of about 4 mg every 6 hours. In various embodiments, the terlipressin is administered intravenously at a dosage from about 6 to about 10 mg every 4 to 6 hours. In various embodiments, the terlipressin is administered intravenously at a dosage of about 6 mg every 6 hours. In various embodiments, the terlipressin is administered intravenously at a dosage of about 8 mg every 6 hours.
• terlipressin is administered intravenously via a bolus injection. In various embodiments, terlipressin is administered intravenously via a bolus injection over from about 1 minute to about 5 minutes. In various embodiments, terlipressin is administered intravenously via a bolus injection over from about 2 minutes to about 3 minutes. In various embodiments, terlipressin is administered intravenously via a bolus injection over about 2 minutes. In various embodiments, the terlipressin is administered intravenously at a dosage from about 1 mg to about 10 mg. In various embodiments, the terlipressin is administered intravenously at a dosage from about 1.5 mg to about 4 mg.
• the terlipressin is administered intravenously at a dosage of about 2 mg. In various embodiments, the terlipressin is administered intravenously at a dosage from about 5 mg to about 8 mg. In various embodiments, the terlipressin is administered intravenously at a dosage of about 6 mg. In various embodiments, the terlipressin is administered intravenously over from about 10 hours to about 30 hours. In various embodiments, the terlipressin is administered intravenously over from about 20 hours to about 25 hours. In various embodiments, the terlipressin is administered intravenously over about 24 hours. [0043] In various embodiments, methods disclosed herein further comprise administering midodrine or octreotide to the individual.
• methods disclosed herein further comprise administering albumin to the individual.
• the dose of the relaxin analogue and the vasopressin analogue are administered simultaneously.
• the dose of the relaxin analogue and the vasopressin analogue are administered in a single composition.
• the dose of the relaxin analogue and the vasopressin analogue are administered in separate compositions.
• the dose of the vasopressin analogue and the relaxin analogue are administered sequentially.
• the combination therapy has a synergistic therapeutic effect.
• the combination therapy achieves an improved response rate incidence, wherein responders are defined according to at least International Club of Acites (ICA) criteria.
• responders comprise full or partial responders, as defined according to ICA criteria, and are alive without renal replacement therapy (RRT) for at least 30 days after start of treatment.
• responders comprise full or partial responders, as defined according to ICA criteria, and are alive without renal replacement therapy (RRT) for at least 10 days after start of treatment.
• full responders are defined as two serum creatinine levels returning to a value within 0.3 mg/dL (26.5 micromolar/L) of a baseline serum creatinine value at least 2 hours apart.
• partial responders are defined as a regression of at least 1 acute kidney injury (AKI) stage with a reduction of serum creatinine greater than or equal to 0.3 mg/dL above a baseline serum creatinine value.
• the combination therapy achieves an improved response rate incidence, wherein response rate incidence is measured according to a return of serum creatinine to a value within 0.3 mg/dL (26.5 micro molar/L) of a baseline value.
• the combination therapy achieves an improved response rate incidence, wherein response rate incidence is measured according to a regression of acute kidney injury (AKI) stage with a reduction of serum creatinine greater than or equal to 0.3 mg/dL above a baseline value.
• the combination therapy achieves an improved response rate incidence, wherein response rate incidence is measured according to two consecutive serum creatinine values at least 2 hours apart being below 1.5 mg/dL.
• the administration of the relaxin analogue mitigates adverse effects associated with vasopressin analogue treatment in the individual.
• the administration of the vasopressin analogue reduces risk of hypotension in the individual associated with treatment with the relaxin analogue.
• the administration of the vasopressin analogue increases renal pressure in the individual.
• a method of preventing or treating renal failure in an individual in need thereof comprising: A) intravenously administering to the individual about a 4.0 mg dose of a relaxin analogue; B) on a same day that step (A) is performed, subcutaneously administering to the individual about a 5.0 mg dose of the relaxin analogue; and C) on a subsequent day different from the day that steps (A) and (B) are performed, subcutaneously administering, to the individual, about a 10 mg dose of the relaxin analogue.
• a method of preventing or treating hepatorenal syndrome in an individual in need thereof comprising: A) intravenously administering to the individual about a 4.0 mg dose of a relaxin analogue; B) on a same day that step (A) is performed, subcutaneously administering to the individual about a 5.0 mg dose of the relaxin analogue; and C) on a subsequent day different from the day that steps (A) and (B) are performed, subcutaneously administering, to the individual, about a 10 mg dose of the relaxin analogue.
• methods disclosed herein further comprise repeating step (c) on a daily basis for up to 13 days.
• methods disclosed herein further comprise: on the same day that step (A) and step (b) are performed, intravenously administering a 1 mg bolus of terlipressin every 6 hours. In various embodiments, methods disclosed herein further comprise: repeating intravenously administering a 1 mg bolus of terlipressin every 6 hours for up to 14 days.
• FIGS. 1 and 2 are the two parts of a schema representing the general method used for synthesizing relaxin peptide analogues.
• FIGs. 3 A and 3B show the change from baseline effective renal plasma flow following administration of Relaxin Agonist.
• FIG. 4 depicts an overall study design of a Phase II Trial Assessing Safety, Tolerability, Efficacy, and Pharmacokinetics of Relaxin Agonist in Combination wth Terlipressin.
• FIG. 5 depicts a design of the open label safety run-in part of the overall study design shown in FIG. 4.
• FIG. 6 depicts a design of the single-blind placebo-conrolled randomized treatment part of the overall study design shown in FIG. 4.
• FIG. 7 depicts a design of the open-label terlipressin non-responder part of the overall study design shown in FIG. 4.
• X y in relaxin peptide analogue formulas is used with y having different values represents an amino acid as defined in the definition of said formulae, y indicates the position of said amino acid in the native B-chain of relaxin-2.
• Xio represents the amino acid in position 10 of the amino acid sequence of the native B-chain of relaxin-2.
• the term "pharmaceutically acceptable carrier” is intended for a fluid, especially a liquid comprising a pharmaceutical compound or combination of pharmaceutical compounds of the invention, such that the pharmaceutical composition is physiologically tolerable, i.e., can be administered to the individual body without toxicity or undue discomfort.
• a “relaxin analogue” refers to a peptide or other compound that is a functional variant of relaxin, e.g., that is able to activate the RXFP1 receptor, and/or a structural analogue of native relaxin-2, e.g., a modified relaxin B chain peptide.
• relaxin analogues can include small molecules that are RXFP1 receptor agonists or display RXFP1 agonistic properties.
• relaxin analogues can also include other modalities such as relaxin coupled to Fc fragments, and RXFP1 agonists such as nanobodies, or monoclonal antibodies.
• vasopressin analogue refers to prodrugs of vasopressin, and peptides structurally similar to vasopressin and/or functional variants of vasopressin, e.g., terlipressin (triglycyl lysine vasopressin), argipressin, desmopressin, felypressin, lypressin, or ornipressin.
• Vasopressin analogues can activate vasopressin receptors Via, V2, and/or V3 (also called Vlb). Small molecule vasopressin analogues are also known to those of ordinary skill in the art and are included within the term ‘vasopressin analogue,’ as used herein.
• agonist in the present context refers to a peptide or small molecule as defined herein, capable of binding to and activating a receptor.
• Full agonists bind to and activate a receptor with the maximum response that an agonist can elicit at the receptor.
• Partial agonists also bind and activate a given receptor but have partial efficacy at the receptor relative to a full agonist, even at maximal receptor occupancy.
• a selective agonist is selective for a specific type or subtype of receptor.
• a “functional variant” of a peptide is a peptide capable of performing essentially the same functions as the peptide it is a functional variant of.
• a functional variant can bind the same molecules, preferably with a similar affinity, as the peptide it is a functional variant of.
• Preventing is intended to mean reducing the risk of manifestation of the phenomenon under consideration. This reduction may be total or partial, i.e., results in a degree of risk that is lower than that pre-existing the use according to the invention.
• 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 if 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 if there is at least 5% improvement, preferably 10% improvement, more preferably at least 25%, even more preferably at least 50%, such as at least 75%, and most preferably at least 100% improvement.
• the change can be based on improvements in the severity of the treated condition in an individual, or on a difference in the frequency of improved conditions in populations of individuals with and without treatment with the bioactive agent or bioactive agents.
• a treatment according to the invention can be prophylactic, ameliorating and/or curative.
• a “bioactive agent” i.e., a biologically active substance/agent
• a biologically active substance/agent is any agent, drug, compound, composition of matter or mixture which provides some pharmacologic, often beneficial, effect that can be demonstrated in vivo or in vitro. It refers to the peptide sequences defined herewith, compounds or compositions comprising these and nucleic acid constructs encoding said peptides. As used herein, this term further includes any physiologically or pharmacologically active substance that produces a localized or systemic effect in an individual.
• a ‘bioactive agent’ as used herein denotes collectively a peptide, a nucleic acid construct encoding said peptide, and a composition comprising a peptide.
• “Pharmacologically effective amount”, “pharmaceutically effective amount”, “physiologically effective amount”, or “effective amount” of a “bioactive agent” is the amount of a bioactive agent or combination of bioactive agents present in one or more pharmaceutical compositions as described herein that is needed to provide a desired level of active agent or agents in the bloodstream or at the site of action in an individual (e.g. the hepatic system, the renal system, the circulatory systyem, the lungs, the gastrointestinal system, the colorectal system, etc.) to be treated to give an anticipated physiological response when such composition is administered.
• an individual e.g. the hepatic system, the renal system, the circulatory systyem, the lungs, the gastrointestinal system, the colorectal system, etc.
• Co-administering or “co-administration” as used herein refers to the administration of two or more bioactive agents.
• the two or more components can be administered separately, sequentially, or simultaneously.
• the term “individual” refers to vertebrates, particular members of the mammalian species, preferably primates including humans. As used herein, ‘subject’ and ‘individual’ may be used interchangeably. Treatment of animals, such as mice, rats, dogs, cats, cows, horses, sheep and pigs, is, however, also within the scope of the present invention.
• an “individual in need thereof’ refers to an individual who may benefit from treatment.
• said individual in need thereof is a diseased individual, wherein said disease may be a renal disorder.
• Reference to "about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X” includes description of "X.”
• a method of preventing or treating a renal disorder e.g., hepatorenal syndrome (HRS)
• a renal disorder e.g., hepatorenal syndrome (HRS)
• HRS hepatorenal syndrome
• a vasopressin analogue such as terlipressin, functions as a vasoconstrictor.
• Terlipressin causes vasoconstriction in the splanchnic circulation, and also in the systemic circulation.
• terlipressin has a poor safety profile.
• splanchnic vasoconstrictors such as terlipressin . . .
• Terlipressin has limited selectivity for splanchnic vasoconstriction over systemic vasoconstriction including hepatic and renal vasoconstriction, which would further exacerbate HRS.
• use of splanchnic vasoconstrictors such as terlipressin can result in various adverse effects that could compromise kidney function and patient safety, and is therefore not optimal for treatment of renal dysfunction in cirrhosis.
