Modified urocortin 3

Abstract

Provided is a modified Urocortin 3 (Ucn3) comprising a covalently attached polymer comprising amino acids, wherein the polymer inhibits degradation and / or elimination of the modified Ucn3 in a subject, and wherein the modified Ucn3 retains corticotropin-releasing factor receptor type 2 (CRFR2) agonist activity. Also provided is a nucleic acid molecule encoding the modified Ucn3. Additionally provided is a vector comprising the above-described nucleic acid molecule. Further provided is a cell comprising the above-described vector. A method of treating a subject suffering from or diagnosed with a disease, disorder, or medical condition mediated by CRFR2 activity is also provided. A method of preparing the above-described modified Ucn3 is additionally provided. Further provided is a method of preparing the above-described modified Ucn3. Use of the above-described modified Ucn3, the above-described nucleic acid, the above-described vector, and the above-described cell for the manufacture of a medicament for the treatment of a disease, disorder, or medical condition mediated by CRFR2 activity is also provided.

Description

[0001] MODIFIED UROCORTIN 3

[0002] BACKGROUND OF THE INVENTION

[0003] The present application generally relates to therapeutic peptides. More specifically, the invention is directed to modified Urocortin 3 (Ucn3) having decreased degradation and / or elimination in mammals.

[0004] Urocortin 3 (Ucn3) is a 38-amino acid peptide that functions as an agonist at corticotropinreleasing factor receptor type 2 (CRFR2), with minimal agonist activity at corticotropin-releasing factor receptor type 1 (CRFR1). The Ucn3 sequence is:

[0005] FTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI (SEQ ID NO: 1).

[0006] Stresscopin (Sen) is a 40-amino acid peptide having essentially the same activity as Ucn3. The sequence of Sen is identical to Ucn3, with an additional threonine and lysine at the amino terminus, thus having the sequence:

[0007] TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI (SEQ ID NO: 2).

[0008] Since Sen is Ucn3 with an additional TK at the amino terminus, Sen can be considered to be modified Ucn3, or “TK-Ucn3”.

[0009] Ucn3 and Sen are useful for treatment of cancer, infectious diseases, fibrotic diseases, inflammatory diseases, neurodegenerative diseases, autoimmune diseases, and heart and vascular diseases, including heart failure (WO 2009 / 040027; US 2011 / 0105397).

[0010] Since peptides like Ucn3 and Sen are rapidly cleared when administered to mammals, Sen has been conjugated to polyethylene glycol (PEG) to extend plasma half-life of the peptide (US 2011 / 0105397). However, it has been increasingly recognized that treating patients with PEGylated drugs can lead to the formation of antibodies that specifically recognize and bind to PEG (i.e., anti- PEG antibodies). Anti-PEG antibodies are also found in patients who have never been treated with PEGylated drugs but have consumed other products containing PEG. Consequently, treating patients who have acquired anti-PEG antibodies with PEGylated drugs results in accelerated blood clearance, low drug efficacy, hypersensitivity, and, in some cases, life-threatening side effects (Thi et al., 2020). In practice, PEGylation has been shown to be an effective means of extending the half-life of Sen in studies involving na(De pre-clinical species (e.g., rats, dogs, and non-human primates). However, PEGylated Sen provides for lower than predicted drug exposure in healthy human subjects (Nnane et al., 2016).

[0011] Thus, there is a need to provide modified Ucn3 and Sen with increased plasma half-life that is not PEGylated. The present invention satisfies that need.

[0012] SUMMARY OF THE INVENTION

[0013] Provided is a modified Urocortin 3 (Ucn3) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to SEQ ID NO :1 , the modified Ucn3 further comprising a covalently attached polymer comprising amino acids, wherein the polymer inhibits degradation and / or elimination of the modified Ucn3 in a subject, wherein the modified Ucn3 retains corticotropin-releasing factor receptor type 2 (CRFR2) agonist activity. In some embodiments, the modified Ucn3 further comprises a threonine and a lysine at the N terminus of SEQ ID NO: 1 , such that the modified Ucn3 comprises a stresscopin (Sen) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity SEQ ID NO: 2.

[0014] In some embodiments, the polymer comprises amino acids consisting of proline and alanine residues (PA) or amino acids consisting of proline, alanine, and serine residues (PAS). In some embodiments, the polymer is PA. In some embodiments, the polymer is PAS. In some embodiments, the polymer comprises at least 100 amino acids. In some embodiments, the polymer comprises the amino acid sequence selected from the group consisting of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 3), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 4), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 5), SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 6), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 7), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 8), ASAAAPAAASAAASAPSAAA (SEQ ID NO: 9), APAAPAPAPAAPAPAPA (SEQ ID NO: 10), AAPAPAPAAPAPAPAAP (SEQ ID NO: 11), APPPAPPPAP (SEQ ID NO: 12), PAPPPAPPPA (SEQ ID NO: 13), AAPAAPAPPAAAPAAPAPPA (SEQ ID NO: 14), and AAAAPAAAAAAAPAAA (SEQ ID NO: 15), or permuted or circular permuted versions or multimer(s) of these sequences as a whole or parts of these sequences. In some embodiments, the polymer is terminated by a proline. In some embodiments, the polymer comprises SEQ ID NO: 3 repeated at least ten times. In some embodiments, the polymer comprises SEQ ID NO: 3 repeated at least thirty times. In some embodiments, the polymer is terminated by a proline. In some embodiments, the PA or PAS polymer is covalently bound to the N-terminus or the C-terminus of the Ucn3 or Sen.

[0015] In some embodiments, the polymer is covalently linked to at least one internal amino acid residue of the Ucn3 or Sen. In some embodiments, the at least one internal amino acid residue covalently linked to the polymer is residue 10, 16, 17, 19, 21 , 23, 26, 29, 30, 32, 33, 36, and / or 37 of SEQ ID NO: 1 or residue 12, 18, 19, 21 , 23, 25, 28, 31 , 32, 34, 35, 38, and / or 39 of SEQ ID NO: 2. In some embodiments, the modified Unc3 further comprises a linker between the modified Ucn3 or Sen and the polymer.

[0016] In some embodiments, the modified Ucn3 comprises more than one polymer. In some embodiments, the more than one polymer is independently a PA or PAS polymer. In some embodiments, the more than one polymer comprises a polymer at a terminus of the modified Ucn3 or Sen and a polymer linked to at least one internal amino acid residue of the Ucn3 or Sen.

[0017] In some embodiments, the modified Ucn3 is in a pharmaceutically acceptable carrier. In some embodiments, the modified Ucn3 is in a formulation that can be aerosolized.

[0018] Also provided is a nucleic acid molecule encoding the above-described modified Ucn3. Additionally provided is a vector comprising the above-described nucleic acid molecule. Further provided is a cell comprising the above-described vector. In some embodiments, the cell is capable of expressing the modified Ucn3.

[0019] A method of treating a subject suffering from or diagnosed with a disease, disorder, or medical condition mediated by CRFR2 activity is also provided. The method comprises administering to a subject in need of such treatment a therapeutically effective amount of the modified Ucn3 described above. A method of preparing the above-described modified Ucn3 is additionally provided. The method comprises obtaining the above-described cell and expressing the modified Ucn3. In some embodiments, the disease, disorder, or medical condition is a cancer, an autoimmune disease, a fibrotic disease, an inflammatory disease, a neurodegenerative disease, an infectious disease, a lung disease, a heart disease, a vascular disease, or a metabolic disease. In some embodiments, the disease, disorder, or medical condition is vasculitis and / or excessive angiogenesis in an autoimmune disorder, systemic sclerosis, multiple sclerosis, Sjogren's disease, a vascular malformation in a blood and / or lymph vessel, portal vein hypertension, liver ascites, pulmonary hypertension, idiopathic pulmonary hypertension, atrial hypertension, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary fibrosis, DiGeorge syndrome, hereditary hemorrhagic telangiectasia, cavernous hemangioma, cutaneous hemangioma, a lymphatic malformation, transplant adenopathy, atherosclerosis, vascular anastomoses, adipose tissue in obesity, allograft rejection, a skin disease, psoriasis, warts, allergic dermatitis, scar keloids, pyogenic granulomas, blistering disease, Kaposi sarcoma in an AIDS patient, systemic sclerosis, an eye disease, persistent hyperplastic vitreous syndrome, diabetic retinopathy, retinopathy of prematurity, choroidal neovascularization, pulmonary hypertension, asthma, nasal polyps, rhinitis, chronic airway inflammation and obstruction, cystic fibrosis, acute lung injury, bronchiolitis obliterans organizing pneumonia, a gastrointestinal tract disease, inflammatory bowel disease, periodontal disease, ascites, peritoneal adhesions, liver cirrhosis, a reproductive system disease, endometriosis, uterine bleeding, ovarian cysts, ovarian hyperstimulation, a bone or joint disease, arthritis, synovitis, osteomyelitis, osteophyte formation, HIV- induced bone marrow angiogenesis, kidney disease, or early diabetic nephropathy. In some embodiments, the disease, disorder, or medical condition is metabolic disease, pulmonary disease, or heart failure. In some embodiments, the disease, disorder, or medical condition is heart failure. In some embodiments, the administration is by injection. In some embodiments, the modified Ucn3 is aerosolized and is administered by inhalation.

