WO2021067387A1 - Elabela-derived conjugates and methods of use - Google Patents

Abstract

Described herein are compositions comprising an elabela peptide or fragment thereof covalently linked to a fatty acid acyl and methods and uses thereof for treating a subject having or at risk for developing heart failure, pulmonary arterial hypertension, and/or ischemia or reperfusion-induced renal fibrosis.

Description

ELABELA-DERIVED CONJUGATES AND METHODS OF USE

Sequence Listing

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 29, 2020, is named 51338-005W02_Sequence_Listing_9_29_20_ST25 and is 22,889 bytes in size.

Background

Heart failure (HF) and pulmonary arterial hypertension (PAH) represent a growing health concern in the US, with an estimated 5.7 million and 20 per million individuals affected by these diseases, respectively.

HF represents an end stage phenotype which typically occurs secondary to pathological stresses, and is most commonly caused by ischemic heart disease, particularly left ventricular (LV) dysfunction after myocardial infarction (Ml). Although current interventional therapies have greatly improved acute post Ml survival, heart failure after Ml remains a major cause of morbidity and mortality.

PAH is a progressive disease with a mortality rate approximating 50% at 5 years. The primary pathological changes underlying PAH include endothelial cell proliferation, as well as pulmonary artery smooth muscle cell (PASMC) hyperplasia and hypertrophy, leading to vascular wall thickening.

Increased resistance of the pulmonary vasculature results in excessive right ventricle (RV) cardiac afterload with eventual death from RV failure. Although recently introduced new drug therapies can improve PAH symptoms and hemodynamics, reversal of the disease is rarely achieved.

Ischemia/reperfusion is a major cause of acute kidney injury and can result in poor long-term graft function and is a risk factor for increased morbidity and mortality. Although most patients with acute kidney injury recover their renal function, a significant portion of patients suffer from progressive deterioration of renal function, including an increase in renal fibrosis, which often results in a progressive loss of renal function. A true reversal of the disease can rarely be achieved with existing strategies, ultimately leading to end-stage renal failure and a requirement for dialysis or kidney transplantation. Accordingly, there remains an urgent need for novel and effective therapies that reduce specific HF risk factors (e.g., hypertension and ischemia), halt the progression of ischemia or reperfusion-induced renal fibrosis and improve kidney function, attenuate the development of abnormal pulmonary vasculature in PAH, improve RV function, and attenuate cardiac remodeling and dysfunction post Ml.

Summary of the Invention

The present invention features compositions and methods for the treatment of ischemia or reperfusion-induced renal fibrosis, heart failure, and pulmonary arterial hypertension.

In one aspect, the invention features a conjugate, or a pharmaceutically acceptable salt thereof, comprising a fatty acid acyl covalently linked through a linker to an elabela peptide or a fragment thereof comprising at least 9 contiguous amino acid residues. In another aspect, the invention features a conjugate, or a pharmaceutically acceptable salt thereof, comprising a fatty acid acyl covalently linked through a linker to an elabela peptide or a fragment thereof comprising at least 10 contiguous amino acid residues. In another aspect, the invention features a conjugate, or a pharmaceutically acceptable salt thereof, comprising a fatty acid acyl covalently linked through a linker to an elabela peptide or a fragment thereof comprising at least 11 contiguous amino acid residues. In another aspect, the invention features a conjugate, or a pharmaceutically acceptable salt thereof, comprising a fatty acid acyl covalently linked through a linker to an elabela peptide or a fragment thereof comprising at least 12 contiguous amino acid residues. In another aspect, the invention features a conjugate, or a pharmaceutically acceptable salt thereof, comprising a fatty acid acyl covalently linked through a linker to an elabela peptide or a fragment thereof comprising at least 13 contiguous amino acid residues. In another aspect, the invention features a conjugate, or a pharmaceutically acceptable salt thereof, comprising a fatty acid acyl covalently linked through a linker to an elabela peptide or a fragment thereof comprising at least 14 contiguous amino acid residues.

In one embodiment, the elabela peptide or fragment thereof comprises an amino acid sequence from N-terminus to C-terminus of formula (I) (SEQ ID NO: 25):

Glnoi-Argo2-Proo3-Valo4-Asno5-Leuo6-Thro7-Meto8-Argo9-Argio-Lysii-Leui2-Argi3-Lysi4-Hisi5-Asni6-Cysi7-

Leui8-Glni9-Arg20-Arg2i-Cys22-Met23-Pro24-Leu25-HiS26-Ser27-Arg28-Val29-Pro30-Phe3i-Pro32 (l).

In some embodiments, one or more of Gl g to Pro3₂ is substituted with Lys, Ala, Val, pyroglutamic acid (Pyr), or Gly. In another embodiment, one or more of Gl g to Pro3₂ is substituted with Lys. In some embodiments, Arg_åi is substituted with Lys. In other embodiments, Pro_å4 is substituted with Lys. In other embodiments, one or more of Gl g to Pro3₂ is substituted with Ala. In some embodiments, Arg_åi is substituted with Ala. In some embodiments, Arg_åo is substituted with Ala. In some embodiments, wherein Arg_ås is substituted with Ala. In other embodiments, one or more of Gl g to Pro3₂ is substituted with Pyr. In some embodiments, Gl g is substituted with Pyr.