• a relaxin analogue such as a long-lasting peptidyl RXFP1 agonist disclosed herein, functions as a vasodilator with a degree of functional selectivity for renal vasodilation.
• vasoconstrictor e.g., a vasopressin analogue, such as terlipressin
• vasodilator e.g., a relaxin analogue
• a pharmaceutical composition comprising, separately or together, a relaxin peptide analogue and a vasopressin analogue, for use in a method of treating a renal disorder, for example HRS, such as hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI), or preserving renal function in perioperative liver transplantation.
• HRS hepatorenal syndrome type 1
• HRS-NAKI type 2
• a pharmaceutical composition comprising, separately or together, a peptide analogue of the B-chain of relaxin-2 able to activate the RXFP1 receptor and a vasopressin analogue able to activate the vasopressin VI (Via) receptor, for use in a method of treating a renal disorder, such as HRS, e.g., hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI), or preserving renal function in peri-operative liver transplantation.
• a renal disorder such as HRS, e.g., hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI
• a pharmaceutical composition comprising, separately or together, a relaxin peptide analogue and terlipressin, for use in a method of treating a renal disorder, including HRS, such as hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI), or preserving renal function in peri-operative liver transplantation.
• HRS such as hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI
• a pharmaceutical composition comprising, separately or together, a peptide analogue of the B-chain of relaxin-2 able to activate the RXFP1 receptor and terlipressin, for use in a method of treating a renal disorder, including HRS, such as hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI), or preserving renal function in peri-operative liver transplantation.
• HRS such as hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI
• a method of treating a renal disorder including hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI), comprising administering a therapeutically effective amount of a composition comprising, separately or together, a relaxin peptide analogue, such as peptide analogue of the B-chain of relaxin-2 able to activate the RXFP1 receptor, and a vasopressin analogue, such as terlipressin able to activate the VI (Via) receptor, to an individual in need thereof.
• HRS-AKI hepatorenal syndrome type 1
• HRS-NAKI type 2
• a composition comprising, separately or together, a relaxin peptide analogue, such as peptide analogue of the B-chain of relaxin-2 able to activate the RXFP1 receptor, and a vasopressin analogue, such as terlipressin able to activate the VI (Via) receptor, to an individual in need thereof.
• the renal disorder is renal dysfunction in cirrhosis, including HRS, such as hepatorenal syndrome type 1 (HRS-AKI) and type 2 (HRS-NAKI), and/or perioperative liver transplantation, chronic kidney disease and acute kidney injury.
• HRS hepatorenal syndrome type 1
• HRS-NAKI type 2
• Vasopressin Analogues such as Terlipressin
• vasopressin analogues such as terlipressin are used in combination with relaxin analogues for treatment of disorders associated with renal dysfunction.
• a vasopressin analogue is terlipressin, argipressin, desmopressin, felypressin, lypressin, or omipressin.
• the vasopressin analogue is terlipressin, or a pharmaceutically acceptable salt of terlipressin.
• Terlipressin also known as triglycyl lysine vasopressin
• Terlipressin is a synthetic analogue of the human neuropeptide hormone vasopressin.
• Terlipressin is a prohormone of lysine-vasopressin (triglycyl lysine vasopressin TGLVP), for example as described in Rittig et al., Movement Disorders, 1991, Vol. 6(1), p 21-28.
• terlipressin itself has weak intrinsic vasopressive activity but is transformed to the fully active lysine vasopressin (LVP) by endothelial endopeptidases.
• LVP lysine vasopressin
• Terlipressin is also known by its tradenames Teripress and Glypressin.
• Terlipressin has a molecular weight of 1227.37 g/mol and is represented by the formula:
• Terlipressin administered intravenously has been used, for example as a vasoactive drug in the management of hypotension (low blood pressure) and for example for treatment of bleeding esophageal varices, septic shock, HRS and ascites.
• terlipressin is available in two forms: lyophilized powder for reconstitution or as a liquid (0.2 milligrams/mL) in vials.
• the lyophilized version is typically supplied in vials containing 1 mg terlipressin powder for reconstitution using the provided ampoule of 5 mL of saline solution (e.g., Glypressin, Ferring Pharmaceuticals is supplied as one vial containing 1 mg terlipressin acetate for reconstitution in 5 mL solution) to deliver 0.17 mg/mL terlipressin (0.2 mg/mL terlipressin acetate) solution for injection.
• saline solution e.g., Glypressin, Ferring Pharmaceuticals
• Administering this product requires two or three prior steps: reconstituting the powder with diluent, withdrawing the solution, and possible further dilution, then injection by slow bolus dose directly into the patient or into the patient’s intravenous line or IV bag.
• Glypressin requires refrigerated storage at a temperature of 2°C to 8°C.
• the liquid, terlipressin acetate 0.2 milligrams/mL solution for injection (Terlipressin acetate, Ever Pharma) is also not stable at room temperature (RT) and requires refrigerated storage at a temperature of 2°C to 8°C. It is supplied in vials containing 5 mL or 10 mL of solution. This solution is withdrawn into a syringe for administration via bolus injection. Current formulations use acetic acid to adjust the pH of the terlipressin acetate solution.
• aqueous composition comprising terlipressin acetate or a pharmaceutically acceptable salt thereof, wherein the composition comprises from about 0.2 to about 10.0 mg/ml terlipressin acetate or a pharmaceutically acceptable salt thereof, wherein the pH of the composition is from about 3.4 to about 5.0.
• aqueous refers to a solution in which the solvent is water.
• the solvent can be sterile water suitable for injection.
• the solvent can be bacteriostatic water.
• the solvent can be mixtures of water with other pharmaceutically acceptable solvents or pharmaceutically acceptable alcohols or other bacteriostatic agents (e.g., benzyl alcohol).
• the concentration of terlipressin in the liquid 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.
• the composition comprises from 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, from 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, terlipressin acetate or a pharmaceutically acceptable salt thereof.
• the pH of the composition can be from about 3.4 to about 5.0, from about 3.5 to about 5.0, from about 3.6 to about 5.0, from about 3.7 from about 5.0, from about 3.8 to about 5.0, from about 3.9 to about 5.0, from about 4.0 to about 5.0, from about 4.1 to about 5.0, from about 4.2 to about 5.0, from about 4.3 to about 5.0, from about 4.4 to about 5.0, from about 4.5 to about 5.0, from about 4.6 to about 5.0, from about 4.7 to about 5.0, from about 4.8 to about 5.0, or from about 4.9 to about 5.0.
• the pH of the composition can 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.
• the composition can be for any route of drug administration, e.g., buccal, nasal, transdermal (e.g., patch technology), parenteral, intravenous, intramuscular or subcutaneous injection, intracistemal, intraperitoneal.
• the composition is for intravenous administration, e.g., by continuous infusion or by a bolus IV dose.
• the methods described herein provide about 1 to about 1000 pg/mL terlipressin concentration in a subject. In some embodiments, the methods described herein provide about 10 to about 600 pg/mL terlipressin concentration in a subject. In some embodiments, the methods described herein provide about 20 to about 200 pg/mL terlipressin concentration in a subject. In some embodiments, the methods described herein provide about 60 pg/mL terlipressin concentration in a subject.
• the methods described herein provide about 1 to about 200 pg/mL (lysine-)vasopressin concentration in a subject; where (lysine-)vasopressin is the active metabolite of terlipressin. [0092] In some embodiments, the methods described herein provide about 2 to about 100 pg/mL vasopressin analogue concentration in a subject, e.g., in the plasma of the subject. In some embodiments, the methods described herein provide about 3 to about 30 pg/mL vasopressin analogue concentration in a subject, e.g., in the plasma of the subject. In some embodiments, the methods described herein provide about 10 pg/mL vasopressin analogue concentration in a subject, e.g., in the plasma of the subject.
• the method comprises administration up to 2 times per day. In some embodiments, the method comprises administration up to 3 or 4 times per day.
• the dosage regimen utilizing the compounds is selected in accordance with a variety of factors including age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
• Another aspect of this invention is a method for treating a subject suffering from ascites (e.g., caused by liver cirrhosis), the method comprising administration to the subject of a therapeutically effective amount of a vasopressin analogue, such as terlipressin and a relaxin analogue.
• a vasopressin analogue such as terlipressin and a relaxin analogue.
• an aspect of the invention is a method of treating a subject suffering from ascites, the method comprising administration to the subject of a therapeutically effective amount of a vasopressin analogue, such as terlipressin, and a relaxin analogue.
• Relaxin analogues or pharmaceutically acceptable salts thereof are useful in combination with vasopressin analogues, such as terlipressin, in the present invention.
• the relaxin analogue is a long-acting, relaxin peptide analogue that functions as an RXFP1 receptor agonist.
• the disclosure further relates to compositions including the same and their use in treating renal conditions, diseases or disorders in combination with vasopressin analogues.
• amino acids in the relaxin peptide analogues disclosed herein can each independently be L-amino acids or D-amino acids.
• amino acids are L-amino acids.
• this amino acid is an L-amino acid.
• Nter and Cter are conventional labels used to indicate, respectively, the N-terminal end of the peptide and the C-terminal end of the relaxin peptide analogues.
• a relaxin peptide analogue is a modified relaxin-2 B chain peptide.
• the modified relaxin B chain peptide comprises formula (I) (SEQ ID NO 105): Nter-Ac-X10-E-G-R-E-Xl 5 -V-R-Xl 8 -X19-I-X21-X22-E-G-X25-S-X27-X28-X29-X30-X31-X32-X 3 3- NH 2 -Cter , wherein:
• Nter represents the N-terminal end of the peptide
• Cter represents the C-terminal end of the peptide
• Xio represents an amino acid selected from the group consisting of leucine, 2-amino- isobutyric acid, Ns-acetyl-lysine and a-methyl-leucine;
• E represents glutamic acid
• G represents glycine
• R represents arginine
• X15 represents an amino acid selected form the group consisting of lysine, arginine, homolysine, homoarginine, ornithine, glutamine, phenylalanine and leucine;
• V represents valine
• Xis represents an amino acid selected from the group consisting of alanine, 2-amino- isobutyric acid, leucine, Ns-acetyl-lysine and glutamine;
• X19 represents an amino acid selected from the group consisting of lysine, Ns-acetyl- lysine, citrulline, glutamine, alanine and 2-amino-isobutyric acid;
• I represents isoleucine
• X21 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and alanine;
• X22 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid and isoleucine;
• X25 represents the following structure: in which:
• Z represents a group of formula (II):
• PEGxx independently represents a polyethylene glycol derivative selected from the group consisting of PEG2, PEG2DGA, and TTDS;
• gE represents y-glutamic acid;
• Cd represents a linear saturated C12-C22 acyl group
• X27 represents an amino acid selected from the group consisting of threonine, lysine, arginine and glutamine;
• X28 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-naphtylalanine, 2-naphtylalanine, a-methyl -tryptophan, a- methyl-phenylalanine and 5 -hydroxy -tryptophan;
• X29 represents an amino acid selected from the group consisting of serine, D-serine, 2-amino-isobutyric acid, threonine, a-methyl-serine, Ns-acetyl-lysine and valine;
• X30 represents an amino acid selected from the group consisting of 2-amino-isobutyric acid, a-methyl-lysine, D-lysine, lysine, homolysine, ornithine, arginine and a-methyl- arginine;
• X31 represents an amino acid selected from the group consisting of arginine, No-methyl- arginine, alanine, No ,No ’ -dimethyl -arginine and citrulline;
• X32 represents an amino acid selected from the group consisting of lysine, alanine, arginine, Ns-acetyl-lysine and Ns,N8,Ns-tri-methyl-lysine;
• X33 represents an amino acid selected from lysine, Ns-acetyl-lysine, leucine, arginine and alanine; or a salt or solvate thereof.