[0020] Further provided is a method of preparing the above-described modified Ucn3. In some embodiments, the method comprises obtaining the above-described cell and expressing the modified Ucn3. In some embodiments, the method comprises expressing a modified Ucn3 from the abovedescribed cell or produced by solution or solid phase techniques, then covalently attaching a polymer using chemical methods.

[0021] Additionally provided is the use of the above-described modified Ucn3, the above-described nucleic acid, the above-described vector, or the above-described cell for the manufacture of a medicament for the treatment of a disease, disorder, or medical condition mediated by CRFR2 activity. In some embodiments, the disease, disorder, or medical condition is metabolic disease, pulmonary disease, or heart failure. In some embodiments, the disease, disorder, or medical condition is heart failure.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way. FIGS. 1 A and 1 B are illustrations of a generalized depiction of a native Sen bound to its receptor. FIG. 1A shows the initial binding of a urocortin with a corticotropin-releasing factor receptor type 2 (CRFR2). FIG. 1 B shows the conformational change in the CRFR2 induced by the binding. In both FIG. 1 A and FIG. 1 B, the region above the lipid bilayer is the extracellular region and the region below the lipid bilayer is the intracellular region. NECD: N-terminal extracellular domain.

[0024] FIG. 2 is an illustration of PASylated Sen derivatives with varying PAS lengths.

[0025] FIGS. 3A and 3B are graphs illustrating the bioanalytical characterization of PAS200-A28C- Scn with size-exclusion chromatography (FIG. 3A) and ESI-mass spectroscopy (FIG. 3B).

[0026] FIG. 4A is a graph showing the activation of corticotrophin-releasing factor receptor type 2 (CRFR2) by Sen and Sen derivatives (PAS200-A28C-Scn, PAS400-A28C-Scn, PAS600-A28C-Scn, and PAS800-A28C-Scn). FIG. 4B is a typical cAMP standard curve. Data are presented as mean ± SEM.

[0027] FIG. 5 is a graph showing the plasma concentrations of PAS400, PAS600, and PAS800 over time following single subcutaneous bolus dosing of PAS400 at 0.1 mg / kg, PAS600 at 0.144 mg / kg, and PAS800 at 0.188 mg / kg in dogs (N=3 ± SEM).

[0028] FIG. 6 is a graph showing the one-compartment model fit of canine plasma concentrations of PAS600-A28C-Scn over time following subcutaneous bolus dosing at 0.144 mg / kg.

[0029] FIG. 7 is a graph showing the plasma concentrations of PAS200, PAS400, PAS600, and PAS800 over time following single subcutaneous bolus dosing of PAS200 at 0.5 mg / kg, PAS400 at 0.892 mg / kg, PAS600 at 1 .285 mg / kg, and PAS800 at 1 .677 mg / kg in rats (N=4 ± SEM).

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] Abbreviations and Definitions

[0032] Urocortin 3 (Ucn3): As used herein, the terms “Urocortin 3” and “Ucn3” refer to a 38-amino acid peptide that functions as an agonist at corticotropin-releasing factor receptor type 2 (CRFR2), with minimal agonist activity at corticotropin-releasing factor receptor type 1 (CRFR1). Ucn3 has the sequence: FTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI (SEQ ID NO: 1). The terms “modified Urocortin 3” and “modified Ucn3” refer to Ucn3 that has a covalently attached polymer. The polymer may consist of proline and alanine residues (PA) or proline, alanine, and serine residues (PAS). The polymer inhibits degradation and / or elimination of the modified Ucn3, thereby increasing its plasma half-life. Modified Ucn3 has a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to SEQ ID NO: 1 .

[0033] In some embodiments, modified Ucn3 has an additional threonine and lysine at the amino terminus, thus having the sequence of Stresscopin. As used herein, the terms “Stresscopin,” “Sen,” and “TK-Ucn3” refer to a 40-amino acid peptide having essentially the same activity as Ucn3. Sen has the sequence: TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI (SEQ ID NO: 2). The terms “modified Stresscopin” and “modified Sen” refer to Sen that has a covalently attached polymer. The polymer may be PA or PAS. The polymer inhibits degradation and / or elimination of the modified Sen, thereby increasing its plasma half-life. Modified Sen has a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to SEQ ID NO: 2. In some embodiments, modified Ucn3 has a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to SEQ ID NO: 2.

[0034] Conservative changes: As used herein, when referring to mutations in a nucleic acid molecule, "conservative changes” are those in which at least one codon in the protein-coding region of the nucleic acid has been changed such that at least one amino acid of the polypeptide encoded by the nucleic acid sequence is substituted with another amino acid having similar characteristics. Examples of conservative amino acid substitutions are ser for ala, thr, or cys; lys for arg; gin for asn, his, or lys; his for asn; glu for asp or lys; asn for his or gin; asp for glu; pro for gly; leu for ile, phe, met, or val; val for ile or leu; ile for leu, met, or val; arg for lys; met for phe; tyr for phe or trp; thr for ser; trp for tyr; and phe for tyr.

[0035] Functional activity: As used herein, the term "functional activity" refers to the biological effect of a substance on a living cell or organism. Accordingly, the terms "functional protein," “functional peptide,” and "functional polypeptide" as used herein relate to proteins, peptides, and polypeptides that are capable of inducing, for example, a biological activity of Ucn3.

[0036] Isolated polypeptide: As used herein, the term “isolated polypeptide” refers to a polypeptide molecule that is present in a form other than found in nature in its original environment with respect to its association with other molecules. The term “isolated polypeptide” encompasses a “purified polypeptide” which is used herein to mean that a specified polypeptide is in a substantially homogenous preparation, substantially free of other cellular components, other polypeptides, viral materials, or culture medium, or when the polypeptide is chemically synthesized and substantially free of chemical precursors or by-products associated with the chemical synthesis. A “purified polypeptide” can be obtained from natural or recombinant host cells by standard purification techniques or by chemical synthesis. The term “isolated polypeptide” also encompasses a “recombinant polypeptide,” which is used herein to mean a hybrid polypeptide produced by recombinant DNA technology or chemical synthesis having a specified polypeptide molecule covalently linked to one or more polypeptide molecules which do not naturally link to the specified polypeptide.

[0037] PASylation or PASylated and PAylation or PAylated: As used herein, the terms “PASylation” and “PASylated” are broadly defined to include Ucn3 or Sen conjugated to conformationally disordered polymer sequences comprised of the amino acids Pro, Ala, and Ser (each a “PAS” group). Where only Pro and Ala polymers are conjugated, the Ucn3 or Sen is PAylated with “PA” groups. Those of skill in the art will recognize that a PAS or PA group may contain conservative substitutions, and the entire random coil comprising the Pro, Ala, and Ser, or Pro and Ala amino acids may also include conservative substituents. Hence, the terms “PASylation” and “PAylation” refer to attachment of a solvated random chain with large hydrodynamic volume to the Ucn3 or Sen peptide. This amino acid string adopts a bulky random coil structure, which significantly increases the size of the resulting modified peptide. By virtue of the significantly increased size of the modified peptide, typically rapid clearance of the biologically active component, usually via kidney filtration, is retarded by one to two orders of magnitude. Similarly, the bulk of the random coil structure may prevent the enzymatic degradation of the biologically active component.

[0038] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.

[0039] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents. Water is a preferred carrier when a compound is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A compound, if desired, can also combine minor amount of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates, or phosphates. Antibacterial agents such as a benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compounds in combination with carriers are known to those of skill in the art.

[0040] Pharmaceutically acceptable salt: As used herein, the term “pharmaceutically acceptable salt” includes those salts of a pharmaceutically acceptable compound formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, and tartaric acids, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, and procaine. If the compound is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Such acids include acetic, benzene-sulfonic (besylate), benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic, and the like. Particularly preferred are besylate, hydrobromic, hydrochloric, phosphoric, and sulfuric acids. If the compound is acidic, salts may be prepared from pharmaceutically acceptable organic and inorganic bases. Suitable organic bases include, but are not limited to, lysine, N,N’-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylene diamine, meglumine (N-methyl-glucamine), and procaine. Suitable inorganic bases include, but are not limited to, alkaline and earth-alkaline metals such as aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Methods for synthesizing such salts are known to those of skill in the art.