In some embodiments, more of Glnoi to Leuis is substituted with Lys, Ala, Val, Pyr, or Gly, or is absent. In some embodiments, Glnoi to Leuis is absent. In other embodiments, Glnoi to Lysn is absent. In further embodiments, Glnoi is substituted with Pyr.

In some embodiments, the elabela peptide or fragment thereof comprises at least one lysine residue. In some embodiments, the at least one lysine residue, is at least two lysine residues. In some embodiments, the at least two lysine residues, is at least three lysine residues. In some embodiments, the at least three lysine residues is at least four lysine residues.

In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 7-24. In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 8, 9, 11 , 12, 14, 15, 16, or 20. In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the elabela peptide or fragment thereof comprises an amino acid sequence of SEQ ID NO: 20.

In some embodiments, the linker comprises the structure: X1-L-X2, wherein Xi is one or more amino acids selected from Gly, Ala, Asn, Cys, and combinations thereof, or is absent; L is a linker selected from the group consisting of a b alanine linker, a b alanine-2x glycine linker, a y-glutamyl linker, a bis-y-glutamyl linker, an animo-3,6-dioxaoctanoic acid (OEG) linker, a y-glutamyl-2x oligo ethylene glycol linker, 2-(2-(2-AminoEthoxy)Ethoxy)Acetic acid, Aminoethylethanolamine, (PEG)8, glutamine, glutamic acid, benzophenone-4-lsothiocyanate, bis-((N-lodoacetyl)Piperazinyl)Sulfonerhodamine, succinimidyl 2-(2-Pyridyldithio)Propionate, 4-Azido-2,3,5,6-Tetrafluorobenzoic acid, (N-((2- Py ridy Id ith io)ethy l)-4- Azidosalicylamide), succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1- carboxylate, and N-(t-BOC)-aminooxyacetic acid; and X2 is one or more amino acids selected from Gly, Ala, Asn, Cys, and combinations thereof, or is absent.

In some embodiments, L is a b alanine linker. In some embodiments, L is a b alanine-2x glycine linker. In some embodiments, L is a g-glutamyl linker. In some embodiments, L is a y-glutamyl-2x oligo ethylene glycol linker. In some embodiments, L is a 2-(2-(2-AminoEthoxy)Ethoxy)Acetic acid linker. In some embodiments, L is a (PEG)s linker.

In some embodiments, Xi is absent. In other embodiments, X2 is absent. In other embodiments, both Xi and X2 are absent. In some embodiments Xi and/or X2 are one or more amino acid residues selected from Ala, Gly, Asn, Cys, and combinations thereof. In some embodiments, Xi and/or X2 comprise Ala-Ala-Ala. In other embodiments, Xi and/or X2 comprise Ala-Gly. In some embodiments, Xi and/or X2 comprise Asn-Gly-Asn-Gly. In some embodiments, Xi and/or X2 independently comprise between one and five amino acids. In some embodiments, Xi and/or X2 comprise one amino acid. In some embodiments, Xi and/or X2 comprise two amino acids. In some embodiments, Xi and/or X2 comprise three amino acids. In some embodiments, Xi and/or X2 comprise four amino acids. In some embodiments, Xi and/or X2 comprise five amino acids.

In some embodiments, the elabela peptide or fragment thereof comprises a lysine residue (for example, an introduced Lys substitution, for example at position 21 or 24) having a side chain covalently linked by the linker to the fatty acid acyl. In some embodiments, the fatty acid acyl is covalently linked through a linker to the N-terminus of the elabela peptide or fragment thereof. In some embodiments, the fatty acid acyl comprises 8 to 26 carbon atoms. In some embodiments, the fatty acid acyl is derived from a fatty acid selected from the group consisting of palmitic acid, palmitoyl, myristic acid, stearic acid, palmitoleic acid, oleic acid, cholesterol, DPPE, GM1 , GM2, GM3, a-Linolenic acid, EPA, DHA, DPPC, DOPS, and DOPC. In some embodiments, the fatty acid acyl is palmitate or palmitoyl. In some embodiments, the fatty acid acyl is octadecandioate.

In some embodiments, the elabela peptide or fragment thereof further comprises a C-terminal amidation modification. In some embodiments, the elabela peptide or fragment thereof further comprises an N-terminal acylation modification.

In another aspect, the invention features a pharmaceutical composition comprising a conjugate as described herein and one or more pharmaceutically acceptable carriers or excipients

In another aspect, the invention features a method of agonizing the apelin receptor in a cell. In some embodiments, the method involves contacting the cell with an effective amount of a conjugate as described herein. In other embodiments, the method involves contacting the cell with an effective amount of a pharmaceutical composition as described herein. In some embodiments, the cell is a cell within the vasculature. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the cell is a renal cell. In some embodiments, the cell is a human cell.