• the relaxin peptide analogues disclosed herein also includes salts of the peptides of the formulae (I) or (la) defined herein, in one embodiment pharmaceutically acceptable salts, for example salts as acid adduct with inorganic acids such as hydrochloric acid, sulphuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid and boric acid; or with organic acid 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; and salts with metals such as alkali metal
• the salts of the peptides are pharmaceutically acceptable salts, for example acid adducts with hydrochloric acid, sulphuric 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 such as alkali metal, e.g. sodium, potassium, lithium and zinc.
• salts such as alkali metal, e.g. sodium, potassium, lithium and zinc.
• the relaxin peptide analogues disclosed herein also includeds solvates, and in one embodiment pharmaceutically acceptable solvates, of the peptides of the above formulae (I) or (la).
• Solvates mean complexes of the compounds of the relaxin peptide analogues or salts thereof with solvent molecules, e.g., organic solvent molecules and/or water. [00103] In keeping with standard polypeptide nomenclature (J. Biol. Chem., 243:3552-59
• X28 representing an amino acid selected from the group of tryptophan, 5 -fluoro-tryptophan, 5-chloro-tryptophan, 5 -methoxy -tryptophan, tyrosine, phenylalanine, 4-fluoro-phenylalanine, 1-naphtylalanine, 2-naphtylalanine, a- methyl-tryptophan, a-methyl-phenylalanine and 5 -hydroxy -tryptophan, the N-terminus or amine group of said amino acid is linked to the amino acid represented by X27 and the C- terminus or carboxyl group of said amino acid is linked to the amino acid represented by X29.
• a relaxin peptide analogue is substituted with an acetyl group (Ac): CH3C(O)— .
• a relaxin peptide analogue comprises the following structure:
• This structure corresponds to a lysine amino acid wherein:
• the alpha (a) nitrogen atom (*) is bound to the previous part of the peptide, i.e., with the group in Nter of X25 on the basis of the Nter-Cter orientation represented in the relaxin peptide analogue formulae, through a covalent bond, for example through a peptide bond, i.e., with the glycine in position 24 (X24);
• the carbon atom of the carboxyl group (*) is bound to the part of the peptide following position X25, i.e., with the group in Cter of X25 on the basis of the Nter-Cter orientation represented in the relaxin peptide analogue formulae, through a covalent bond, for example through a peptide bond, i.e., with the serine in position 26 (X26);
• This Z group is defined as being of formula (II):
• [00112] b and c independently represent 1, 2, 3, 4 or 5, in particular 2, 3, 4 or 5.
• b represents 2, 3, 4 or 5.
• c represents 2, 3 or 4.
• b represents 2, 3, 4 or 5 and c independently represents 2, 3 or 4.
• PEG XX in the relaxin peptide analogue formulae independently represents a polyethylene glycol derivative selected from the group consisting of PEG2, PEG2DGA and TTDS
• Said groups are defined as follows: in which, in (PEG xx )b of formula (II), represents a covalent bond with the (gE) c group and represents a covalent bond linking the group of formula (II) with the nitrogen atom of lateral chain of the lysine structure in X25.
• (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)?.
• gE which can also be represented as yE, gGlu or yGlu, represents a y-glutamic acid.
• This amino acid has the following structure: in which, when represented in formula (II) as (gE) c , represents a covalent bond with Cd and O represents a covalent bond with the (PEG xx )b group.
• Cd represents a linear saturated C12-C22 acyl group, for example a linear saturated acyl group selected from the group consisting of C12 (Lau), C14 (Myr), C15 (Penta), Ci6 (Palm), C 17 (Hepta), Cis (Stea), C20 (Eico) and C22 (Doco) acyl group.
• a linear saturated acyl group selected from the group consisting of C12 (Lau), C14 (Myr), C15 (Penta), Ci6 (Palm), C 17 (Hepta), Cis (Stea), C20 (Eico) and C22 (Doco) acyl group.
• Cd represents a linear saturated acyl group selected from the group consisting of C12 (Lau), C14 (Myr), C15 (Penta), Ci6 (Palm), C17 (Hepta) or Cis (Stea) acyl group, for example a linear saturated C14, Ci6 or Cis acyl group, or for example a linear Ci6 or Cis acyl group.
• Cd represents a linear saturated C12 acyl group.
• a linear saturated C12 acyl group is a lauroyl group (also represented as "Lau” in the present text).
• Cd represents a linear saturated C14 acyl group.
• a linear saturated C14 acyl group is a Myristoyl group (also represented as "Myr" in the present text).
• Cd represents a linear saturated C15 acyl group.
• a linear saturated C15 acyl group is a pentadecanoyl group (also represented as "Penta" in the present text).
• Cd represents a linear saturated Ci6 acyl group.
• a linear saturated Ci6 acyl group is a palmitoyl group (also represented as "Palm” in the present text).
• Cd represents a linear saturated C17 acyl group.
• a linear saturated C17 acyl group is a heptadecanoyl group (also represented as "Hepta” in the present text).
• Cd represents a linear saturated Cis acyl group.
• a linear saturated Cis acyl group is a stearoyl group (also represented as "Stea" in the present text).
• Cd represents a linear saturated C20 acyl group.
• a linear saturated C20 acyl group is a Eicosanoyl group (also represented as "Eico" in the present text).
• Cd represents a linear saturated C22 acyl group.
• a linear saturated C22 acyl group is a Docosanoyl group (also represented as "Doco" in the present text).
• Z is selected from the group consisting of -(TTDS)2-(y glutamic acid)3-Palmitoyl (-(TTDS)2-(gE)3-Palm), -(TTDS)3-(Y glutamic acid)3-Palmitoyl (- (TTDS)3-(gE)3-Palm), -(PEG2DGA)3-(Y glutamic acid)3-Palmitoyl (-(PEG2DGA)3-(gE)3- Palm), -(PEG 2 ) 4 -(Y glutamic acid)3-Palmitoyl (-(PEG2)4-(gE)3-Palm), -(TTDS)2-(Y glutamic acid)2-Palmitoyl (-(TTDS)2-(gE)2-Palm), -(TTDS)2-(Y glutamic acid)3-Stearoyl (-(TTDS)2- (gE)3-Palm), -
• the first — symbol represents the covalent bond between the Z group and nitrogen atom of the lateral chain of the lysine X25 structure.
• the Z group is for example represented as being -(TTDS)2-(Y glutamic acid)3-Palmitoyl (also represented as -(TTDS)2-(gE)3-Palm)
• the first TTDS group is bound to the nitrogen atom of the lateral chain of the X25 structure.
• a covalent bond also binds the second TTDS group while another covalent bond binds this second TTDS group to the first Y-glutamic acid (gE).
• This first y-glutamic acid (gE) is itself bonded through a covalent bond to the second y-glutamic acid (gE), this second y-glutamic acid (gE) is bonded through another covalent bond to the third y-glutamic acid (gE) and this third y-glutamic acid (gE) group is further linked by a covalent bond to a palmitoyle (Palm) group.
• a relaxin peptide analogue is of formula (la) (SEQ ID NO 106): Nter-Ac-L-E-G-R-E-Xi 5 -V-R-Xi8-Xi9-I-Aib-Aib-E-G-X25-S-T-X28-X29-X30-R-X32-X33-NH 2 - Cter wherein:
• Nter represents the N-terminal end of the peptide
• Cter represents the C-terminal end of the peptide
• Ac represents acetyl group
• L represents leucine
• E represents glutamic acid
• G represents glycine
• R represents arginine
• X15 represents an amino acid selected form the group consisting of lysine, arginine, homolysine, glutamine, phenylalanine and leucine;
• V represents valine
• Xis represents an amino acid selected from the group consisting of alanine and 2- amino-isobutyric acid;
• X19 represents an amino acid selected from the group consisting of lysine, Ns-acetyl- lysine, glutamine and citrulline;
• I represents isoleucine
• Aib represents 2-amino-isobutyric acid
• X25 represents the following structure: in which:
• - * represents a covalent bond with the serine following X25 in formula (la); and Z is selected from the group consisting of a -(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) 3 -Stea, -(PEG 2 DGA) 3 -(gE) 4 -Stea, -(PEG 2 ) 3 -(gE) 3
• Palm represents Palmitoyl and Stea represents Stearoyl
• S represents serine
• T represents threonine
• X28 represents an amino acid selected from the group consisting of tryptophan and phenylalanine
• X29 represents an amino acid selected from the group consisting of serine, D-serine and 2-amino-isobutyric acid;
• X30 represents an amino acid selected from the group consisting of 2-amino- isobutyric acid, a-methyl-lysine, D-lysine and lysine;
• X32 represents an amino acid selected from the group consisting of lysine, alanine and arginine;
• X33 represents an amino acid selected from lysine and Ns-acetyl-lysine; or a salt or solvate thereof.
• a relaxin peptide analogue has an amino acid sequence selected from the group consisting of the amino acid sequences of reference SEQ ID NO: 1-97.
• a relaxin peptide analogue has an amino acid sequence selected from the group consisting of the amino acid sequences of reference SEQ ID NO: 1-32, 34-37, 39, 42, 44, 45, 47-49, 51 and 54-97.
• X19 represents an amino acid selected from the group consisting of Ns-acetyl-lysine and citrulline;
• X25 represents the following structure: in which:
• - * represents a covalent bond with the serine following X25 in formula (la); and Z is selected from the group consisting of a -(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 ) 3 - (gE)3-Palm, -(PEG2)4-(gE)3-Stea and -(TTDS)2-(gE)4-Palm, wherein gE represents y-glutamic acid Palm represents Palmitoyl and Stea represents Stearoyl; X32 represents an amino
• a relaxin peptide analogue has an amino acid sequence selected from the group consisting of the amino acid sequences of reference SEQ ID NO: 1-30.