[0041] As used herein, the terms “polypeptide,” “protein,” and “peptide” interchangeably refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms “polypeptide,” “protein”, and “peptide” also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms are glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, mimetics (Mason, 2010), and the like. Modifications also include intra-molecular crosslinking and covalent attachment of various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, and the like. In addition, modifications may also include cyclization, branching, and cross-linking. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide. The terms “protein” and “polypeptide” may also encompass a “purified” polypeptide that is substantially separated from other polypeptides in a cell or organism in which the polypeptide naturally occurs (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, and 100% free of contaminants).

[0042] As used herein, the terms “primer,” “probe,” and “oligonucleotide” may interchangeably refer to a relatively short nucleic acid fragment or sequence. They can be DNA, RNA, or a hybrid thereof, or chemically modified analogs or derivatives thereof. Typically, they are single-stranded. However, they can also be double-stranded having two complementing strands that can be separated by denaturation. In certain aspects, they are of a length of from about 8 nucleotides to about 200 nucleotides, preferably from about 12 nucleotides to about 100 nucleotides, and more preferably about 18 nucleotides to about 50 nucleotides. They can be labeled with detectable markers or modified in any conventional manners for various molecular biological applications.

[0043] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” refers to those amounts that, when administered to a particular subject in view of the nature and severity of that subject’s disease or condition, will have a desired therapeutic effect, e.g., an amount that will cure, prevent, inhibit, or at least partially arrest or partially prevent a target disease or condition.

[0044] Transformed, transfected or transgenic: A cell, tissue, or organism into which a foreign nucleic acid, such as a recombinant vector, has been introduced is considered “transformed,” “transfected,” or “transgenic.” A “transgenic” or “transformed” cell or organism also includes progeny of the cell or organism, including progeny produced from a breeding program employing such a “transgenic” cell or organism as a parent in a cross.

[0045] Treatment: As used herein in the context of modified Ucn3 or Sen administration, the terms "treat", "treatment", and the like, refer to relief from or alleviation of pathological processes mediated by modified Ucn3 or Sen administration. In the context of the present invention insofar as it relates to any of the other conditions recited herein below, the terms "treat", "treatment", and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.

[0046] Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”.

[0047] Linker: As used herein, the term “linker” refers to a short amino acid sequence that separates multiple domains of a polypeptide. Modified Urocortin 3 and Stresscopin

[0048] Provided are Ucn3 and Sen that further comprise amino acid polymers, for example, polymers consisting of proline and alanine residues (PA) or proline, alanine, and serine residues (PAS).

[0049] Thus, in some embodiments, a modified Ucn3 is provided. The modified Ucn3 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to SEQ ID NO: 1 . In these embodiments, the modified Ucn3 further comprises a covalently attached polymer comprising amino acids, wherein the polymer inhibits degradation and / or elimination of the modified Ucn3 in a subject, and wherein the modified Ucn3 retains corticotropin-releasing factor receptor type 2 (CRFR2) agonist activity.

[0050] In various embodiments, the modified Ucn3 further comprises a threonine and a lysine at the N terminus of SEQ ID NO: 1 such that the modified Ucn3 comprises a Sen having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to SEQ ID NO: 2.

[0051] The polymers of these embodiments can be a variety of lengths and molecular weights. In some embodiments, the polypeptide forms a random coil structure. The polymers can have any length. In some embodiments, the polymer length is less than 200 amino acids. In other embodiments, the polymer length is 200 to 1000 amino acids, including in multiples of 200. Each 200 amino acid biopolymer unit confers a calculated molecular weight of about 17 kDa to the molecule to which it is attached. The use of modified amino acids or amino acid mimetics in these polymers is also envisioned.

[0052] The polymer of some of these modified Ucn3 comprises amino acids consisting of proline and alanine residues (i.e., is PAylated) or proline, alanine, and serine residues (i.e., is PASylated). In some embodiments, the polymer comprises any one or any combination of the following amino acid sequences:

[0053] ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 3),

[0054] AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 4),

[0055] APSSPSPSAPSSPSPASPSS (SEQ ID NO: 5),

[0056] SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 6),

[0057] SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 7),

[0058] AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 8),

[0059] ASAAAPAAASAAASAPSAAA (SEQ ID NO: 9),

[0060] APAAPAPAPAAPAPAPA (SEQ ID NO: 10),

[0061] AAPAPAPAAPAPAPAAP (SEQ ID NO: 11),

[0062] APPPAPPPAP (SEQ ID NO: 12),

[0063] PAPPPAPPPA (SEQ ID NO: 13),

[0064] AAPAAPAPPAAAPAAPAPPA (SEQ ID NO: 14), and AAAAPAAAAAAAPAAA (SEQ ID NO: 15), or permuted or circular permuted versions or multimer(s) of these sequences as a whole or parts of these sequences. It has been discovered that terminating the polymer with a proline aids in the subsequent purification of the modified Ucn3 in some cases. Thus, in various embodiments, the polymer is terminated by a proline.

[0065] Particularly useful polymers comprise SEQ ID NO: 3, repeated at least ten to thirty times, optionally terminated by a proline.

[0066] The polymer or polymers (e.g., the PA and / or the PAS polymer or polymers) of the modified Ucn3 can be covalently bound to the N-terminus or the C-terminus of the Ucn3 or Sen. Additionally or alternatively, the polymer or polymers can be bound to any amino acid residues of Ucn3 or Sen, including the side chain of residue 1 or 38 of Ucn3 or the side chain of residue 1 or 40 of Sen, and / or at least one internal amino acid residue of Ucn3 or Sen.

[0067] To determine which amino acid residues could be bound to an amino acid polymer without losing CRFR2 agonist activity, the inventors evaluated structural data and which residues could be PEGylated without loss of activity, as disclosed in US 2011 / 0105397.

[0068] Ucn3 and Sen appear to be typical hormones for a Class B G-protein coupled receptor (GPCR), existing mainly in the form of a helical secondary structure. Where studied, hormones for Class B bind with their C-terminus initially via interacting with the N-terminal extracellular domain (NECD) of the receptor, and their N-terminus with the juxta-membrane portion of the transmembrane helical bundle (FIG. 1 A). Once bound in this manner, it is thought that a conformational change is induced in the receptor which permits coupling of the G-protein, ultimately leading to signal transduction (FIG. 1 B). From FIGS. 1A and 1 B (adapted from Hoare, 2007), it is evident that a substantial portion of the hormone is solvent exposed and hence potentially tolerant of hydrophilic residues. Modification with amino acid chains (e.g., PA or PAS) should be useful in prolonging the in vivo half-life of the polypeptide whilst maintaining some or all of its efficacy.

[0069] The amino acid polymers can be added to any residue of Ucn3 or Sen. In order to identify other residues where addition of amino acid polymers might be best tolerated in Ucn3 and Sen, US 2011 / 0105397, which provides a full PEG Cys scan, was referred to. This reference provides the following 13 residues in Sen (SEQ ID NO: 2): residues 12, 18, 19, 21 , 23, 25, 28, 31 , 32, 34, 35, 38, and 39. These residues of Sen correspond to residues 10, 16, 17, 19, 21 , 23, 26, 29, 30, 32, 33, 36, and 37 of Ucn3 (SEQ ID NO: 1).

[0070] Thus, in various embodiments, the polymer in the modified Ucn3 is covalently linked to residue 10, 16, 17, 19, 21 , 23, 26, 29, 30, 32, 33, 36, and / or 37 of SEQ ID NO: 1 or residue 12, 18, 19, 21 , 23, 25, 28, 31 , 32, 34, 35, 38, and / or 39 of SEQ ID NO: 2.

[0071] To facilitate conjugation of amino acid polymers to Ucn3 or Sen, a linker between the Ucn3 or Sen and the polymers may be utilized. Any linker known in the art that can facilitate this conjugation may be utilized. Examples are provided in US 2011 / 0105397, e.g., at para. 135, and references cited therein. In some embodiments, the linker moiety is N-(ethylcarbonyl)succinimide or methylcarbonyl. Those linkers have the following structures:

[0072] N-(ethylcarbonyl)succinimide methylcarbonyl

[0073] The modified Ucn3 described herein can comprise any number of polymers, including 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or 13 polymers. Where more than one polymer is on the modified Ucn3, the polymers can be the same or different in composition and / or length.

[0074] In some embodiments, a polymer is at the N- or C-terminus of the Ucn3 or Sen. Such terminal polymers can be produced genetically, e.g., by coding the polymer with the Ucn3 or Sen in a DNA sequence and expressing that sequence. Thus, a nucleic acid molecule encoding the modified Ucn3 or Sen having an amino acid polymer at either or both of the N- and / or C-terminus is also provided herein, as is a vector comprising that nucleic acid molecule. A cell comprising that vector, including a cell capable of expressing that modified Ucn3 or Sen, is also provided herein.