In another aspect, the invention features a method of preventing pulmonary arterial hypertension (PAH) in a subject identified as at risk for developing PAH. In some embodiments, the method involves administering to the subject a therapeutically effective amount of a conjugate as described herein. In other embodiments, the method involves administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein. In some embodiments, the administering comprises subcutaneous, intravenous, percutaneous coronary intervention via cardiac catheter, aerosolized inhalation, or nasal administration of the conjugate or the pharmaceutical composition to the subject.

In another aspect, the invention features a method of treating PAH in a subject in need thereof.

In some embodiments, the method involves administering to the subject a therapeutically effective amount of a conjugate as described herein. In other embodiments, the method involves administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein. In some embodiments, the administering comprises subcutaneous, intravenous, percutaneous coronary intervention via cardiac catheter, aerosolized inhalation, or nasal administration of the conjugate or the pharmaceutical composition to the subject.

In another aspect, the invention features a method of preventing heart failure in a subject identified as at risk for developing heart failure. In some embodiments, the method involves administering to the subject a therapeutically effective amount of a conjugate as described herein. In other embodiments, the method involves administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein. In some embodiments, the administering comprises subcutaneous, intravenous, percutaneous coronary intervention via cardiac catheter, aerosolized inhalation, or nasal administration of the conjugate or the pharmaceutical composition to the subject. In some embodiments, prior to the administration, the subject has had an acute myocardial infarction,

In another aspect, the invention features a method of treating heart failure in a subject in need thereof. In some embodiments, the method involves administering to the subject a therapeutically effective amount of a conjugate as described herein. In other embodiments, the method involves administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein. In some embodiments, the administering comprises subcutaneous, intravenous, percutaneous coronary intervention via cardiac catheter, aerosolized inhalation, or nasal administration of the conjugate or the pharmaceutical composition to the subject.

In another aspect, the invention features a method of preventing ischemia or reperfusion-induced renal fibrosis in a subject identified as at risk for developing ischemia or reperfusion-induced renal fibrosis. In some embodiments, the method involves administering to the subject a therapeutically effective amount of a conjugate as described herein. In other embodiments, the method involves administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein. In some embodiments, the administering comprises subcutaneous, intravenous, percutaneous coronary intervention via cardiac catheter, aerosolized inhalation, or nasal administration of the conjugate or the pharmaceutical composition to the subject. In another aspect, the invention features a method of treating ischemia or reperfusion-induced renal fibrosis in a subject in need thereof. In some embodiments, the method involves administering to the subject a therapeutically effective amount of a conjugate as described herein. In other embodiments, the method involves administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein. In some embodiments, the administering comprises subcutaneous, intravenous, percutaneous coronary intervention via cardiac catheter, aerosolized inhalation, or nasal administration of the conjugate or the pharmaceutical composition to the subject.

Brief Description of the Drawings

FIG. 1 is a graph showing that Ela-14 conjugated to a fatty acid acyl exhibits conserved agonist efficacy and potency compared to unconjugated apelin-13 and unconjugated Ela-14.

FIG. 2 is a pair of graphs showing that Ela-14 conjugated to a fatty acid acyl exhibits resistance to protease degradation (left panel) compared to unconjugated Ela-14 (right panel).

Definitions

In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; and (iii) the terms “including” and “includes” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps.

As used herein, the terms “about” and “approximately” refer to a value that is within 10% above or below the value being described. For example, the term “about 5 mg” indicates a range of from 4.5 to 5.5 mg.

As used herein, the term “administration” refers to the administration of a composition (e.g., a compound or a pharmaceutical preparation that includes a therapeutic agent as described herein e.g., an elabela-derived conjugate) to a subject or system. Administration to an animal subject (e.g., to a human) may be by any appropriate route.

As used herein, the term “affinity” refers to the strength of a binding interaction between two molecules, such as a ligand and a receptor (e.g., between elabela and the apelin receptor). The term “Kd,” as used herein, is intended to referto the dissociation constant, which can be obtained, for example, from the ratio of the rate constant for the dissociation of the two molecules (kd) to the rate constant for the association of the two molecules (ka) and is expressed as a molar concentration (M). Kd values for peptide-protein interactions can be determined, e.g., using methods established in the art. Methods that can be used to determine the Kd of a peptide-protein interaction include surface plasmon resonance, e.g., through the use of a biosensor system such as a BIACORE® system, as well as fluorescence anisotropy and polarization methods and calorimetry techniques known in the art, such as isothermal titration calorimetry (ITC).

As used herein, the terms “apelin receptor” and “APJ” referto the apelin receptor, a G protein- coupled receptor (GPCR) expressed in the pulmonary vasculature and in cardiomyocytes. The apelin receptor is encoded by the APLNR gene. The amino acid sequence of an exemplary protein encoded by human APLNR is shown under Uniprot Accession No. P35414, or in SEQ ID NO: 1 . The nucleic acid sequence of exemplary human APJ is shown under NCBI Reference Sequence: NM_005161.4, or in SEQ ID NO: 2. The term “apelin receptor” also refers to natural variants of the wild-type apelin receptor protein, such as proteins having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% identity, or more) to the amino acid sequence of wild-type apelin receptor, which is set forth in SEQ ID NO: 1 .