• a relaxin peptide analogue of formula (la) is such that: Nter represents the N-terminal end of the peptide;
• Cter represents the C-terminal end of the peptide
• L represents leucine
• E represents glutamic acid
• G represents glycine
• R represents arginine
• X15 represents an amino acid selected form the group consisting of lysine and homolysine
• V represents valine
• Xis represents alanine
• X19 represents an amino acid selected from the group consisting of Ns-acetyl-lysine and citrulline;
• I represents isoleucine
• Aib represents 2-amino-isobutyric acid
• X25 represents the following structure: in which:
• - * represents a covalent bond with the glycine preceding X25 in formula (la); - * represents a covalent bond with the serine following X25 in formula (la); and 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, - (PEG 2 DGA) 3 -(gE) 3 -Palm, -(TTDS) 2 -(gE) 3 -Stea, -(TTDS) 3 -(gE) 3 -Stea and - (PEG2) 4 -(gE) 3 -Stea, wherein gE represents y-glutamic acid Palm represents Palmitoyl and Stea represents Stearoyl;
• T represents threonine
• X28 represents an amino acid selected from the group consisting of tryptophan and phenylalanine
• X29 represents serine
• X 3 o represents an amino acid selected from the group consisting of 2-amino- isobutyric acid and a-methyl-lysine;
• X 3 2 represents an amino acid selected from the group consisting of lysine, alanine and arginine;
• X 33 represents an amino acid selected from lysine and Ns-acetyl-lysine; or a salt or solvate thereof.
• a relaxin peptide analogue of formula (la) is such that: Nter represents the N-terminal end of the peptide;
• Cter represents the C-terminal end of the peptide
• L represents leucine
• E represents glutamic acid
• G represents glycine
• R represents arginine
• X15 represents lysine
• V represents valine
• Xis represents alanine
• X19 represents Ns-acetyl-lysine
• I represents isoleucine
• Aib represents 2-amino-isobutyric acid
• X25 represents the following structure:
• - + represents a covalent bond with the serine following X25 in formula (la); and 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 represents y-glutamic acid Palm represents Palmitoyl and Stea represents Stearoyl;
• T represents threonine
• X28 represents an amino acid selected from the group consisting of tryptophan and phenylalanine
• X29 represents serine
• X30 represents an amino acid selected from the group consisting of 2-amino- isobutyric acid and a-methyl-lysine;
• X32 represents an amino acid selected from the group consisting of lysine, alanine and arginine;
• X33 represents an amino acid selected from lysine and Ns-acetyl-lysine; or a salt or solvate thereof.
• a relaxin peptide analogue is of formula (lb): Nter-Ac-X10-E-G-R-E-Xl 5 -V-R-Xl 8 -X19-I-X21-X- 22-E-G-X25-S-X27-X28-X 2 9-X30-R-X32-X33- NEE-Cter wherein:
• Nter represents the N-terminal end of the peptide
• Cter represents the C-terminal end of the peptide
• X10 represents an amino acid selected form the group consisting of leucine, Ns-acetyl- lysine and 2-amino-isobutyric acid;
• E represents glutamic acid
• G represents glycine
• R represents arginine
• X15 represents an amino acid selected form the group consisting of lysine, arginine, homolysine, glutamine, phenylalanine and leucine;
• V represents valine
• Xis represents an amino acid selected from the group consisting of alanine, 2-amino- isobutyric acid and Ns-acetyl-lysine;
• X19 represents an amino acid selected from the group consisting of lysine, Ns-acetyl- lysine, glutamine and citrulline; I represents isoleucine;
• X21 represents an amino acid selected from the group consisting of 2-amino- isobutyric acid and alanine;
• X22 represents an amino acid selected from the group consisting of 2-amino- isobutyric acid and isoleucine;
• X25 represents the following structure: in which:
• - * represents a covalent bond with the serine following X25 in formula (la); and Z is selected from the group consisting of a -(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) 3 -Stea, -(PEG 2 DGA) 3 -(gE) 4 -Stea, -(PEG 2 ) 3 -(gE) 3
• S represents serine
• X27 represents an amino acid selected from the group consisting of threonine, glutamine, arginine and lysine;
• X28 represents an amino acid selected from the group consisting of tryptophan, phenylalanine, 5-Chlorotryptophan, a-Methyl-phenylalanine, 4-Fluoro-phenylalanine and 5 -Fluorotryptophan;
• X29 represents an amino acid selected from the group consisting of serine, D-serine, 2-amino-isobutyric acid, Ns-acetyl-lysine, threonine and valine;
• X30 represents an amino acid selected from the group consisting of 2-amino- isobutyric acid, a-methyl-lysine, D-lysine, lysine, homolysine and arginine;
• X32 represents an amino acid selected from the group consisting of lysine, alanine, arginine and Ns-acetyl-lysine;
• X33 represents an amino acid selected from lysine, Ns-acetyl-lysine and arginine; or a salt or solvate thereof.
• Z can be selected from the group consisting of a -(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 )3-(gE)3-Palm, -(PEG 2 )4-(gE)3-Stea, -(PEG2)5-(gE) 3 -Palm, -(TTDS) 3 -(gE) 4 -Stea, - (TTDS)
• a relaxin peptide analogue has an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 6, 7, 9, 10, 11, 12, 13, 20, 21, 22, 26, 28 and 30.
• a relaxin peptide analoguean amino acid sequence selected from the group consisting of SEQ ID NO: 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 indicated here-above and illustrated in the enclosed examples, these peptides have an EC50 lower or equal to 1 nM.
• a relaxin peptide analogue has an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 6, 7, 13, 20, 26 and 30.
• a relaxin peptide analogue has an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 6, 7, 20, 26, 30-34, 45, 48, 49, 51, 54-61, 67, 71, 73, 75-79, 81, 83-92 and 97.
• the peptides according to this embodiment all have an EC50 ⁇ 0.5 nM in the in vitro OVCAR5 cell line cyclic- adenosine monophosphate (cAMP) assay, where OVCAR5 cells express endogenous human RXFP1 (see Example 3).
• relaxin peptide analogues display improved solubility at pH 4.5 or pH 7.5, improved rat and human plasma or blood stability and in-vivo pharmacokinetic half-lives.
• Such properties allow relaxin peptide analogue formulations in broad concentration ranges for use as medicament that will retain in-vivo efficacy for longer period of time (i.e., long-acting), permitting once a day administration by the intravenous or subcutaneous route.
• a relaxin peptide analogue 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 particularly has the amino acid sequence of reference SEQ ID NO: 3.
• relaxin analogues described herein can be used in combination with vasopressin analogues, as described herein.
• Such disclosure of relaxin analogues can be found in PCT Publication No. WO 2019/149782, “Modified Lipidated Relaxin B Chain Peptides and Their Therapeutic Use,” published August 8, 2019 as a publication of International Application No.
• PCT/EP2019/052298 PCT Publication No. WO 2019/149780, “Modified Relaxin B Chain Peptides and Their Therapeutic Use,” published August 8, 2019 as a publication of International Application No. PCTZEP2019/052296; and PCT Publication No. WO 2019/149781, “Modified Lipidated Relaxin B Chain Peptides and Their Therapeutic Use,” published August 8, 2019 as a publication of International Application No.
• PCTZEP2019/052297 incorporated by reference in its entirety.
• other relaxin peptide analogues known in the art can be used in combination with a vasopressin analogue, such as terlipressin, for treating an individual in need thereof as described herein.
• RXFP1 agonist relaxin peptide analogues are disclosed in PCT Publication WO 2015/157829, “Modified relaxin B chain peptides,” published October 22, 2015 as a publication of International Application No. PCT/AU2015/050184, incorporated by reference herein in its entirety.
• a relaxin peptide analogue may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides, by standard techniques for production of polypeptides, even if non-natural amino acids are used or according to the methods described herein.
• the relaxin peptide analogues can be synthesized using well-known solid phase method, for example using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, Calif., Gyros Protein technologies, Arlington, Ariz. or CEM corporation, Matthews, N.C.) and following the manufacturer's instructions.
• a commercially available peptide synthesis apparatus such as that made by Applied Biosystems, Foster City, Calif., Gyros Protein technologies, Arlington, Ariz. or CEM corporation, Matthews, N.C.
• the relaxin analogue is a small molecule with orthosteric or allosteric agonist activity at RXFP1.
• small molecule agonists are known to those of ordinary skill in the art, such as ML290 and ML290 analogues (see, e.g., McBride, A., Hoy, A.M., Bamford, M.J. 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, S.Y. et al. ML290 is a biased allosteric agonist at the relaxin receptor RXFP1. Sci Rep 7, 2968 (2017)).
• a method for preventing or treating renal failure in an individual in need thereof comprising co-administering a dose of a relaxin analogue from about 1.0 mg to about 5.0 mg and an effective amount of a vasopressin analogue to the individual.
• 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.
• hepatorenal syndrome is HRS-AKI (hepatorenal syndrome type 1).
• the relaxin analogue is an RXFP1 agonist, such as a long-acting peptidyl RXFP1 agonist.
• one or more doses of the relaxin analogue are administered to an individual.
• 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 analogue are administered to an individual.
• a single dose of the relaxin analogue is administered to an individual.
• two or more doses of the relaxin analogue are administered on the same day. In various embodiments, two doses of the relaxin analogue are administered to the individual on the same day, followed by administration of one or more doses of the relaxin analogue on subsequent days.
• the subsequent description refers to a dose of the relaxin analogue.
• the subsequent description is similarly applicable to each of the individual doses of the relaxin analogue administered to the individual.
• the relaxin analogue is administered to an individual at a fixed dose. In various embodiments, the relaxin analogue is administered to an individual at a dose from about 0.1 mg to about 100 mg. In various embodiments, the relaxin analogue is administered to an individual at a dose from about 0.5 mg to about 50 mg. In various embodiments, the relaxin analogue is administered to an individual at a dose from about 1.0 mg to about 50 mg.
• the relaxin analogue is administered to an individual at a dose from about 1 mg to about 25 mg, from about 2 mg to about 20 mg, from about 3 mg to about 19 mg, from about 4 mg to about 18 mg, from about 5 mg to about 15 mg, from about 6 mg to about 14 mg, from about 7 mg to about 13 mg, from about 8 mg to about 12 mg, or from about 9 mg to about 11 mg.
• the relaxin analogue is administered to an individual at a dose from about 1 mg to about 10 mg, from about 1 mg to about 9 mg, from about 1 mg to about 8 mg, from about 1 mg to about 7 mg, from about 1 mg to about 6 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 6 mg, or from about 1 mg to about 2 mg.
• the relaxin analogue is administered to an individual at a dose from about 1 mg to about 10 mg, from about 2 mg to about 8 mg, from about 3 mg to about 6 mg, from about 3 mg to about 5 mg, or from about 4 mg to about 5 mg.
• the relaxin analogue is administered to an individual at a dose from about 1 mg to about 9 mg, from about 2 mg to about 6 mg, from about 3 mg to about 5 mg, or from about 3.5 mg to about 4.5 mg. [00159] In various embodiments, the relaxin analogue is administered to an individual at a dose from about 10 mg to about 20 mg, from about 10.5 mg to about 18 mg, from about 11 mg to about 16 mg, from about 11.5 mg to about 14 mg, or from about 12 mg to about 13 mg.