[0075] Pharmaceutical Preparations and Methods of Administration

[0076] In some embodiments, the modified Ucn3 or Sen described above is formulated in a pharmaceutically acceptable carrier. Those compositions can be administered to a subject at therapeutically effective doses to treat any disease, disorder, or medical condition mediated by CRFR2 activity. The subject can be any mammal, reptile, or avian, including but not limited to horses, cows, dogs, cats, sheep, pigs, chickens, and humans.

[0077] Therapeutically Effective Dosage

[0078] Toxicity and therapeutic efficacy of such compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are preferred. While compositions exhibiting toxic side effects may be used, care should be taken to design a delivery system that targets such compositions to the site affected by the disease or disorder in order to minimize potential damage to unaffected cells and reduce side effects.

[0079] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans and other mammals. The dosage of such compositions lies preferably within a range of circulating plasma or other bodily fluid concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dosage may be formulated in animal models to achieve a circulating plasma concentration range that includes the EC50 (the concentration of the test composition that achieves a half-maximal effect) as determined in cell culture. Such information can be used to more accurately determine useful dosages in humans and other mammals. Composition levels in plasma may be measured, for example, by high performance liquid chromatography.

[0080] The amount of a composition that may be combined with pharmaceutically acceptable carriers to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of a composition contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses. The selection of dosage depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of those skilled in the art.

[0081] The dosage regime for treating a disease or condition with the compositions and / or composition combinations of this invention is selected in accordance with a variety of factors including the type, age, weight, sex, diet, and medical condition of the patient, the route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic, and toxicology profiles of the particular composition employed, whether a composition delivery system is utilized, and whether the composition is administered as a pro-drug or part of a drug combination. Thus, the dosage regime actually employed may vary widely from subject to subject.

[0082] Formulations and Use

[0083] The compositions of the present invention may be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, inhaled, and ophthalmic routes. The individual compositions may also be administered in combination with one or more additional compositions of the present invention and / or together with other biologically active or biologically inert agents ("composition combinations"). Such biologically active or inert agents may be in fluid or mechanical communication with the composition(s) or attached to the compositions) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces. It is preferred that administration is localized in a subject, but administration may also be systemic.

[0084] The compositions or composition combinations may be formulated by any conventional manner using one or more pharmaceutically acceptable carriers and / or excipients. Thus, the compositions and their pharmaceutically acceptable salts and solvates may be specifically formulated for administration, e.g., by parenteral, inhalation or insufflation (either through the mouth or the nose), oral, buccal, parenteral, or rectal administration. The composition or composition combinations may take the form of charged, neutral, and / or other pharmaceutically acceptable salt forms. Examples of pharmaceutically acceptable carriers are those described in Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000).

[0085] The compositions may also take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, controlled- or sustained-release formulations, and the like. Such compositions will contain a therapeutically effective amount of the composition, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0086] Parenteral Administration

[0087] The composition or composition combination may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form in ampoules or in multi-dose containers with an optional preservative added. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or the like. The composition may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0088] For example, a parenteral preparation may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent (e.g., as a solution in 1 ,3-butanediol). Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the parenteral preparation.

[0089] Alternatively, the composition may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use. For example, a composition suitable for parenteral administration may comprise a sterile isotonic saline solution containing between 0.1 % and 90% weight per volume of the composition or composition combination. By way of example, a solution may contain from about 5% to about 20%, more preferably from about 5% to about 17%, more preferably from about 8% to about 14%, and still more preferably about 10% of the composition. The solution or powder preparation may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Other methods of parenteral delivery of compositions will be known to the skilled artisan and are within the scope of the invention.

[0090] Other Systems of Administration

[0091] Various other delivery systems are known in the art and can be used to administer the compositions of the invention. Moreover, these and other delivery systems may be combined and / or modified to optimize the administration of the compositions of the present invention. In some embodiments, the formulation can be aerosolized.

[0092] Active Ingredient Kits

[0093] In various embodiments, the present invention can also involve kits. Such kits can include the compositions of the present invention and, in certain embodiments, instructions for administration. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit longterm storage without losing activity of the components. In addition, if more than one route of administration is intended or more than one schedule for administration is intended, the different components can be packaged separately and not mixed prior to use. In various embodiments, the different components can be packaged in one composition for administration together.

[0094] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain lyophilized phosphatases and in a separate ampule, sterile water, or sterile saline, each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that, upon removal, permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0095] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium, such as a thumb drive, CD-ROM, DVD-ROM, video, audio, and the like. Detailed instructions may not be physically associated with the kit. Instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0096] Methods of Treatment

[0097] A method of treating a subject suffering from or diagnosed with a disease, disorder, or medical condition mediated by CRFR2 activity is also provided. The method comprises administering to a subject in need of such treatment a therapeutically effective amount of any of the modified Ucn3 or Sen described above.

[0098] In some embodiments, the disease, disorder, or medical condition is a cancer, an autoimmune disease, a fibrotic disease, an inflammatory disease, a neurodegenerative disease, an infectious disease, a lung disease, a heart disease, a vascular disease, or a metabolic disease. In some of these embodiments, the disease, disorder, or medical condition is vasculitis and / or excessive angiogenesis in an autoimmune disorder, systemic sclerosis, multiple sclerosis, Sjogren's disease, a vascular malformation in a blood and / or lymph vessel, portal vein hypertension, liver ascites, pulmonary hypertension, idiopathic pulmonary hypertension, atrial hypertension, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary fibrosis, DiGeorge syndrome, hereditary hemorrhagic telangiectasia, cavernous hemangioma, cutaneous hemangioma, a lymphatic malformation, transplant adenopathy, atherosclerosis, vascular anastomoses, adipose tissue in obesity, allograft rejection, a skin disease, psoriasis, warts, allergic dermatitis, scar keloids, pyogenic granulomas, blistering disease, Kaposi sarcoma in an AIDS patient, systemic sclerosis, an eye disease, persistent hyperplastic vitreous syndrome, diabetic retinopathy, retinopathy of prematurity, choroidal neovascularization, pulmonary hypertension, asthma, nasal polyps, rhinitis, chronic airway inflammation and obstruction, cystic fibrosis, acute lung injury, bronchiolitis obliterans organizing pneumonia, a gastrointestinal tract disease, inflammatory bowel disease, periodontal disease, ascites, peritoneal adhesions, liver cirrhosis, a reproductive system disease, endometriosis, uterine bleeding, ovarian cysts, ovarian hyperstimulation, a bone or joint disease, arthritis, synovitis, osteomyelitis, osteophyte formation, HIV-induced bone marrow angiogenesis, kidney disease, or early diabetic nephropathy. In some specific embodiments, the disease, disorder, or medical condition is metabolic disease, pulmonary disease, or heart failure.

[0099] As discussed above, the compositions can be administered by any appropriate method known in the art. In some embodiments, the administration is by injection. In other embodiments, the modified Ucn3 or Sen is aerosolized and is administered by inhalation.

[0100] Methods of Preparation

[0101] The above-described compositions can be prepared by any appropriate method known in the art. Where the polymer is at the N- or C-terminus of the Ucn3 or Sen, the above-described cell comprising a vector encoding the modified Ucn3 or Sen can express the modified Ucn3 or Sen.

[0102] Where the polymer is to be conjugated to one or more internal amino acid residues of Ucn3 or Sen, the modified Ucn3 or Sen can be produced by solution or solid phase techniques, then covalently attaching a polymer using chemical methods. Such techniques and methods are well known in the art.

[0103] Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.