As used herein, the term “apelin” refers to an endogenous ligand for the apelin receptor that is expressed at the surface of cells in a variety of tissues including the heart, lung, kidney, liver, adipose tissue, gastrointestinal tract, brain, adrenal glands, endothelium, and plasma. Apelin is encoded by the APLN gene. The amino acid sequence of an exemplary protein encoded by human APLN is shown under Uniprot Accession No. Q9ULZ1 , or in SEQ ID NO: 3. The nucleic acid sequence of exemplary human APLN is shown under NCBI Reference Sequence: NM_017413.5, or in SEQ ID NO: 4. The term “apelin” also refers to natural variants of the wild-type apelin protein, such as proteins having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% identity, or more) to the amino acid sequence of wild-type apelin, which is set forth in SEQ ID NO: 3.

As used herein in the context of conjugates, the term “linked” refers to the covalent joining of one molecule, such as a peptide, or fragment thereof (e.g., an elabela peptide or a fragment thereof), to another molecule, such as a fatty acid acyl. Two molecules that are “linked” to one another as described herein may be linked by way of a linker. Exemplary linkers include synthetic linkers as well as peptidic linkers, such as those that contain one or more glycine, serine, and/or threonine residues. Exemplary linkers are included herein in Table 3.

As used herein, a “combination therapy” and “administered in combination” mean that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent and the agents may be co-formulated. In some embodiments, the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen. In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent can be affected by any appropriate route including, but not limited to subcutaneous, intravenous, via percutaneous coronary intervention (via cardiac catheter), aerosolized inhalation, nasal application. The therapeutic agents can be administered by the same route or by different routes.

As used herein, the term “conjugate” refers to a molecule containing two or more regions from distinct sources that are ligated together (e.g., by a covalent bond) to form a single compound.

As used herein, the terms “conservative mutation,” “conservative substitution,” and “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. It is appreciated that conservative amino acid families include (i) G, A, V, L and I; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg).

As used herein, the terms “effective amount,” “therapeutically effective amount,” and a “sufficient amount” of a conjugate or composition described herein refer to a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of treating or preventing ischemia/reperfusion induced renal fibrosis, heart failure (e.g., post myocardial infarction heart failure), and pulmonary arterial hypertension, it is an amount of the composition sufficient to achieve a treatment response as compared to the response obtained without administration of the composition. The amount of a given composition described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, a “therapeutically effective amount” of a composition of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of a composition of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regime may be adjusted to provide the optimum therapeutic response.

As used herein, the terms “elabela,” “apela,” and “toddler” refer to apelin receptor endogenous ligands, peptide hormones that binds to the Apelin receptor. Elabela is encoded by the APELA gene.

The amino acid sequence of an exemplary protein encoded by human APELA is shown under UniProt Accession No. P0DMC3, or in SEQ ID NO: 5. The nucleic acid sequence of exemplary human APELA is shown under NCBI Reference Sequence: NM_001297550.2, or in SEQ ID NO: 6. The term “elabela” also refers to natural variants of the wild-type elabela protein, such as proteins having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% identity, or more) to the amino acid sequence of wild-type elabela, which is set forth in SEQ ID NO: 1 .

The term “fatty acid,” as used herein, represents a linear, saturated, acyclic aliphatic carboxylic acid having 4 to 28 carbon atoms. A “short chain fatty acid” is a fatty acid having 5 or fewer carbon atoms. A “medium chain fatty acid” is a fatty acid having 6 to 12 carbon atoms. A “long chain fatty acid” is a fatty acid having 13 to 21 carbon atoms. A “very long chain fatty acid” is a fatty acid having 22 or more carbon atoms. Non-limiting examples of long chain fatty acids include palmitic acid, myristic acid, and stearic acid.

The term “fatty acid acyl,” as used herein, represents a monovalent group that is a fatty acid having a carboxylic hydroxyl replaced with a valency.

A “fragment,” when used in reference to a peptide, refers to a portion of the peptide. As used herein, a fragment of a peptide is of sufficient length such that the interaction of interest (e.g., between a peptide and its cognate receptor) is maintained. The terms “peptide” and “polypeptide” are used interchangeably herein to mean a compound that contains a sequence of amino acids bonded to each other through peptidic bonds. A polypeptide or peptide includes at least 10 amino acids.

“Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.

As used interchangeably herein, the terms “subject,” “patient,” and “individual” refer to any organism to which a therapeutic agent in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals, such as mice, rats, cats, dogs, pigs, horses, rabbits, non-human primates, and humans). A subject may seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition.