• the relaxin analogue is administered to an individual at a dose from about 20 mg to about 30 mg, from about 21 mg to about 29 mg, from about 22 mg to about 28mg, from about 23 mg to about 26 mg, or from about 24 mg to about 25 mg. In various embodiments, the relaxin analogue is administered to an individual at a dose from about 40 mg to about 50 mg, from about 42 mg to about 49.5 mg, from about 44 mg to about 49 mg, from about 46 mg to about 48.5 mg, or from about 47 mg to about 48 mg.
• the relaxin analogue is administered to an individual at a dose of about 2.0 mg. In particular embodiments, the relaxin analogue is administered to an individual at a dose of about 4.0 mg. In particular embodiments, the relaxin analogue is administered to an individual at a dose of about 5.0 mg. In particular embodiments, the relaxin analogue is administered to an individual at a dose of about 10.0 mg. In particular embodiments, the relaxin analogue is administered to an individual at a dose of about 12.0 mg. In particular embodiments, the relaxin analogue is administered to an individual at a dose of about 15.0 mg. In particular embodiments, the relaxin analogue is administered to an individual at a dose of about 24.0 mg. In particular embodiments, the relaxin analogue is administered to an individual at a dose of about 48.0 mg.
• the relaxin analogue is administered subcutaneously. In various embodiments, the relaxin analogue is administered intravenously. In various embodiments, the relaxin analogue is administered intravenously as a bolus injection. In various embodiments, the relaxin analogue is administered intravenously over from about 10 minutes to about 24 hours, from about 30 minutes to about 16 hours, from about 45 minutes to about 12 hours, from about 1 hour to about 10 hours, from about 1.5 hours to about 9 hours, from about 2 hours to about 8 hours, from about 2.5 hours to about 7 hours, from about 3 hours to about 6 hours, from about 3.5 hours to about 5 hours, or from about 4 hours to about 4.5 hours. In particular embodiments, the relaxin analogue is administered intravenously over about 4 hours.
• the second dose (e.g., an additional dose) of the relaxin analogue is administered to the individual between from about 1 hour to about 24 hours after administration of the first dose of the relaxin analogue.
• the second dose of the relaxin analogue is administered to the individual between from about 2 hours to about 22 hours, from about 3 hours to about 20 hours, from about 4 hours to about 19 hours, from about 5 hours to about 18 hours, from about 6 hours to about 17 hours, from about 7 hours to about 16 hours, from about 8 hours to about 15 hours, from about 9 hours to about 14 hours, from about 10 hours to about 13 hours, or from about 11 hours to about 12.5 hours after administration of the first dose of the relaxin analogue.
• timing of the second dose of the relaxin analogue is measured in relation to the completion of the administration of the first dose of the relaxin analogue. For example, assuming the first dose of the relaxin analogue is a 4 hour continuous intravenous administration, the timing of the second dose of the relaxin analogue is measured in relation to the completion of the four hour continuous intravenous administration. In various embodiments, timing of the second dose of the relaxin analogue is measured in relation to the initiation of the administration of the first dose of the relaxin analogue. For example, assuming the first dose of the relaxin analogue is a 4 hour continuous intravenous administration, the timing of the second dose of the relaxin analogue is measured in relation to the initiation of the four hour continuous intravenous administration.
• the first dose and second dose of the relaxin analogue are administered to the individual through different administration routes.
• the first dose of the relaxin analogue may be administered intravenously to the individual and the second dose of the relaxin analogue may be administered subcutaneously to the individual.
• the first dose of the relaxin analogue may be administered subcutaneously to the individual and the second dose of the relaxin analogue may be administered intravenously to the individual.
• the first dose and the second dose of the relaxin analogue are administered ot the individual through the same administration route.
• both the first dose and the second dose of the relaxin analogue are administered to the individual intravenously.
• both the first and the second dose of the relaxin analogue are administered to the individual subcutaneously.
• the second dose of the relaxin analogue is higher than the first dose of the relaxin analogue.
• the second dose of the relaxin analogue is at least 100% higher than the first dose of the relaxin analogue.
• the first dose may be about 2.0 mg and the second dose may be about 5.0 mg.
• the first dose may be about 4.0 mg and the second dose may be about 10.0 mg.
• the second dose of the relaxin analogue is less than 30% higher than the first dose of the relaxin analogue.
• the second dose of the relaxin analogue may be 25% higher than the first dose of the relaxin analogue.
• the first dose may be about 4.0 mg and the second dose may be about 5.0 mg.
• a third dose e.g., a yet additional dose
• the third dose of the relaxin analogue is administered to the individual.
• the third dose of the relaxin analogue is administered to the individual between from about 12 hours to about 48 hours after administration of the first dose of the relaxin analogue.
• the third dose of the relaxin analogue is administered to the individual between from about 13 hours to about 45 hours, from about 14 hours to about 42 hours, from about 15 hours to about 39 hours, from about 16 hours to about 36 hours, from about 17 hours to about 33 hours, from about 18 hours to about 30 hours, from about 19 hours to about 28 hours, from about 20 hours to about 26 hours, from about 22 hours to about 25 hours, or from about 23 hours to about 24.5 hours after administration of the first dose of the relaxin analogue.
• timing of the third dose of the relaxin analogue is measured in relation to the completion of the administration of the first dose of the relaxin analogue.
• timing of the third dose of the relaxin analogue is measured in relation to the completion of the four hour continuous intravenous administration.
• timing of the third dose of the relaxin analogue is measured in relation to the initiation of the administration of the first dose of the relaxin analogue.
• the timing of the third dose of the relaxin analogue is measured in relation to the initiation of the four hour continuous intravenous administration.
• the first dose and the third dose of the relaxin analogue are administered to the individual through different administration routes.
• the first dose of the relaxin analogue may be administered intravenously to the individual and the third dose of the relaxin analogue may be administered subcutaneously to the individual.
• the first dose of the relaxin analogue may be administered subcutaneously to the individual and the third dose of the relaxin analogue may be administered intravenously to the individual.
• the first dose and the third dose of the relaxin analogue are administered ot the individual through the same administration route.
• both the first dose and the third dose of the relaxin analogue are administered to the individual intravenously.
• both the first and the third dose of the relaxin analogue are administered to the individual subcutaneously.
• the third dose of the relaxin analogue is higher than the first dose of the relaxin analogue.
• the third dose of the relaxin analogue is at least 100% higher than the first dose of the relaxin analogue.
• the first dose may be about 2.0 mg and the third dose may be about 5.0 mg.
• the first dose may be about 4.0 mg and the third dose may be about 10.0 mg.
• the third dose of the relaxin analogue is less than 30% higher than the first dose of the relaxin analogue.
• the third dose of the relaxin analogue may be 25% higher than the first dose of the relaxin analogue.
• the first dose may be about 4.0 mg and the third dose may be about 5.0 mg.
• a dose beyond the third dose is administered to the individual.
• the dose beyond the third dose replicates the quantity and administration route of the third dose. For example, assuming the third dose includes a 10 mg subcutaneous administration of the relaxin analogue, the dose beyond the third dose also includes a 10 mg subcutaneous administration of the relaxin analogue.
• the dose beyond the third dose is administered daily. In various embodiments, the dose beyond the third dose is administered daily from about 5 days to about 25 days. In various embodiments, the dose beyond the third dose is administered daily from about 6 days to about 24 days, from about 7 days to about 23 days, from about 8 days to about 22 days, from about 9 days to about 21 days, from about 10 days to about 20 days, from about 11 days to about 18 days, from about 12 days to about 16 days, or from about 13 days to about 15 days. In particular embodiments, the dose beyond the third dose is administered daily for 13 days. In particular embodiments, the dose beyond the third dose is administered daily for 14 days. In particular embodiments, the dose beyond the third dose is administered daily for 15 days.
• the dose beyond the third dose is administered daily. In various embodiments, the dose beyond the third dose is administered daily from about 3 days to about 15 days. In various embodiments, the dose beyond the third dose is administered daily from about 4 days to about 13 days, from about 5 days to about 11 days, or from about 6 days to about 9 days. In particular embodiments, the dose beyond the third dose is administered daily for 6 days. In particular embodiments, the dose beyond the third dose is administered daily for 7 days. In particular embodiments, the dose beyond the third dose is administered daily for 8 days. In particular embodiments, the dose beyond the third dose is administered daily for 9 days.
• methods for preventing or treating renal failure in an individual in need thereof and/or methods for preventing or treating hepatorenal syndrome in an individual comprise: about 4 hour intravenous administration of a first dose of 2.0 mg of a relaxin analogue, a subcutaneous administration of 5.0 mg of the relaxin analogue at 12 hours after initiation of the first dose, a subcutaneous administration of 5.0 mg of the relaxin analogue at 24 hours after initiation of the first dose, and thereafter daily subcuitaneous administration of 5.0 mg of the relaxin analogue for up to 12 to 15 days.
• methods for preventing or treating renal failure in an individual in need thereof and/or methods for preventing or treating hepatorenal syndrome in an individual comprise: about 4 hour intravenous administration of a first dose of 4.0 mg of a relaxin analogue, a subcutaneous administration of 10.0 mg of the relaxin analogue at 12 hours after initiation of the first dose, a subcutaneous administration of 10.0 mg of the relaxin analogue at 24 hours after initiation of the first dose, and thereafter daily subcuitaneous administration of 5.0 mg of the relaxin analogue for up to 12 to 15 days.
• methods for preventing or treating renal failure in an individual in need thereof and/or methods for preventing or treating hepatorenal syndrome in an individual comprise: about 4 hour intravenous administration of a first dose of 4.0 mg of a relaxin analogue, a subcutaneous administration of 5.0 mg of the relaxin analogue at 12 hours after initiation of the first dose, a subcutaneous administration of 10.0 mg of the relaxin analogue at 24 hours after initiation of the first dose, and thereafter daily subcuitaneous administration of 5.0 mg of the relaxin analogue for up to 12 to 15 days.
• methods for preventing or treating renal failure in an individual in need thereof and/or methods for preventing or treating hepatorenal syndrome in an individual comprise co-administering an effective amount of a relaxin analogue and a vasopressin analogue to the individual.
• the vasopressin analogue is terlipressin or a pharmaceutically acceptable salt thereof.
• the terlipressin is administered intravenously at a dosage from about 0.5 to about 10 mg. In various embodiments, the terlipressin is administered intravenously at a dosage from about 0.6 mg to about 5 mg, from about 0.7 mg to about 3 mg, from about 0.8 mg to about 2 mg, or from about 0.9 mg to about 1.5 mg. In particular embodiments, the terlipressin is administered intravenously at a dosage of about 1 mg.
• the terlipressin is administered intravenously at a dosage from about 0.8 mg to about 6 mg, from about 1 mg to about 5 mg, from about 1.2 mg to about 4 mg, from about 1.4 mg to about 3 mg, from about 1.6 mg to about 2.5 mg, from about 1.8 mg to about 2.2 mg, or from about 1.9 mg to about 2.1 mg.
• the terlipressin is administered intravenously at a dosage of about 2 mg.