[0104] EXAMPLES

[0105] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein can be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0106] Example 1. Administration of Sen

[0107] Study Objective

[0108] The study determines that long-term (3 month) treatment with Sen protein with induced random coiling in dogs with ischemia-induced, progressive, irreversible heart failure is associated with: (1) preservation and / or improvement of LV structure and function, (2) no change in or long-term reduction in biomarkers of myocardial injury, and (3) absence of significant de novo ventricular arrhythmias or increased susceptibility for malignant arrhythmias compared to placebo (vehicle). Study Protocol

[0109] The study analyzes 24 dogs with advanced heart failure (HF) produced by multiple sequential intracoronary microembolizations (LV ejection fraction < 25%). Dogs are randomized into 3 study groups. Group I (n=8) receives subcutaneous vehicle injections for 3 months, while serving as a placebo control. Group II (n=8) receives chronic therapy with Sen derivative (5 pg / kg, Q5d) for 3 months. Group III (n=8) receives chronic therapy with Sen derivative (15 pg / kg, Q5d) for 3 months. All dosing is performed at the same time of the day on a 20 cm x 24 cm area. Within the 20 cm x 24 cm area that is shaved on the anterior dorsal scapular region (scruff) of the animal’s neck, six regions are outlined with the center of each region being 12 cm apart. The regions are numbered as outlined and the order of injection is region 1 , 5, 3, 6, 2, 4. Hemodynamic, angiographic, and echocardiographic measurements are performed during a left and right heart catheterization under general anesthesia. A left and right heart catheterization are performed at baseline, 7 days prior to placebo vehicle or Sen derivative injection, 24 hrs following the first Sen derivative injection, 24 hrs following the third Sen derivative injection (day 10), 24 hrs following the fifth Sen derivative injection (day 20), 24 hrs following the seventh Sen derivative injection (day 30), 24 hrs following the twelfth Sen derivative injection (day 60), and 24 hrs following the eighteenth Sen derivative injection (day 90). Following the hemodynamic and ventriculographic measurements on day 90, the chest is rapidly opened and a 0.5- 1 .0 g section of left ventricle is quickly removed and flash frozen with Wollenberger clamps cooled in liquid nitrogen for myocardial cyclic guanosine monophosphate (cGMP) analysis. The levels of cGMP in plasma are also analyzed. Then samples for histomorphometric measurements, myocardial receptor and ion channel measurements, and RNA gene chip analysis are removed. Venous blood samples are obtained at the same time of the day in conscious dogs prior to each cardiac catheterization and echocardiographic measurement including days -7, 0-16, 22, 30, 38, 45, 53, 60, 68, 75, 83, and 90. Blood samples (at least 9 mL - 3 x 3 mL) are collected in plastic tubes containing EDTA and Complete protease inhibitor (Roche Biosciences). From a stock solution of the following composition of 1 Complete protease inhibitor tablet dissolved in 2 mL normal saline, each EDTA blood collection tube, contains 40 pL of Complete protease inhibitor per mL of whole blood. Whole blood samples are collected with EDTA and protease inhibitor. The whole blood samples collected with EDTA and protease inhibitor are immediately placed on ice and centrifuged at 3000 rpm for 10 min within 30 min of collection. The plasma is placed in cryostorage tubes and stored upright at -70 “C until analysis to determine Sen derivative plasma concentration. Samples of the dosing solution (2 mL) are placed in cryostorage tubes and stored upright at -70 “C. Separate venous blood samples (serum) are drawn at baseline and at the end of each cardiac catheterization for determination of serum electrolytes, including creatinine, to estimate renal glomerular filtration rate (eGFR). Venous blood is collected at baseline and at the end of each cardiac catheterization for plasma biomarkers. The dog’s body weight is measured monthly just prior to each cardiac catheterization.

[0110] Hemodynamic and Angiographic Measurements

[0111] All hemodynamic measurements are made during left and right heart catheterizations in anesthetized dogs at each specified study time point. The following parameters are evaluated in all dogs: (1) aortic and LV pressures using catheter tip micromanometers (Millar Instruments); (2) peak rate of change of LV pressure during isovolumic contraction (peak +dP / dt) and relaxation (peak - dP / dt); (3) LV end diastolic pressure; (4) cardiac output; (5) stroke volume; (6) cardiac index; and (7) systemic vascular resistance.

[0112] Left ventriculograms (LV) are performed on the dogs during cardiac catheterization after completion of the hemodynamic measurements. The dogs are placed on its right side such that the left ventriculograms are recorded on digital media at 30 frames / sec during a power injection of 20 mL of contrast material (RENO M 60, Squibb Diagnostics). Correction for image magnification is made using a radiopaque grid placed at the level of the LV. LV end systolic and end diastolic volumes are calculated from angiographic silhouettes using the area length method. Premature beats and post- extrasystolic beats are excluded from the analysis. LV ejection fraction is calculated as the ratio of the difference of end diastolic (EDI) and end systolic (ESY) volumes to end diastolic volume times 100:

[0113] LV ejection fraction = [(VolumeEDI - VolumeESY) / VolumeEDI] x 100

[0114] Echocardiographic and Doppler Measurements

[0115] Echocardiographic and Doppler studies are performed in all dogs at all specified study time points using a VIVID 7 ultrasound system (General Electric) with a 3.5 megahertz (MHz) transducer. All echocardiographic measurements are made with the dog placed in the right lateral decubitus position and recorded on a Panasonic 6300 VHS recorder for subsequent offline analysis.

[0116] LV fractional area of shortening (FAS) and LV systolic function are measured from a short axis view at the level of the papillary muscles. LV major and minor semi-axes are measured and used for calculation of LV end-diastolic circumferential wall stress.

[0117] Wall stress is calculated as indicated below:

[0118] Wall Stress = Pb / h(1-h / 2b)(1-hb / 2a2) where P is LV end-diastolic pressure, a is LV major semi-axis, b is LV minor semi-axis, and h is LV wall thickness.

[0119] Global longitudinal strain (GLS) is measured by speckle tracking.

[0120] Mitral inflow velocity is measured by pulsed-wave Doppler echocardiography to assess LV diastolic function. The velocity waveforms is used to calculate: (i) peak mitral flow velocity in early diastole (PE); (ii) peak mitral inflow velocity during LA contraction (PA); (iii) ratio of PE to PA (PE / PA); (iv) time-velocity integral of the mitral inflow velocity waveform representing early filling (Ei); (v) timevelocity integral representing LA contraction (Ai); (vi) ratio of Ei / Ai (Ei / Ai); and (vii) deceleration time of early mitral inflow velocity (DT). Color flow Dopplers assess the presence and severity of functional mitral regurgitation (i.e., regurgitant jet). The severity of the regurgitation, when present, is quantified as the ratio of the area of the regurgitant jet to the area of the left atrium.

[0121] A 24-hour ambulatory ECG Holter monitoring, as performed at all pre-specified time points (baseline, 1 , 2, 14, 30, 60, and 90 days), assesses: (1) peak; (2) average and minimum heart rate; and (3a) average number per hour of single premature beats (PVC’s), (3b) couplets, and (3c) triplets and (3c) episodes of ventricular tachycardia (VT) (> 3 beats). An episode of non-sustained VT is defined as an episode lasting less than 30 seconds. An episode lasting more than 30 seconds is defined as “sustained VT”.

[0122] Circulating Plasma Biomarkers

[0123] Venous blood samples, as obtained at baseline and at each follow-up timepoint (at baseline and following each cardiac catheterization), quantify the following plasma biomarkers: (1) Troponin-I; (2) myoglobin; (3) Big-endothelin (Big-ET); (4) angiotensin-ll (ANG II); (5) norepinephrine (NE); (6) N- Terminal pro-BNP (NT-pro-BNP); (7) atrial natriuretic peptide (proANP); (8) tumor necrosis factoralpha (TNF-a); (9) interleukin-6 (IL-6); (10) C-reactive protein (CRP); (11) procollagen type 1 C- terminal propeptide (PICP); (12) CK-MB; and (13) cyclic guanosine monophosphate (cGMP). Blood samples from 6 normal dogs are compared.

[0124] Histomorphometric Measurements

[0125] From each heart, 3 transverse slices (approximately 3 mm thick) are obtained such that there is one each from basal, middle, and apical thirds of the LV. From each slice, transmural tissue blocks are obtained and embedded in paraffin blocks. Transmural tissue blocks, as obtained from the free wall segment of the slice, are: (i) mounted on cork using Tissue-Tek embedding medium; (ii) rapidly frozen in isopentane pre-cooled in liquid nitrogen; and (iii) stored at -70 “C until used up. The volume fraction of replacement fibrosis (VFRF), volume fraction of interstitial fibrosis (VFIF), myocyte cross- sectional area (MCSA), a measure of cardiomyocyte hypertrophy, capillary density (CD), and oxygen diffusion distance (ODD) are measured as previously described. LV tissue from 6 normal dogs is processed in an identical manner as above and the results used for comparisons.

[0126] Myocardial Receptor and Ion Channel Measurements

[0127] From each heart, ~1-5 g samples of LV anterior free wall are rapidly removed, dissected, and flash frozen at -80 °C for radioligand binding. The density and affinity of beta adrenoceptors and sarcoplasmic reticular calcium release channels are quantified by analyzing saturation isotherms from the specific binding of [3H]-dihydroalprenolol and [3H]-ryanodine to enriched sarcolemmal and sarcoplasmic reticular membranes.

[0128] RNA Gene Chip Analysis

[0129] RNA gene chip analysis, as used with the compositions and methods herein, involves expression profiling, samples taken, treatment groups, and tissues. Whereby there are two samples per round (1 for RNA, 1 for protein), the tissues are stored in RNA later, with half kept at -70 “C for protein.