As used herein, “treatment” and “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. For any term presented in the art which is identical to any term expressly defined in this disclosure, the term’s definition presented in this disclosure will control in all respects. The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

Detailed Description

Described herein are compositions and methods for the prevention or treatment of i) ischemia or reperfusion-induced renal fibrosis, (ii) heart failure (HF) (e.g., post myocardial infarction heart failure), and (iii) pulmonary arterial hypertension (PAH) in a subject (such as a mammalian subject, for example, a human). Using the compositions and methods described herein, one can prevent or treat ischemia/reperfusion induced renal fibrosis, HF (e.g., post myocardial infarction HF), and/or PAH in a subject (e.g., a human subject) by administering an elabela-derived conjugate or a composition thereof. For example, described herein are compositions containing an elabela peptide or fragment thereof that has been modified to improve potency, stability, and efficacy. The sections that follow describe the compositions and methods useful for the prevention or treatment of ischemia/reperfusion induced renal fibrosis, HF (e.g., post myocardial infarction HF), and/or PAH in further detail.

Heart Disease

At least 5.7 million adults in the US suffer from heart failure (HF). The combined direct and indirect cost of HF has been estimated to be approximately $30.7 billion each year. The most common cause of HF is coronary artery disease resulting in myocardial infarction (Ml), with subsequent chronic cardiac dysfunction, and ultimately development of the HF syndrome. This chronic cardiac dysfunction subsequent to Ml is induced by cellular damage or death of heart muscle secondary to ischemia caused by an imbalance of oxygen supply and demand in a specific region, such as that which occurs as a result of reduced blood flow to the area. Post-MI left ventricular (LV) dysfunction represents a well-established prognostic factor, which is regarded as a major contributor to eventual HF and mortality. Congestive heart failure (CHF) is characterized by an inability of the heart to generate sufficient cardiac output to meet the body’s demands. Patients having CHF experience signs and symptoms of intravascular and interstitial volume overload, including shortness of breath, rapid heart rate, fluid in the lungs, and edema, along with indicators of inadequate tissue perfusion, including fatigue and/or poor exercise tolerance. While the introduction of percutaneous coronary intervention has greatly improved the immediate outcome of myocardial infarction, additional treatments are needed to address the associated cardiac dysfunction and HF resulting from ischemia/reperfusion injury.

Pulmonary Arterial Hypertension (PAH)

The prevalence of pulmonary arterial hypertension (PAH) in the US has been estimated at ~20 per million individuals. The annual direct medical cost of treating PAH exceeds $100,000 per patient, with a much higher indirect economic burden of this disease. PAH presents with dyspnea on exertion, fatigue, chest pain, and fainting leading to progressive disability. Despite recent therapeutic advances, idiopathic PAH, the most common form in the western world, has a mortality of 50% within 5 years, with death usually occurring from right ventricular (RV) failure. In its secondary forms, pulmonary hypertension (PH) complicates 14-80% of chronic lung and heart diseases and is a major contributor to morbidity and mortality in these patients.

The histopathological changes of PAH include endothelial cell proliferation, as well as pulmonary artery smooth muscle cell (PASMC) hyperplasia and hypertrophy, leading to vascular wall thickening. Current therapeutic options, mainly aimed at improving symptoms and hemodynamics, include endothelin receptor antagonists, prostacyclin analogs, enhancers of nitric oxide signaling, or a combination of such compounds. However, a true reversal of the disease can rarely be achieved with existing strategies. Therefore, there is an urgent need to develop more effective therapies, that will target the abnormal pulmonary vasculature and, at the same time, improve RV function.

Ischemia or Reperfusion-Induced Renal Fibrosis

Ischemia/reperfusion is a major cause of acute kidney injury and can result in poor long-term graft function and is a risk factor for increased morbidity and mortality. Although most patients with acute kidney injury recover their renal function, a significant portion of patients suffer from progressive deterioration of renal function, including an increase in renal fibrosis. The extent of renal damage is influenced by the onset of hypertension, local inflammation, and ongoing nephrotoxic medication. Renal scarring due to fibrosis characterized as a progressive detrimental connective tissue deposition on the kidney parenchyma results in a progressive loss of renal function.

A true reversal of the disease can rarely be achieved with existing strategies, ultimately leading to end-stage renal failure and a requirement for dialysis or kidney transplantation. Therefore, there is an urgent need to develop more effective therapies that will halt the progression of ischemia or reperfusion- induced renal fibrosis and, at the same time, improve kidney function.

Compositions

In general, the present invention provides conjugates comprising an elabela peptide or a fragment thereof covalently linked through a linker to a fatty acid acyl. Advantageously, conjugates of the invention may exhibit superior pharmacokinetic properties as compared to a wild type elabela peptide or fragment thereof, which are rapidly degraded by serum proteases in vitro and in vivo. Additionally, a conjugate of the invention may elicit reduced adverse immune reactions as compared to, for example an elabela Fc-fusion protein. Without wishing to be bound by theory, it is believed that a conjugate of the invention binds to and agonizes the apelin receptor resulting in the attenuation of pathological pulmonary vasculature remodeling and the induction of complementary vasodilatory, inotropic, and cardioprotective effects.