• the terlipressin is administered intravenously at a dosage from about 1 mg to about 8 mg, from about 2 mg to about 6 mg, from about 3 mg to about 5 mg, from about 3.2 mg to about 4.8 mg, from about 3.4 mg to about 4.6 mg, from about 3.6 mg to about 4.4 mg, or from about 3.8 mg to about 4.2 mg.
• the terlipressin is administered intravenously at a dosage of about 4 mg.
• the terlipressin is administered intravenously at a dosage from about 2 mg to about 9 mg, from about 4 mg to about 8 mg, from about 5 mg to about 7 mg, from about 5.2 mg to about 6.8 mg, from about 5.4 mg to about 6.6 mg, from about 5.6 mg to about 6.4 mg, or from about 5.8 mg to about 6.2 mg.
• the terlipressin is administered intravenously at a dosage of about 6 mg.
• the terlipressin is administered intravenously every 2 to 10 hours. In various embodiments, the terlipressin is administered intravenously every 3 to 8 hours, or every 4 to 6 hours. In various embodiments, the terlipressin is administered intravenously every 6 hours. In various embodiments, terlipressin is administered intravenously via a bolus infusion. In various embodiments, terlipressin is administered intravenously via a bolus injection over from about 30 seconds to about 10 minutes, from about 1 minute to about 5 minutes, or from about 2 minutes to about 3 minutes. In various embodiments, terlipressin is administered intravenously via a bolus injection over about 2 minutes.
• from about 0.5 mg to about 2 mg terlipressin is admininstered intravenously every 4 to 6 hours.
• about 1.0 mg terlipressin is admininstered intravenously every 6 hours.
• from about 2 mg to about 6 mg terlipressin is administered intravenously every 4 to 6 hours.
• about 4.0 mg terlipressin is administered intravenously every 6 hours.
• from about 6 mg to about 10 mg of terlipressin is administered intravenously every 4 to 6 hours.
• about 6.0 mg terlipressin is administered intravenously every 6 hours.
• about 8.0 mg terlipressin is administered every 8 hours.
• the terlipressin is administered intravenously over from about 8 hours to about 36 hours. In various embodiments, the terlipressin is administered intravenously over from about 10 hours to about 30 hours, from about 15 hours to about 28 hours, or from about 20 hours to about 25. In particular embodiments, the terilipressin is administered intravenously over about 24 hours.
• the terlipressin is administered intravenously at a dosage from about 1 mg to about 10 mg. In various embodiments, the terlipressin is administered intravenously at a dosage from from about 1.5 mg to about 4.0 mg. In various embodiments, the terlipressin is administered intravenously at a dosage of about 2.0 mg. In various embodiments, the terlipressin is administered intravenously at a dosage from from about 5.0 mg to about 8.0 mg. In various embodiments, the terlipressin is administered intravenously at a dosage of about 6.0 mg.
• methods for preventing or treating renal failure in an individual in need thereof and/or methods for preventing or treating hepatorenal syndrome in an individual comprises: one of (1A), (IB), or (1C) in combination with (2), where:
• methods for preventing or treating renal failure in an individual in need thereof and/or methods for preventing or treating hepatorenal syndrome in an individual comprises: one of (1A), (IB), or (1C) in combination with (2), where:
• methods for preventing or treating renal failure in an individual in need thereof and/or methods for preventing or treating hepatorenal syndrome in an individual comprises: one of (1A), (IB), or (1C) in combination with (2), where:
• the present application also relates to a medicament or a pharmaceutical composition
• a medicament or a pharmaceutical composition comprising separately or in combination, vasopressin analogues and relaxin analogues as described above, or pharmaceutically acceptable salts or solvates thereof, and at least one pharmaceutically acceptable carrier.
• the relaxin analogue and/or vasopressin analogue is present in a medicament or pharmaceutical composition of the invention as active principle.
• a composition or a medicament of the invention is in a form suitable for administration to an individual in need thereof.
• a composition or a medicament of the invention can be administered, for example, parenterally, intravenously, subcutaneously, rectally, transdermally, topically or by inhalation.
• a composition according to the invention is administered by the intravenous or subcutaneous route.
• the pharmaceutically acceptable carrier of a composition of the invention is suitably selected from the group consisting of an injectable carrier liquid such as sterile water for injection; and an aqueous solution such as saline.
• a composition or a medicament of the invention can comprise a content of peptides of the invention comprised between 0.01 mg/mL and 30 mg/mL, in particular between 0.3 mg/mL and 3 mg/mL.
• a medicament or a pharmaceutical composition of the invention can comprise at least one peptide of the invention as sole active principle or can also comprise at least one other active principle, as long as said other active principle does not prevent the biological activity of the peptide according to the invention.
• a pharmaceutical composition or a medicament according to the invention can further comprise at least one antioxidant, dispersant, emulsifier, antifoam, flavouring, preservative, solubilizer and/or colour, as long as this/these additional substances do not prevent the biological properties of the peptides according to the invention.
• compositions of the invention for parenteral administration may in particular embodiments be aqueous or non-aqueous solutions, suspensions or emulsions.
• Solvents or vehicles that can be used include water, propylene glycol, a polyethylene glycol, plant oils, for example olive oil, injectable organic esters, for example ethyl oleate, or other suitable organic solvents.
• These compositions may also comprise adjuvants, for example wetting agents, tonicity agents, emulsifiers, dispersants and stabilisers.
• the sterilisation may be performed in several ways, for example by aseptic filtration, by incorporating sterilising agents into the composition, by irradiation or by heating. They may also be prepared in the form of sterile solid compositions that may be dissolved at the time of use in sterile water or any other injectable sterile medium.
• compositions for topical administration may be, for example, nasal drops or aerosols.
• the peptides of the invention used are converted, if desired with the substances customary for this purpose, such as solubilizers, emulsifiers or other excipients, into a solution, suspension or emulsion.
• suitable solvents are: water, physiological saline or alcohols, e.g. ethanol, propanol, glycerol, as well as sugar solutions such as glucose or mannitol solutions, or else a mixture of the various solvents mentioned.
• a composition of the invention, a medicament of the invention, or a peptide of the invention, or one of its pharmaceutically acceptable salt or solvate thereof is administered to an individual by the parenteral route, and is in particular transdermaly, intravenously, subcutaneously or intramuscularly, in particular intravenously or subcutaneously administered.
• compositions of the invention or of a peptide of the invention to an individual can be a systemic administration or an administration localized to a tissue, organ and/or site of the individual organism.
• the present invention relates to a combination therapy for treatment of an individual in need thereof of a vasopressin analogue and a relaxin analogue, and associated compositions comprising these bioactive agents, as well as their pharmaceutically acceptable salts or solvates thereof.
• the invention also relates to one or more pharmaceutical compositions according to the invention for use as a combination therapy including a vasopressin analogue and relaxin analogue bioactive agents.
• the present invention relates to peptides of the invention, pharmaceutically acceptable salts or solvates thereof, or a pharmaceutical composition of the invention for its use in combination therapy for the treatment and/or prevention of various diseases or conditions implicating the RXFP1 receptor and/or the Via vasopressin receptor, more particularly in 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 in particular can be used for the treatment and/or prevention of HRS, in particular HRS-AKI (hepatorenal syndrome type 1) or HRS-NAKI (hepatorenal syndrome type 2).
• a combination therapy of a relaxin analogue and vasopressin analogue peptide, pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition or compositions including one or more of these bioactive agents is administered once a day, in particular by the intravenous or subcutaneous route.
• the dosage of the relaxin analogue and vasopressin analogue peptide, or of its pharmaceutically acceptable salt or solvate thereof, to be administered, and the frequency of administration, depend on the desired effect, the potency and duration of action of the compounds used; additionally, also on the nature and severity of the disease or condition to be treated and on the sex, age, weight and individual responsiveness of the individual to be treated. In general, the physician determines the appropriate dosage as a function of the age and weight and all the other factors specific to the individual to be treated.
• Also provided herein is a method for preventing and/or treating 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 comprising co-administering to an individual in need of said prevention and/or treatment a combination of relaxin analogue and vasopressin analogue peptides of the invention, pharmaceutically acceptable salts or solvates thereof or a pharmaceutical composition or compositions of the invention comprising vasopressin analogue and/or relaxin analogue bioactive agents according to the invention or a therapeutically effective amount of the combination of vasopressin analogue and relaxin analogue peptides, pharmaceutically acceptable salts or solvates thereof, or a pharmaceutical composition or compositions of the invention comprising vasopressin analogue and/or relaxin analogue bioactive agents according to the invention.
• articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
• the invention includes embodiments in which exactly one member of the 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 members are present in, employed in, or otherwise relevant to a given product or process.
• Fmoc (fluorenylmethyloxy carbonyl) protected natural amino acids were purchased from different sources, i.e., Protein Technologies Inc., Merck Biosciences, Novabiochem, Iris Biotech, Bachem, Chem-Impex International or MATRIX Innovation.
• Standard heating protocol irradiation at 170 watts, 75° C., 15 sec. then irradiation at 30 watts, 90° C., 120 sec.
• Heating protocol for deprotection irradiation at 170 watts, 75° C., 15 sec. then irradiation at 30 watts, 90° C., 50 sec.
• each amino-acid required double couplings at 90° C. for 120 seconds.
• 2-aminoisobutyric acid at positions X21 and X22 and serine at position X29 of the formula (I) a triple coupling at 90° C. for 2 minutes was used.
• the Z groups was attached to the side chain nitrogen of the X25 lysine on solid support according to the following steps: [00235] When the lysine in position X25 was protected on its side chain by Dde or iv-Dde, resin-peptide was transferred into a 50 ml polypropylene syringe. 80 ml of a solution of hydrazine 5% in DMF was percolated through the resin followed by DMF wash (3 times). The reaction was monitored by Kaiser Test.
• Fmoc-Glu-OtBu ((4S)-5-tert-butoxy-4-(9H-fluoren-9-yl methoxy carbonyl amino)-5-oxo-pentanoic acid
• gE present in the Z group of the relaxin peptide analogue was introduced by performing single coupling with 3 eq. of amino acid for 18 h with 3 eq. of DIC and 3 eq. of HOAt monitoring the reactions by Kaiser Test.
• the side chain was lipidated using 3 eq. of Ck lauric acid (C12), myristic acid (C14), pentadecanoic acid (C15), palmitic acid (Cie), heptadecanoic acid (C17), stearic acid (Cis), eicosanoic acid (C20) or docosanoic acid (C22) using 3 eq. DIC and 3 eq. HO
• Ck lauric acid C12
• myristic acid C14
• pentadecanoic acid C15
• palmie palmitic acid
• heptadecanoic acid C17
• stearic acid Cis
• eicosanoic acid C20
• docosanoic acid C22
• cleavage reagent B TFA/phenol/H2O/TIPS (87.5%/5%/5%/2.5%/25 ml) for 3 h.