[0130] Method for collection

[0131] Sections, which are 5 mm3, undergo dissection followed by RNALater rinsing and storage in 1 mL RNALater in labeled 1 .5 mL polypropylene Eppendorf tubes. Vascular tissue (artery or vein) are collected as 1 cm lengths.

[0132] Once the data above is analyzed, it is compared to data obtained from native Sen administration and Sen derivative administration. It is expected that the half-life of the Sen protein is increased without the unwanted immunogenic properties which are produced by PEGylation.

[0133] Example 2. CRFR2 Agonist Activity of PASylated Sen

[0134] Study Objective

[0135] The objective of this study is to evaluate the potential functional effects of test compounds on hCRFR2 (the membrane-bound receptor of Sen) under agonist mode by detection of cAMP levels using a HTRF cAMP kit (LANCE ULTRA PerkinElmer).

[0136] Study Protocol

[0137] Materials

[0138] DMEM:HAM F12 (D6421), Penicillin-Streptomycin (P0781), DPBS (D8537), DMSO (276855- 1 L), HBSS (H8264), BSA, (A7030), HEPES (H3375), IBMX (I5879), and G 418 disulfate salt (A1720) were obtained from Sigma Aldrich. PBS (21 -040-CV) was obtained from Corning. OptiMEM (31985- 062) and TrypLE Express (12604-013) were obtained from Gibco. The LANCE UltracAMP kit (TRF0262) and 384-well proxiplate plate were obtained from PerkinElmer. PEI (23966-1) was obtained from Polysciences Inc. Human stresscopin was supplied by CHI Scientific. PAS200-A28C- hScn, PAS400-A28C-hScn, PAS600-A28C-hScn, and PAS800-A28C-hScn were supplied by XL- protein. hCRFR2 receptor plasmid was obtained from GenScript (UK) (clone ID OHu10803, NM_001883.5, in pcDNA3.1-C-(k)DYK vector).

[0139] Development of the CHO-hCRFR2 receptor containing cell line

[0140] CHO cells were cultured in a t75 cm2flask at 37 “C, 5% (v / v) CO2. On reaching 70% confluence (day 2), the growth medium was discarded, 5 mL of Opti-MEM I reduced-serum medium was added to the cells, and the flask was returned to the incubator. The hCRFR2 receptor plasmid DNA / PEI reagent mixture was prepared at a weight ratio of 1 :3. The DNA / PEI reagent mixture was incubated at room temperature for 20 min before being added to the cells. The cells were further cultured at 37 “C in a humidified atmosphere with 5% (v / v) CO2 for 16 hours (day 3). At this point, the medium was supplemented with complete culture medium DMEM:HAM F12 with 10% FBS and 100 U / mL Pen-Strep. The transfected cells were then further cultured at 37 “C in a humidified atmosphere with 5% (v / v) CO2 for 24 hours before the addition of G418 to establish a CHO-hCRFR2 receptorcontaining cell line.

[0141] CHO-hCRFR2 receptor cAMP assay

[0142] CHO-hCRFR2 cells were grown in complete culture medium DMEM:HAM F12 with 10% FBS and 100 U / ml Pen-Strep in a t75 flask. The growth medium on the cells was discarded and the cells were washed once with 10 mL of D-PBS. 2 mL of TrypLE was then added to the cells for 2 min, the point at which the cells round up. Complete growth medium (8 mL) was added to stop the reaction and the cells were transferred to a sterile 25 mL centrifuge tube (Universal). The cells were counted using a Countess cell counter. An appropriate amount of cells (-1.33 million) was then centrifuged at 350 g for 3 min. The cell pellet was resuspended in 2 mL of complete assay buffer (HBSS buffer, 0.1% BSA, 5mM HEPES supplemented with 0.5 mM IBMX) and transferred to a 384-well proxiplate plate in a final density of 3000 cells / well in a total volume of 4.5 pL. This cell density was chosen based on an initial cell dilution experiment. The plates were then centrifuged at 1000 rpm for 30 seconds and incubated at 37 °C for 15 min.

[0143] The reference compound (human stresscopin) was initially dissolved in water at a concentration of 1 mM, and test compounds were dissolved in PBS to make 100 pM stock solutions. Human stresscopin was further diluted in PBS to a concentration of 100 pM, and all further dilutions of peptides were made in assay buffer. Specifically, peptides were serially diluted in assay buffer in 3- fold steps in low-bind polypropylene tubes and transferred (0.5 pL) to the assay plate. The plates were then agitated at 600 rpm for 30 seconds before being incubated at 37 “C for 60 min. cAMP Standard was prepared according to the kit manufacturer’s instructions in assay buffer with 5 pL of each dilution added to the assay plate. 2.5 pL / well of Eu-cAMP tracer working solution and 2.5 pL / well of ULight™ anti-cAMP working solution were then added to each well of the plate. The plate was centrifuged at 1000 rpm for 30 seconds and then agitated (Eppendorf MixMate) at 600 rpm before being incubated at 25 “C for 1 hour.

[0144] The plate and the samples were then transferred to the Pherastar FSX with HTRF ratios (Fl 665 / 620 *10,000) monitored using 10 flashes for up to 7 cycles. Associated data was exported to Excel and analysed in Prism. Concentration response data was plotted in Prism as % response to human stresscopin (10 mM) and pECso and Emax value were calculated. The upper asymptotes, midpoint slope, and potency (EC50) are determined by fitting the % of activation as a function of compound concentrations to a four-parameter general logistic function using the GraphPad Prism™ software.

[0145] Results

[0146] The standard human stresscopin supplied by CHI scientific was diluted in cAMP assay buffer (0.51 nM-10 mM) for the detection of cAMP in cell samples. Initial experiments were carried out in HEK293-T17 cells, transiently expressing hCRFR2. However, the potency of human stresscopin was considered too high to allow an accurate quantification of its effects (steep Hill slope), most likely due to the very high receptor density. The decision was made to change to the CHO cell line, which stably expresses hCRFR2. The following results describe the cAMP data obtained in the CHO cell line for the control human stresscopin and the PAS analogues.

[0147] The standard purified PAS-hScn powders supplied by Antlia Bioscience and produced by XL- protein were made up in PBS and diluted in cAMP assay buffer (57 pM-10 mM) for the detection of cAMP in CHO-hCRFR2 cell samples. FIG. 4A represents the mean normalised cAMP responses of hScn, PAS200-A28C-hScn, PAS400-A28C-hScn, PAS600-A28C-hScn, and PAS800-A28C-hScn, measured in CHO cells stably expressing hCRFR2 with absolute cAMP levels calculated from the cAMP Standard curve, a typical example of which is shown in FIG. 4B. Each assay point represents the results of 6 separate determinations performed in singlet.

[0148] Table 1 . Potency and relative efficacy of human stresscopin and its PAS analogues. Data are presented as mean ± SEM.

[0149] Discussion

[0150] This example established that PASylated Sen has agonist activity at human CRFR2. The PAS200 analogue was 2.6-fold less potent than the native peptide, hScn, at CRFR2. Surprisingly, doubling the PAS size did not significantly further reduce the potency - the PAS400 analogue was only 3.2-fold less potent than the native peptide. Similarly, increasing the PAS size further to 1 .5-fold (PAS600) and 2-fold (PAS800) did not produce any further loss of potency.

[0151] Example 3. A Single Dose Pharmacokinetic Study of Sen Derivatives Following Subcutaneous Administration in Beagle Dogs

[0152] Study Objective

[0153] The objective of this study was to determine the pharmacokinetic profile of PAS400-A28C- Scn, PAS600-A28C-Scn, and PAS800-A28C-Scn for 14 days following a single subcutaneous bolus dose in Beagle dogs.

[0154] Study Protocol

[0155] Dosing

[0156] Compounds were dissolved in PBS at a concentration of 0.2 mg / mL (PAS400), 0.288 mg / mL (PAS600), and 0.376 mg / mL (PAS800), and dosed according to Table 2 in equimolar amounts.

[0157] Table 2. Dosing of PASylated Sen in dogs (three groups of six dogs).

[0158] Whole blood and plasma sampling

[0159] Following dosing, serial whole blood samples from a jugular vein (0.5 mL per sample) were collected into individual K2EDTA and IBMX (0.5mM) treated containers. Sample timepoints were alternated between A and B cohorts to reduce the total number of bleeds per dog.

[0160] Samples were collected at the following times: pre-dose, post-dose at 0.25, 1 , 2, 4, 8, 16, 24 (8 samples on day 1), 36, 48, 60, 72, 84, 96, 108, and 120 hours (8 samples on days 2-5), then once daily until day 10 (4 samples on days 6-10), and thereafter every two days until day 14.

[0161] Blood samples were placed into a cooling block before being centrifuged at 3000g for 10 minutes at 4 “C to allow withdrawal of the plasma. Samples were centrifuged within 15 minutes of collection. The remaining blood pellets were discarded.