Elabela Peptides

Elabela peptides that can be used in conjunction with the compositions and methods described herein include those that bind to and agonize the apelin receptor. The elabela peptide may be a full- length elabela peptide, or a fragment thereof comprising at least 9 (e.g., at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, or more) contiguous amino acid residues. In some embodiments, the elabela peptide comprises at least 10 contiguous amino acid residues. In some embodiments, the elabela peptide comprises at least 11 contiguous amino acid residues. In some embodiments, the elabela peptide comprises at least 12 contiguous amino acid residues. In some embodiments, the elabela peptide comprises at least 13 contiguous amino acid residues. In some embodiments, the elabela peptide comprises at least 14 contiguous amino acid residues.

The elabela peptide or fragment thereof may include or may be an amino acid sequence from N- terminus to C-terminus of formula (I) (SEQ ID NO: 25):

Glnoi-Argo2-Proo3-Valo4-Asno5-Leuo6-Thro7-Meto8-Argo9-Argio-Lysii-Leui2-Argi3-Lysi4-Hisi5-Asni6-

Cysi7-Leui8-Glni9-Arg20-Arg2i-Cys22-Met23-Pro24-Leu25-HiS26-Ser27-Arg28-Val29-Pro30-Phe3i-Pro32

(I)·

The invention additionally provides for conjugates comprising elabela peptides, or fragments thereof of formula (I) in which one or more of Glnoi to Pro3₂ are substituted with a different amino acid residue. These substitutions may serve to confer resistance to protease degradation, or improve binding affinity to the apelin receptor as compared to a wild-type elabela peptide. For example, in some embodiments, one or more of Gl g to Pro32 (e.g., one or more of Gl g, Arg2o, Argåi, Cys22, Met23, Pro24, I_eu25, HiS26, Serå7 , Arg28, Val29, Pro3o, Phe3i, and/or Pro32) is substituted with Lys, Ala, Val, pyroglutamic acid (Pyr), or Gly. In particular embodiments, one or more of Gl g to Pro3₂ (e.g., one or more of Gl g, Arg2o, Argåi, Cys22, Met23, Pro24, Leu25, HiS26, Serå7 , Arg28, Val29, Pro3o, Phe3i, and/or Pro32) is substituted with Lys. In other particular embodiments, Arg_åi or Pro_å4 is substituted with Lys.

In other embodiments, one or more of Gl g to Pro3₂ (e.g., one or more of Gl g, Arg2o, Arg_åi, Cys22, Met23, Pro24, Leu25, HiS26, Serå7 , Arg28, Val29, Pro3o, Phe3i, and/or Pro32) is substituted with Ala. In particular embodiments, Arg_åi is substituted with Ala. In other embodiments, Arg_åo is substituted with Ala. In other embodiments, both Arg2oand Arg_åi are substituted with Ala. In other embodiments, Arg_ås is substituted with Ala. In still other embodiments, each of Arg2o, Arg_åi, and Arg_ås is substituted with Ala.

In other embodiments, one or more of Gl g to Pro3₂ (e.g., one or more of Gl g, Arg2o, Arg_åi, Cys22, Met23, Pro24, Leu25, HiS26, Ser27 , Arg28, Val29, Pro3o, Phe3i, and/or Pro32) is substituted with Val.

In other embodiments, one or more of Gl g to Pro3₂ (e.g., one or more of Gl g, Arg2o, Arg_åi, Cys22, Met23, Pro24, Leu25, HiS26, Ser27 , Arg28, Val29, Pro3o, Phe3i, and/or Pro32) is substituted with Pyr. In particular embodiments, Gl g is substituted with Pyr.

In other embodiments, one or more of Gl g to Pro3₂ (e.g., one or more of Gl g, Arg2o, Arg_åi, Cys22, Met23, Pro24, Leu25, HiS26, Ser27 , Arg28, Val29, Pro3o, Phe3i, and/or Pro32) is substituted with Gly.

In some embodiments, one or more of Glnoi to Leuis (e.g., one or more of Glnoi, Argo2, Proo3, Valo4, Asno5, Leuo6, Thro7, Metos, Argo9, Argio, Lysn, Leui2, Argi3, Lysu, Hisis, Asni6, Cysi7, and/or Leuis) is substituted with Lys, Ala, Val, Pyr, or Gly. In particular embodiments, one or more of Glnoi to Le s (e.g., one or more of Glnoi, Argo2, Proo3, Valo4, Asnos, Leuo6, Thro7, Metos, Argo9, Argio, Lysn, I_eui2, Argi3, Lysi4, Hisi5, Asni6, Cysi7, and/or Leuis) is substituted with Lys. In other embodiments, one or more of Glnoi to Leui8 (e.g., one or more of Glnoi, Argo2, Proo3, Valo4, Asnos, Leuo6, Thro7, Metos, Argo9, Argio,