• TIPS stands for tri-isopropylsilane
• a dithiol such as 1,2-ethane dithiol or DODT (2,2'- (ethylenedioxy)diethanethiol) may be advantageous (e.g. cleavage reagent K).
• cleavage reagent K 2,2'- (ethylenedioxy)diethanethiol
• the desired product was precipitated with ice-cold MTBE (methyl tert-butyl ether) or diethyl ether and centrifuged at 3000 rpm for 30 min. The centrifuged pellet was then washed with ice-cold diethyl ether and centrifuged. This process was repeated three times.
• MTBE methyl tert-butyl ether
• diethyl ether diethyl ether
• peptide 80 mg were dissolved in 1.5 mL DMSO and purified by Reverse Phase High Pressure Liquid Chromatography (RP-HPLC). The RP-HPLC was described hereunder.
• the ion exchange was performed using a TOYOPEARL.RTM. DEAE 650 C grade resin (a weak anion exchange resin).
• the column (5 ml bed volume) was connected to a peristaltic pump set at 48 (4.5 ml/min) and before loading the peptide it was washed with 50 ml (10 column volumes) of H2O, 100 ml (20 column volumes) of a 1 M solution of sodium acetate, 150 ml (30 column volumes) of H2O and with 50 ml (10 column volumes) of 0.16 M solution of acetic acid.
• TTDS spacer units were introduced by single coupling by performing three times the following procedure: To the resin a solution of Fmoc-TTDS-OH (1.62 g, 3 mmol) in 30 mL of DMF were added 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). Then to the resin, 30 mL of 20% v/v of piperidine in DMF was added. The syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated a second time and the resin was washed with DMF (2 x 30 mL) and dichloromethane (3 x 30 mL).
• 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 a second time.
• the reaction was monitored by Kaiser Test.
• the resin was washed with DMF (2 x 30 mL).
• 30 mL of 20% v/v of piperidine in DMF was added.
• the syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated a second time and the resin was washed with DMF (3 x 30 mL) and di chloromethane (3 x 30 mL).
• the peptide was acylated with palmitic acid (768 mg, 3 mmol), HO At (5 ml of a 0.6 M solution in DMF, 3 mmol) and DIC (1 ml, 6 mmol) activation in DMF (30 mL) for 2.5 h.
• the resin was washed with DMF (2 x 30 mL) and dichloromethane (3 x 30 mL) and dried under vacuum.
• the peptide was acylated by adding a solution of stearoyl chloride (362 mg, 1.2 mmol) and DIPEA (0.255 ml, 1.5 mmol) in 20 ml of DCM for 2.5 h. The resin was washed with DMF (2 x 30 mL) and dichloromethane (3 x 30 mL) and dried under vacuum.
• Two TTDS spacer units were introduced by single coupling by performing twice the following procedure: To the resin a solution of Fmoc-TTDS-OH (163 mg, 0.3 mmol) HOAt (42 mg, 0.3 mmol) and DIC (77 pL, 0.5 mmol) 7 mL of DMF was added. The syringe was agitated on an orbital table for 18 h. The reaction was monitored by Kaiser Test. When needed, a double coupling was performed. The resin was washed with DMF (2 x 10 mL). Then to the resin, 10 mL of 20% v/v of piperidine in DMF was added. The syringe was agitated on an orbital table for 20 min.
• the peptide was acylated with Stearoyl chloride (62 mg, 0.2 mmol) and DIPEA (54 pL, 0.3 mmol) in 5 ml DCM for 2.5 h.
• the resin was washed with DMF (2 x 30 mL) and dichloromethane (3 x 30 mL) and dried under vacuum.
• the cleavage of the peptide from the resin was performed using a solution phenol (0.5 g), water (0.5 mL) and TIPS (0.2 mL) in TFA (QSP 10 mL) for 2.5 hours at room temperature.
• the resin was filtered off and washed with 2 x 4 mL TFA.
• the combined filtrates were transferred to a 100 mL round bottom flask and partially concentrated under vacuum at T ⁇ 30°C and the peptide was precipitated by the addition of 50 mL ice-cold MTBE and centrifuged at 3600 rpm for 30 minutes.
• the Aloe group on Lys25 side chain was removed by adding to the resin, under argon atmosphere, a solution of 1 ml (8.33 mmol) of phenyl silane in 2 ml of degazed DCM and a solution of 10 mg (25.96 pmoles) of tetrakis-(triphenylphosphine) palladium in 4 ml DCM.
• the resin was shaken on an orbital table for 60 min and the reaction media was replaced with fresh reagents twice and shaken 60 min each time.
• 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 a second time.
• the reaction was monitored by Kaiser Test.
• the resin was washed with DMF (2 x 10 mL).
• 10 mL of 20% v/v of piperidine in DMF was added.
• the syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated a second time and the resin was washed with DMF (3 x 30 mL) and dichloromethane (3 x 30 mL).
• the peptide was acylated 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) activation in DMF (10 mL) for 2.5 h.
• the resin was washed with DMF (2 x 30 mL) and dichloromethane (3 x 30 mL) and dried under vacuum.
• the cleavage of the peptide from the resin was performed using a solution phenol (0.5 g), water (0.5 mL) and TIPS (0.25 mL) in TFA (QSP 10 mL) for 2.5 hours at room temperature.
• the resin was filtered off, and washed with 2 x 5 mL TFA.
• the combined filtrates were transferred to a 250 mL round bottom flask and partially concentrated under vacuum at T ⁇ 30° C. and the peptide was precipitated by the addition of 100 mL ice-cold MTBE and centrifuged at 3600 rpm for 30 minutes.
• Condition A was the following:
• Condition B was the following:
• Condition C was the following:
• Condition D was the following:
• the following relaxin peptide analogue can be prepared respectively as described above: SEQ ID NO 33, 38, 40, 41, 43, 46, 50, 52 and 53.
• OVCAR5 cells expressing endogenous human RXFP1 were used to test RXFP1 agonist properties of relaxin peptide analogues, and in particular of the peptides of sequence SEQ ID NO: 1-97.
• RXFP1 is a Gs coupled GPCR
• increases in cAMP was used as readout of RXFP1 activation.
• IBMX Isobutyl methyl xanthine
• HTRF Homogenous Time Resolved Fluorescence
• OVCAR5 were grown in regular medium (RPMI) containing 10% fetal calf serum (FCS) and 1% antibiotics (penicillin/streptomycin).
• RPMI regular medium
• FCS fetal calf serum
• antibiotics penicillin/streptomycin
• Emax % was determined as the maximal intracellular value of cAMP for test compound (upper limit of cAMP vs concentration curve) divided by the maximal intracellular value of cAMP for human relaxin-2 (H2-Rlx) determined in the same test occasion multiplied by 100.
• Relaxin peptide analogues described herein are potent agonists of human RXFP1, with consistently high Emax values in the in vitro OVCAR5 cAMP assay, comparable to native human relaxin-2.
• the same experiment has been performed with peptides from the prior art (Bathgate et al. WO2015/157829, incorporated by reference in its entirety) known under the names B7- 33 C11.23S, AcB7-33 C11.23S and KKKK(AcB 7-29 C11.23S).
• the relaxin peptide analogues in Table 2 possess very interesting RXFP1 agonist properties and are very effective in activating RXFP1. They are moreover all significantly and unexpectedly superior to the peptides from the prior art.
• Example 4 Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single and Multiple Ascending Dose Ranging Study in Healthy Volunteers to Assess Safety, Tolerability, and Evaluate the Pharmacokinetics and Pharmacodynamics of Relaxin Agonist
• Described in this Example is a Phase 1, randomized, double-blind, pacebo controlled trial for assessing safety, tolerability, PK, and PD of a Relaxin Agonist (i.e., Relaxin peptide analogue of SEQ ID NO: 3).
• a Relaxin Agonist i.e., Relaxin peptide analogue of SEQ ID NO: 3
• Four cohorts received a dose via SC injection only and 1 cohort (Cohort A2, 4.0 mg) was dosed via both SC and IV injection, with at least 2-week washout period between SC and IV injections.
• the 4.0 mg dosing cohort (Cohort A2) received the IV dose after the 12.0 mg SC cohort (Cohort A3) was completed.
• Renal plasma flow (RPF) measurements were approximated based on Para- aminohippurate (PAH) clearance. PAH measurements were performed in MAD (Part B) study only. Baseline measurements were taken on Day 1, and Day 13 measurements were taken at -2.5, -1, and -0.5 h predose and at 4, 5.5, and 6 h postdose, which was the predicted Tmax.
• RPF Para- aminohippurate
• FIGs. 3 A and 3B show the change from baseline effective renal plasma flow following administration of Relaxin Agonist. After multiple dosing with Relaxin Agonist or placebo, RPF assessed using the PAH biomarker tended to be increased in the active dose groups compared to placebo, with a more pronounced effect on Day 13 postdose compared to Day 13 predose for all treatment groups.
• This study is an open label, phase 1, multi-centre, single dose study in severe renal impaired subjects and in matched subjects with normal renal function. In this preliminary evaluation, five subjects with severe renal impairment and two subjects with normal renal function were included.
• This study is a Phase 1, multi-center, single dose study in severe renal impaired subjects and in 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 predose and at 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 h postdose.
• Mean Cmax in the severe renal impaired group is 391.8 ng/mL.
• Mean Cmax in the normal group is 302.0 and in the SAD study after 4mg it was 355.7.
• AUCinf there is a similar increase in the severe renal impaired group as compared to the normal group and compared to the SAD 4mg group.
• mean Cmax there is a 10-30% increase.
• mean AUCinf there is a 14-31% increase.
• Described in this example is a randomized, single blind controlled, two groups, multicenter trial preceeded by a safety run-in, in patients with hepatorenal syndrome (HRS) - Acute Kidney Injury (AKI).
• HRS hepatorenal syndrome
• AKI Acute Kidney Injury
• 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 analogue of SEQ ID NO: 3) combined with terlipressin as compared to terlipressin alone in the treatment of patients with HRS-AKI.
• Efficacy is assessed through the primary endpoint of established hepatorenal syndrome (HRS) reversal. Other important efficacy parameters are assessed.
• Safety data are collected and incorporated into the overall safety assessment of the Relaxin Agonist.
• responders are defined according to the International Club of Ascites (ICA) criteria.
• Full response is defined as two serum creatinine levels returning to a value within 0.3 mg/dL (26.5 micromolar/L) of a baseline serum creatinine value at least 2 hours apart.
• Partial response is defined as a regression of at least 1 acute kidney injury (AKI) stage with a reduction of serum creatinine greater than or equal to 0.3 mg/dL above a baseline serum creatinine value.
• Established HRS reversal (clinical responders) are defined as patients with a Full or Partial response based on serum creatinine levels and AKI stage and are alive without renal replacement therapy (RRT) for at least 30 days after original treatment start.
• the primary objectives of the study include:
• the secondary objectives of the study include:
• the exploratory objectives of the study include:
• FIG. 4 depicts an example study design of a Phase II Trial Assessing Safety, Tolerability, Efficacy, and Pharmacokinetics of Relaxin Agonist in Combination wth Terlipressin.