[0162] The plasma was used for determination of the concentration of each PASylated Sen derivative using a sandwich ELISA setup with the high-affinity monoclonal aPAS antibody Avi-PA(S) 1.1 and a bespoke monoclonal antibody with specificity for the C-terminus of Sen. The antibody was created with an immunogen comprising the terminal 12 amino acids of Sen, ensuring high sensitivity and selectivity. Results

[0163] The plasma concentrations from the ELISA assay of plasma samples following subcutaneous (SC) dosing are shown over time in FIG. 5. The data for each derivative was fitted to a one- compartmental model using Phoenix WinNonlin software (Certara). The model fit of the PAS600- A28C-Scn derivative is shown in FIG. 6. The results of the analysis are shown in Table 3.

[0164] Table 3. Pharmacokinetic parameter values from one-compartmental model fit of the dog plasma values over time for PAS400, PAS600, and PAS800 derivatives of Sen following SC bolus dosing.

[0165] Discussion

[0166] The half-lives following subcutaneous dosing for PAS400, PAS600, and PA800 derivatives are 105-, 123-, and 138-fold longer, respectively, than that of the parent peptide, Sen, which is 15 minutes in dogs (US 2011 / 0105397). The significant increase in Tmax that is observed with the PAS600 and PAS800 derivatives compared to the PAS400 is indicative of the changes beyond a simple half-life extension, suggesting PAS size-dependent changes in the rates of absorption from the site of subcutaneous administration and effects on distribution.

[0167] The behavior of other PASylated proteins shows the effect of increasing PAS size on both agonist receptor binding / potency and pharmacokinetic profile. The overall changes in half-life, AUC, clearance, Cmax, and Tmax following extravascular dosing are highly specific to the compound being PASylated. For example, Table 4 shows the increase in half-life over parent protein achieved with other PASylated proteins.

[0168] Table 4. Half-life increase of PASylated compounds over un-PASylated compounds, in multiples of un-PASylated compound (fold-increase). aThis study;bMorath et al., 2015;cSchlapschy et al., 2015

[0169] Example 4. A Single Dose Pharmacokinetic Study of PAS200, PAS400, PAS600, and PAS800 Derivatives of Sen Following Subcutaneous Administration in Rats

[0170] Study Objective

[0171] The objective of this study was to determine the pharmacokinetic profile of PAS200-A28C- Scn, PAS400-A28C-Scn, PAS600-A28C-Scn, and PAS800-A28C-Scn for 14 days following a single subcutaneous bolus dose in rats. Study Design

[0172] Compounds were dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg / mL (PAS200), 1.785 mg / mL (PAS400), 2.570 mg / mL (PAS600), and 3.354 mg / mL (PAS800). Five groups of four male Sprague Dawley rats (body weight: 250-300g) were dosed according to Table 5 in equimolar amounts.

[0173] Table 5. Study Design

[0174] Whole blood and plasma sampling

[0175] Following dosing, serial whole blood samples from a lateral tail vein (about 0.2 mL per sample) were collected into individual K2EDTA- and IBMX- (0.5mM) treated containers. Samples were collected at the following times: Pre-dose, post-dose at 0.5, 2, 4, 8, 12, and 24 hours, and then 24- hour intervals thereafter until the end of the study period for each treatment group.

[0176] For the vehicle treated group, blood was only collected at 2, 8, and 24 hours and then 24-hour intervals thereafter. Sham bleeds were performed for the non-sample timepoints. The same sampling procedure was conducted during the sham bleeds, except for the tail prick that was not performed.

[0177] Blood samples were placed into a cooling block before being centrifuged at 10,000g for 2 minutes at 4 “C and the resultant plasma drawn off. Samples were centrifuged within 15 minutes of collection. The remaining blood pellets were discarded.

[0178] The plasma was used for determination of the concentration of each PASylated Sen derivative using a sandwich ELISA setup with the high-affinity monoclonal aPAS antibody Avi-PA(S) 1.1 and a bespoke monoclonal antibody with specificity for the C-terminus of Sen. The antibody was created with an immunogen comprising the terminal 12 amino acids of Sen, ensuring high sensitivity and selectivity.

[0179] Results

[0180] The plasma concentrations from the ELISA assay of plasma samples following SC dosing are shown over time in FIG. 7. The data for each derivative was fitted to a one compartmental model using Phoenix WinNonlin software (Certara) and the results of the analysis are shown in Table 6. Table 6. Pharmacokinetic parameter values from one-compartmental model fit of the rat plasma values over time (shown in FIG. 7) for PAS200, PAS400, PAS600, and PAS800 derivatives of Sen following subcutaneous bolus dosing.

[0181] Discussion

[0182] The half-lives following subcutaneous dosing for PAS200, PAS400, PAS600, and PA800 derivatives are 19-, 41 -, 64-, and 84-fold longer, respectively, than that of the parent peptide, Sen, which is 15 minutes in rats (US 2011 / 0105397).

[0183] The PAS-size dependent increase in Tmax is indicative of changes beyond simple half-life extension, suggesting PAS-size dependent changes in rates of absorption from the SC site of administration and effects on distribution.

[0184] SEQUENCES

[0185] SEQ ID NO: 1

[0186] FTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI

[0187] SEQ ID NO: 2

[0188] TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI

[0189] SEQ ID NO: 3

[0190] ASPAAPAPASPAAPAPSAPA

[0191] SEQ ID NO: 4

[0192] AAPASPAPAAPSAPAPAAPS

[0193] SEQ ID NO: 5

[0194] APSSPSPSAPSSPSPASPSS

[0195] SEQ ID NO: 6

[0196] SAPSSPSPSAPSSPSPASPS

[0197] SEQ ID NO: 7

[0198] SSPSAPSPSSPASPSPSSPA

[0199] SEQ ID NO: 8

[0200] AASPAAPSAPPAAASPAAPSAPPA SEQ ID NO: 9

[0201] ASAAAPAAASAAASAPSAAA

[0202] SEQ ID NO: 10

[0203] APAAPAPAPAAPAPAPA

[0204] SEQ ID NO: 11

[0205] AAPAPAPAAPAPAPAAP

[0206] SEQ ID NO: 12

[0207] APPPAPPPAP

[0208] SEQ ID NO: 13

[0209] PAPPPAPPPA

[0210] SEQ ID NO: 14

[0211] AAPAAPAPPAAAPAAPAPPA

[0212] SEQ ID NO: 15

[0213] AAAAPAAAAAAAPAAA

[0214] SEQ ID NO: 16 (PAS200-A28C-Scn):

[0215] TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI-NH2

[0216] Ac-

[0217] ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAP APSAPAASPAAPAPASPAAPAPSAPAASPAAPAPA SPAAPAPSAPAASPAAPAPASPAAPAPSAPAA

[0218] SEQ ID NO: 17 (PAS400-A28C-Scn):

[0219] TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI-NH2

[0220] Ac-

[0221] ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAP

[0222] APSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SP AAP AP AS PAAP APS AP AAS P AAP AP AS P AAPAPS APAAS PAAP AP AS P AAP APS AP AAS P AAP AP AS P AAPAPS APAAS PAAP APAS PAAP APS APAAS P AAP AP AS PAAPAPS APAAS P AAP APAS PAAP A PSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA

[0223] SEQ ID NO: 18 (PAS600-A28C-Scn):

[0224] TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI-NH2 Ac-

[0225] ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAP APSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SP AAP AP AS PAAP APS AP AAS P AAP AP AS P AAPAPS APAAS PAAP AP AS P AAP APS AP AAS P AAP AP AS P AAPAPS APAAS PAAP APAS PAAP APS APAAS P AAP AP AS PAAPAPS APAAS P AAP APAS PAAP A PSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPA SPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAP SAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA

[0226] SEQ ID NO: 19 (PAS800-A28C-Scn):

[0227] TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI-NH2

[0228] Ac-

[0229] ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAP APSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SP AAP APAS PAAP APS APAAS PAAP APAS PAAPAPS APAAS PAAP APAS PAAP APS APAAS P AAP AP AS PAAPAPS APAAS PAAP APAS PAAP APS APAAS PAAP APAS PAAPAPS APAAS PAAP APAS PAAP A PSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPA SPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAP SAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASP AAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPAS PAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPS APAAS PAAP APAS PAAP APSAP AAS P AAP AP ASP AAP APS APAAS PAAP APAS P AAPAPSAP AA

[0230] REFERENCES

[0231] Aghaabdollahian et al. (2019) Scientific Reports 9:2978.

[0232] Green and Sambrook (2012) Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0233] Hoare, E.R.J. (2007) Current Neuropharm. 5:168-179.