Lysn, Leui2, Argi3, Lysu, Hisis, Asni6, Cysi7, and/or Leuis) is substituted with Ala. In further embodiments, one or more of Glnoi to Leuis (e.g., one or more of Glnoi, Argo2, Proo3, Valo4, Asnos, Leuo6, Thro7, Metos, Argo9, Argio, Lysn, Leui2, Argi3, Lysu, Hisis, Asni6, Cysi7, and/or Leuis) is substituted with Val. In still further embodiments, one or more of Glnoi to Leuis (e.g., one or more of Glnoi, Argo2, Proo3, Valo4, Asnos, Leuo6, Thro7, Metos, Argo9, Argio, Lysn, Leui2, Argi3, Lysu, Hisis, Asni6, Cysi7, and/or Leuis) is substituted with Pyr. In particular embodiments, Glnoi is substituted with Pyr. In still further embodiments, one or more of Glnoi to Leuis (e.g., one or more of Glnoi, Argo2, Proo3, Valo4, Asnos, Leuo6, Thro7, Metos, Argo9, Argio, Lysn, Leui2, Argi3, Lysu, Hisis, Asni6, Cysi7, and/or Leuis) is substituted with Gly.

Elabela peptides or fragments thereof may include at least one (e.g., at least one, at least two, at least three, at least four, or more) lysine residues.

In addition to the twenty standard amino acid residues, elabela peptides or fragments thereof may include one or more rare amino acids (e.g., Pyr, D-amino acids, citrulline (Cit), hydroxyproline (Hyp), norleucine (Nle), 3-nitrotyrosine, nitroarginine, ornithine (Orn), naphtylalanine (Nal), Abu, DAB, methionine sulfoxide, and methionine sulfone) or non-natural amino acid derivatives such as homo-amino acids (e.g., amino acids that include a methylene group within the alpha carbon of the amino acid), beta-homo amino acids, and N-methyl amino acids. As can be appreciated by one of skill in the art, the incorporation of non-standard amino acids may enhance the stability of an elabela peptide of the invention.

In some embodiments, one or more of Glnoi to Leuis (e.g., one or more of Glnoi, Argo2, Proo3, Valo4, Asnos, Leuo6, Thro7, Metos, Argo9, Argio, Lysn, Leui2, Argi3, Lysu, Hisis, Asni6, Cysi7, and/or Leuis) is absent. In particular embodiments, Glnoi to Leuis is absent. In other embodiments, Glnoi to Lysn is absent.

In a particular embodiment, elabela peptides or fragments thereof useful in conjunction with the compositions and methods described herein include those containing an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or greater) to an amino acid sequence of any one of SEQ ID NOs 7-24.

Additional elabela peptides or fragments thereof useful in conjunction with the compositions and methods described herein include those containing an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or greater) to one of the foregoing sequences and/or having one or more conservative amino acid substitutions with respect to one of the foregoing sequences. Exemplary elabela peptides and fragments thereof for use in the conjugates of the invention are shown in Table 1. Table 1: Exemplary elabela peptides and fragments

Elabela peptides and fragments thereof may further include modifications to the C- or N-terminus.

Exemplary C-terminal modifications include amidation modifications, and exemplary N-terminal modifications include acylation modifications.

Fatty Acid Acyls A variety of fatty acid acyl entities may be utilized in accordance with the present invention.

In some embodiments, the fatty acid acyl includes 8 to 26 carbon atoms. In some embodiments, the fatty acid acyl is palmitate. In other embodiments, the fatty acid acyl is octadecandioate. Exemplary fatty acids that can be linked to elabela-derived peptides in the form of fatty acid acyls are shown in Table 2.

Table 2: Exemplary Fatty Acids

Linkers

Fatty acid acyls may be covalently linked to the elabela peptide or fragment thereof through a linker. Exemplary linkers include peptides and linking moieties that comprises a peptide. In some embodiments, peptidic linkers may include repeating units (e.g., repeating b alanine-glycine units or repeating glycine-asparagine (GN) units). In some embodiments, the linker is a b alanine linker. In some embodiments, the linker is a b alanine-2x glycine linker. In some embodiments, the linker is a y-glutamyl linker. In other embodiments, the linker is a y-glutamyl-2x oligo ethylene glycol linker. Non-peptidic linkers may also be used in the conjugates of the invention. In one example, the non-peptidic linker is a synthetic polymer. Synthetic polymers useful in the linkers of the invention may be a variety of lengths. Accordingly, in some embodiments, a linker comprising a synthetic polymer comprises a monomer unit of the polymer. In other embodiments, a linker comprising a synthetic polymer comprises two or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more) monomeric units of the polymer. Exemplary non-peptidic linkers for use in the conjugates of the invention include those that comprise at least one ethylene group (for example polyethylene glycol (PEG)). In some embodiments, the non-peptidic linker is (PEG)e.

Exemplary linkers are shown in Table 3.