• terlipressin administered as a 1 mg bolus IV infusion every 6 hours (h), to be increased if clinically appropriate up to 2.0 mg infusion over 6h.
• Terlipressin dosing should continue up to 24 h after achievement of an HRS response (either Partial or Full response) based on Serum Creatinine (SCr)/AKI stage oir up to day 14.
• FIG. 5 depicts a design of the open label safety run-in part of the overall study design shown in FIG. 4.
• three initial cohorts of 3 patients (labeled in FIG. 4 as Cohorts 1, 2, and 3) each are treated open label with the combination of terlipressin and Relaxin Agonist to ascertain its safety.
• Cohort 3 receives terlipressin and 4.0 mg Relaxin Agonist IV over 4 h, followed by 5.0 mg Relaxin Agonist SC at 12 h after initiation of the IV, and 10.0 mg Relaxin Agonist SC at 24 h, and thereafter the same dose (lO.Omg) once a day up to 24 h after achievement of an HRS response (either Partial or Full) based on SCr/AKI stage or up to Day 14; i.e., the same dose and schedule as to be used for Cohort 4 in the randomized part of the trial.
• Cohort 3 finishes dosing before Part B, the Single-Blind Placebo-Controlled Randomized Part starts. Based on safety and tolerability of the various Relaxin Agonist dose schedules in Cohorts 1, 2, and 3, the SRC determines the appropriate Relaxin Agonist dose schedule to be taken forward for Cohorts 4 and 5. This Relaxin Agonist schedule is one of the three treatment schedules from Cohorts 1, 2, or 3.
• FIG. 6 depicts a design of the single-blind placebo-conrolled randomized treatment part of the overall study design shown in FIG. 4.
• FIG. 5 After conclusion of the Open-Label Safety Run-In Part (shown in FIG. 5), and after the appropriate Relaxin Agonist dose schedule is determined, approximately 80 patients are randomized 1 : 1 to Relaxin Agonist plus terlipressin (Cohort 4) or terlipressin with placebo (Cohort 5).
• SIRS systemic inflammatory response syndrome
• Relaxin Agonist is administered to patients in Cohort 4 on the first day as an infusion of 4.0 mg over 4 h, followed by 5.0 mg SC at 12 h after initiation of the infusion, followed by 10.0 mg SC at 24 h and thereafter once a day up to 24 h after achievement of an HRS response (either Partial or Full) based on SCr/ AKI stage or up to Day 14.
• FIG. 7 depicts a design of the open-label terlipressin non-responder part of the overall study design shown in FIG. 4. Patients that do not respond to terlipressin in Cohort 5 are discontinued. After discontinuation, patients are allowed to enter Cohort 6 (Terlipressin Non-Responder Part) to receive Relaxin Agonist with the same dosing and schedule Cohort 4. No patient from any Cohort other than Cohort 5 is allowed in Cohort 6.
• Terlipressin non-responders are defined as follows: if on day 4, the serum creatinine has improved by less than 10% or is at the same level or higher than baseline the patient are considered a non-responder and drop out of Cohort 5. These patients are eligible for entry in Cohort 6. All patients receive albumin standard of care.
• AKI stage 2 or 3 (see Table 11; AKI defined by any of the followings: 1) increase in SCr (SCr) > 0.3 mg/dl (or > 26.5 micromolar/L) within 48 h, or 2) increase > 50% in baseline SCr, which is known or presumed to have occurred within the prior seven days.
• MELD Model for End-Stage Liver Disease
• nephrotoxic drugs e.g., aminoglycosides, amphotericin, cyclosporine, NSAIDS (e.g., ibuprofen, naproxen, celecoxib), significant exposure to radiographic contrast agents (large doses or multiple injections of iodinated contrast media).
• NSAIDS e.g., ibuprofen, naproxen, celecoxib
• Shock hypervolemic-, cardiogenic-, or vasodilatory/distributive shock
• mean arterial blood pressure (MAP) ⁇ 70 mmHg or systolic blood pressure ⁇ 90 mmHg along with hypoperfusion.
• Sepsis or uncontrolled bacterial infection e.g., persisting bacteremia, persisting ascitic fluid leucocytosis, fever, increasing leucocytosis with vasomotor instability
• qSOFA quick sepsis-related organ dysfunction assessment
• Haematuria or microhaematuria (more than 50 red blood cells per high power field).
• Severe cardiovascular and pulmonary diseases including, but not limited to, unstable angina, pulmonary edema, congestive heart failure requiring increasing doses of drug therapy, persisting symptomatic peripheral vascular disease, or any other cardiovascular disease judged by the Investigator to be severe.
• TIPS Transjugular intra-hepatic systemic shunt
• vasopressors including midodrine, unless used for only 48 h before screening; in this case a wash-out period of 8 h before randomization is used.
• Subject is not suitable to participate in the study for any reason (including, but not limited to co-morbidities, history of non-compliance with study visits, procedures, or drug administration) in the opinion of the Investigator.
• terlipressin can be increased up to 8.0 mg per day; i.e., 2.0 mg every six hours.
• terlipressin dose administration across all Cohorts, all patients in all Cohorts (1-6) are treated with terlipressin, administered as a 1.0 mg bolus IV infusion (Img over 2 minutes) every 6 h. Terlipressin dosing should continue up to 24 h after achievement of an HRS response (either Partial or Full) based on SCr/AKI stage or up to day 14.
• terlipressin dose modification across all Cohorts if SCr has decreased by less than 25% of QLY SCr (i.e., less than 25% of the SCr value when the patient was randomized) at day 3 (after 2 days of terlipressin treatment), and if terlipressin shows acceptable safety, terlipressin can be increased to 8.0 mg per day; i.e., 2.0 mg every six hours.
• the dose should not be increased in patients with coronary artery disease or if circulatory overload, pulmonary edema or bronchospasm is present.
• terlipressin can be re-started in the Investigator’s judgment, at an equal or lower dose as per protocol. If the AE consists of cardiac ischemia or mesenteric ischemia, dosing should NOT be restarted.
• terlipressin non-responder definition (only applicable for Cohort 5) if on day 4, SCr has decreased by less than 10% of QLY SCr or is at the same level or higher than QLY SCr, the patient is considered a non-responder and drops out of Cohort 5. These patients are eligible for entry into Cohort 6.
• the first Relaxin Agonist administration commences immediately following the first terlipressin administration.
• Relaxin Agonist should be dosed up to 24 h after achievement of an HRS response (either Partial or Full) based on SCr/AKI stage or up to Day 14.
• the pre-treatment period occurs prior to administration of study drug and includes performing baseline assessments and collection of prior medication information.
• the qualifying SCr value (SCr value at least 48 h after both diuretic withdrawal and the beginning of albumin fluid challenge) is considered the QLY SCr value and is drawn no more than 8 h prior to start of study drug.
• the QLY SCr value should be > 1.5 mg/dL. No subjects should be randomized unless their QLY SCr has been obtained within 8 h prior to randomization and start of study drug. If there is a delay in subject randomization, then the QLY SCr value is redrawn so that the value is collected within 8 h prior to randomization and start of study drug to verify that the subject still meets the inclusion criterion for QLY SCr.
• Other baseline assessments are performed no more than 24 h prior to start of study drug.
• the active study period extends from the initiation of study treatment through Day 14 or discharge from the hospital for any reason, whichever occurs first. Study drug is administered as described above in the Study Design.
• Terlipressin dosing should continue up to 24 h after achievement of an HRS response based on SCr/AKI stage, or up to Day 14. Following the first SCr level returning to a value within 0.3 mg/dl (26.5 micromol/L) of the BL SCr value, or a regression of AKI stage with a reduction of SCr > 0.3 mg/dl above the BL SCr value, the second SCr value is obtained a minimum of 2 h after the first SCr value. Efforts are made to collect these SCr values. All information regarding RRT, TIPS, liver transplant, or open-label vasopressor use are collected.
• the follow-up period begins after the end of the study treatment and concludes 90 days following the start of treatment. All subjects return for follow-up on Day 30 ( ⁇ 2), and 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 first day of study drug administration (or from randomization for those subjects who do not receive 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 SAE assessments are collected.
• Efficacy assessments are evaluated as followed: serum creatinine are collected at baseline, once daily during treatment; and then once daily (regardless of treatment status) until Day 14 or hospital discharge, whichever occurs first. If SCr assessments are performed more than once daily as part of the subject’s medical care, all values obtained each day are recorded on the eCRF. SCr values obtained after RRT, TIPS, liver transplant, or open-label vasopressor use are excluded from the efficacy evaluation.
• the primary efficacy variables include:
• the secondary efficacy variables include:
• the exploratory evaluation criteria include:
• Physical Examination A physical examination including assessment of the following is performed by the investigator or his/her designee: Head, ears, nose and throat, Neck/thyroid, Extremities and the Cardiovascular, Integumentary, Lymphatic, Nervous, Musculoskeletal, Respiratory systems is evaluated at specific timepoints. Any clinically significant change in the physical examination findings during the study is considered an AE and recorded on the eCRF.
• Vital Signs The assessment of vital signs (heart rate, blood pressure, respiratory rate, body temperature, height and weight are recorded.
• Safety/12-lead ECGs 12-lead ECGs are performed in triplicate, one minute apart, at specific timepoints. The ECG results are recorded as normal or abnormal on the eCRF. All abnormal results are be evaluated as either clinically significant or not clinically significant by the investigator. Any new, abnormal, clinically significant ECG result is recorded as an AE. A copy of each ECG tracing remains with the source documents.
• Clinical safety laboratory assessments With the exception of urine pregnancy tests, serum creatinine, CBC, INR and electrolytes, all protocol specified laboratory tests are performed at a central study laboratory. Detailed instructions for the collection, handling and reporting of clinical laboratory samples are provided to sites in a laboratory manual provided by the central laboratory prior to site initiation.
• the abnormal tests may be repeated, if appropriate, to ensure the validity of the abnormal result. If the abnormal, clinically significant abnormal results are valid, the laboratory test(s) are to be repeated every two weeks until the results are within normal limits or are no longer considered clinically significant by the investigator. Any new, abnormal, clinically significant laboratory result is recorded as AE.
• Laboratoratory assessment include: Haemoglobin, haematocrit, RBC, MCV, MCH, MCHC, reticulocytes, haptoglobin, WBC with differential, platelet count and platelet aggregation test, 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, ketones, glucose, assessment of multi-organ dysfunction (e.g., according to CLIF-SOFA score), Encephalopathy Score, Systemic Inflammatory Response Syndrome (SIRS) assessment, Quick sepsis-related organ dysfunction assessment (qSOFA) (
• SEP ID NO: 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—
• SEP ID NO: 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-
• SEP ID NO: 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-
• W-S- Aib-R-K-K(Ac)-NH 2 SEQ-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)-NH 2
• SEQ ID NO: 100 H2-relaxin chain A
• SEQ ID NO: 101 H2-relaxin chain B

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