[0234] Mason (2010) Future Med. Chem. 2:1813-1822.

[0235] Nnane et al. (2016) AAPS Poster Submission 17R0800.

[0236] Rademaker, MT and Richards, AM (2017) Clinica Chimica Acta 474:76-87.

[0237] Thi et al. (2020) Polymers 12:298.

[0238] US 2011 / 0105397.

[0239] WO 2009 / 040027.

[0240] In view of the above, it will be seen that several objectives of the invention are achieved and other advantages attained. As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0241] All references cited in this specification, including but not limited to patent publications and non-patent literature, are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

[0242] As used herein, in particular embodiments, the terms “about” and “approximately,” when preceding a numerical value, indicates the value plus or minus a range of 10%. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. That the upper and lower limits of these smaller ranges can independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0243] The indefinite articles “a” and “an,” as used herein in the specification and in the embodiments, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0244] The phrase “and / or,” as used herein in the specification and in the embodiments, should be understood to mean “either or both” of the elements so conjoined, i.e. , elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements can optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0245] As used herein in the specification and in the embodiments, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the embodiments, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the embodiments, shall have its ordinary meaning as used in the field of patent law.

[0246] As used herein in the specification and in the embodiments, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements can optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.

[0247] Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

Claims

Claims1 . A modified Urocortin 3 (Ucn3) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to SEQ ID NO: 1 , the modified Ucn3 further comprising a covalently attached polymer comprising amino acids, wherein the polymer inhibits degradation and / or elimination of the modified Ucn3 in a subject, and wherein the modified Ucn3 retains corticotropin-releasing factor receptor type 2 (CRFR2) agonist activity.

2. The modified Ucn3 of claim 1 , further comprising a threonine and a lysine at the N-terminus of SEQ ID NO: 1 such that the modified Ucn3 comprises a stresscopin (Sen) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to SEQ ID NO: 2.

3. The modified Ucn3 of claim 1 or 2, wherein the polymer comprises:(a) amino acids consisting of proline and alanine residues (PA); or(b) amino acids consisting of proline, alanine, and serine residues (PAS).

4. The modified Ucn3 of claim 3, wherein the polymer is PA.

5. The modified Ucn3 of claim 3, wherein the polymer is PAS.

6. The modified Ucn3 of claim 4 or 5, wherein the polymer comprises at least 100 amino acids.

7. The modified Ucn3 of any one of claims 4-6, wherein the polymer comprises the amino acid sequence selected from the group consisting of:(a) ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 3);(b) AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 4);(c) APSSPSPSAPSSPSPASPSS (SEQ ID NO: 5);(d) SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 6);(e) SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 7);(f) AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 8);(g) ASAAAPAAASAAASAPSAAA (SEQ ID NO: 9);(h) APAAPAPAPAAPAPAPA (SEQ ID NO: 10);(i) AAPAPAPAAPAPAPAAP (SEQ ID NO: 11);0) APPPAPPPAP (SEQ ID NO: 12);(k) PAPPPAPPPA (SEQ ID NO: 13);(l) AAPAAPAPPAAAPAAPAPPA (SEQ ID NO: 14); and(m) AAAAPAAAAAAAPAAA (SEQ ID NO: 15); or permuted or circular permuted versions or multimers of these sequences as a whole or parts of these sequences.

8. The modified Ucn3 of any one of claims 4-7, wherein the polymer is terminated by a proline.

9. The modified Ucn3 of claim 7 or 8, wherein the polymer comprises SEQ ID NO: 3 repeated at least ten times.

10. The modified Ucn3 of claim 9, wherein the polymer comprises SEQ ID NO: 3 repeated at least thirty times.11 . The modified Ucn3 of claim 9 or 10, wherein the polymer is terminated by a proline.

12. The modified Ucn3 of any one of claims 4-11 , wherein the PA or PAS polymer is covalently bound to the N-terminus or the C-terminus of the Ucn3 or Sen.

13. The modified Ucn3 of any one of claims 1-12, wherein the polymer is covalently linked to at least one internal amino acid residue of the Ucn3 or Sen.

14. The modified Ucn3 of claim 13, wherein the at least one internal amino acid residue covalently linked to the polymer is:(a) residue 10, 16, 17, 19, 21 , 23, 26, 29, 30, 32, 33, 36, and / or 37 of SEQ ID NO: 1 ; or(b) residue 12, 18, 19, 21 , 23, 25, 28, 31 , 32, 34, 35, 38, and / or 39 of SEQ ID NO: 2.

15. The modified Ucn3 of claim 13 or 14, further comprising a linker between the modified Ucn3 or Sen and the polymer.

16. The modified Ucn3 of any one of claims 1-15, wherein the modified Ucn3 comprises more than one polymer.

17. The modified Ucn3 of claim 16, wherein the more than one polymer is independently a PA or PAS polymer.

18. The modified Ucn3 of claim 16 or 17, wherein the more than one polymer comprises a polymer at a terminus of the modified Ucn3 and a polymer linked to at least one internal amino acid residue of the Ucn3.

19. The modified Ucn3 of any one of claims 1 -18, wherein the modified Ucn3 or Sen is in a pharmaceutically acceptable carrier.

20. The modified Ucn3 of claim 19, wherein the modified Ucn3 or Sen is in a formulation that can be aerosolized.

21. A nucleic acid molecule encoding the modified Ucn3 of claim 12.

22. A vector comprising the nucleic acid molecule of claim 21 .

23. A cell comprising the vector of claim 22.

24. The cell of claim 23, wherein the cell is capable of expressing the modified Ucn3.

25. A method of treating a subject suffering from or diagnosed with a disease, disorder, or medical condition mediated by CRFR2 activity, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of the modified Ucn3 of claim 19 or 20.

26. The method of claim 25, wherein the disease, disorder, or medical condition is a cancer, an autoimmune disease, a fibrotic disease, an inflammatory disease, a neurodegenerative disease, an infectious disease, a lung disease, a heart disease, a vascular disease, or a metabolic disease.

27. The method of claim 25, wherein the disease, disorder, or medical condition is vasculitis and / or excessive angiogenesis in an autoimmune disorder, systemic sclerosis, multiple sclerosis, Sjogren's disease, a vascular malformation in a blood and / or lymph vessel, portal vein hypertension, liver ascites, pulmonary hypertension, idiopathic pulmonary hypertension, atrial hypertension, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary fibrosis, DiGeorge syndrome, hereditary hemorrhagic telangiectasia, cavernous hemangioma, cutaneous hemangioma, a lymphatic malformation, transplant adenopathy, atherosclerosis, vascular anastomoses, adipose tissue in obesity, allograft rejection, a skin disease, psoriasis, warts, allergic dermatitis, scar keloids, pyogenic granulomas, blistering disease, Kaposi sarcoma in an AIDS patient, systemic sclerosis, an eye disease, persistent hyperplastic vitreous syndrome, diabetic retinopathy, retinopathy of prematurity, choroidal neovascularization, pulmonary hypertension, asthma, nasal polyps, rhinitis, chronic airway inflammation and obstruction, cystic fibrosis, acute lung injury, bronchiolitis obliterans organizing pneumonia, a gastrointestinal tract disease, inflammatory bowel disease, periodontal disease, ascites, peritoneal adhesions, liver cirrhosis, a reproductive system disease, endometriosis, uterine bleeding, ovarian cysts, ovarian hyperstimulation, a bone or joint disease, arthritis, synovitis, osteomyelitis, osteophyte formation, HIV-induced bone marrow angiogenesis, kidney disease, or early diabetic nephropathy.

28. The method of claim 25, wherein the disease, disorder, or medical condition is metabolic disease, pulmonary disease, or heart failure.

29. The method of claim 25, wherein the disease, disorder, or medical condition is heart failure.

30. The method of any one of claims 25-29, wherein the modified Ucn3 is administered by injection.

31. The method of any one of claims 25-29, wherein the modified Ucn3 is aerosolized and is administered by inhalation.

32. A method of preparing the modified Ucn3 of claim 12, the method comprising obtaining the cell of claim 23 and expressing the modified Ucn3.

33. A method of preparing the modified Ucn3 of any one of claims 1-18, the method comprising expressing a modified Ucn3 from the cell of claim 24 or produced by solution or solid phase techniques, then covalently attaching a polymer using chemical methods.

34. Use of the modified Ucn3 of claim 16 or 17, the nucleic acid of claim 21 , the vector of claim 22, or the cell of claim 24 for the manufacture of a medicament for the treatment of a disease, disorder, or medical condition mediated by CRFR2 activity.

35. The use of claim 34, wherein the disease, disorder, or medical condition is metabolic disease, pulmonary disease, or heart failure.

36. The method of claim 34, wherein the disease, disorder, or medical condition is heart failure.

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