Table 3: Exemplary Linkers

Linkers for use in the compositions of the invention may include those having the structure: Xi-L- X2, where Xi is one or more amino acids, or is absent; L is a linker as described herein (e.g., an exemplary linker from Table 3); and X2 is one or more amino acids, or is absent. In some embodiments, Xi is absent. In other embodiments, X2 is absent. In other embodiments, both Xi and X2 are absent. In some embodiments Xi and/or X2 are one or more amino acid residues selected from Ala, Gly, Asn, Cys, and combinations thereof (for example, Asn-Gly-Asn-Gly). In some embodiments, Xi and/or X2 comprise Ala-Ala-Ala. In other embodiments, Xi and/or X2 comprise Ala-Gly. In some embodiments, Xi and/or X2 comprise Asn-Gly-Asn-Gly. In some embodiments, Xi and/or X2 independently comprise between one and five amino acids. In some embodiments, Xi and/or X2 comprise one amino acid. In some embodiments, Xi and/or X2 comprise two amino acids. In some embodiments, Xi and/or X2 comprise three amino acids. In some embodiments, Xi and/or X2 comprise four amino acids. In some embodiments, Xi and/or X2 comprise five amino acids.

Further linkers suitable for use in the conjugates of the invention can be determined by those of skill in the art.

Methods of Manufacture

Peptide Synthesis

The elabela peptides and fragments thereof may be made by any technique known in the art. For example, elabela peptides can be made using a standard solid phase synthesis (e.g., on Wang resin (0.45 meq/g)) and purified using HPLC purification (e.g., using a reverse-phase C-18 column). Alternatively, elabela peptides can be produced in bacteria utilizing recombinant DNA technologies to obtain relatively large quantities of the purified peptides for human therapeutic use. For example, elabela peptides and fragments thereof can be produced in E. coli and subsequently purified to homogeneity, for example, by chromatography. Elabela peptides and fragments thereof can be produced and isolated according to methods described herein, and known in the art, to the purity, consistency, and potency to be administered to a subject as described herein.

Fatty Acid Acyl Conjugation

Conjugates including elabela peptides or fragments thereof, linkers, and fatty acid acyls may be prepared using techniques and reactions known in the art. For example, the linker and palmitic acid can be coupled using DIC/Oxyma.

A fatty acid acyl may be covalently linked through a linker to a side chain of a lysine residue within the elabela peptide or fragment thereof. Alternatively, a fatty acid acyl may be covalently linked through a linker to the N-terminus of the elabela peptide or fragment thereof.

Methods of Treatment

The elabela-derived conjugates described herein are useful in the methods of invention, and, while not wishing to be bound by theory, are believed to exert their desirable effects through their ability to bind to and agonize the apelin receptor resulting in the attenuation of pathological pulmonary vasculature remodeling and the induction of complementary vasodilatory, inotropic, and cardioprotective effects. An aspect of the present invention relates to methods of agonizing the apelin receptor in a cell. In some embodiments, the method includes contacting the cell with an effective amount of an elabela- derived conjugate, or a pharmaceutical composition thereof. In some embodiments, the cell is a cell within the vasculature. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the cell is a renal cell. In some embodiments, the cell is a human cell. The cell may be contacted in vitro, ex vivo, or in vivo.

Another aspect of the present invention relates to methods of preventing pulmonary arterial hypertension in a subject identified as at risk for developing pulmonary arterial hypertension. In some embodiments, the method includes administering to the subject an effective amount of an elabela-derived conjugate, or a pharmaceutical composition thereof.

Another aspect of the present invention relates to methods of treating pulmonary arterial hypertension in a subject in need thereof. In some embodiments, the method includes administering to the subject an effective amount of an elabela-derived conjugate, or a pharmaceutical composition thereof.

Another aspect of the present invention relates to methods of preventing heart failure in a subject identified as at risk for developing heart failure. In some embodiments, the method includes administering to the subject an effective amount of an elabela-derived conjugate, or a pharmaceutical composition thereof. In some embodiments, prior to the administration, the subject has had an acute myocardial infarction,

Another aspect of the present invention relates to methods of treating heart failure in a subject in need thereof. In some embodiments, the method includes administering to the subject an effective amount of an elabela-derived conjugate, or a pharmaceutical composition thereof.

Another aspect of the present invention relates to methods of preventing ischemia or reperfusion- induced renal fibrosis in a subject identified as at risk for developing ischemia or reperfusion-induced renal fibrosis. In some embodiments, the method includes administering to the subject an effective amount of an elabela-derived conjugate, or a pharmaceutical composition thereof.

Another aspect of the present invention relates to methods of treating ischemia or reperfusion- induced renal fibrosis in a subject in need thereof. In some embodiments, the method includes administering to the subject an effective amount of an elabela-derived conjugate, or a pharmaceutical composition thereof.

In some embodiments, treatment of PAH with an elabela-derived conjugate results in improvement in one or more symptoms (e.g., decreased dyspnea, fatigue, chest pain, and palpitations), enhanced exercise tolerance, improvement in pulmonary function (e.g., as assessed by pulmonary function tests, arterial blood gas measurement, echocardiography, cardiac MRI, right heart catheterization with vasoreactivity testing, and six-minute walk test (to assess severity of PAH)), and/or prolonged survival.

In some embodiments, treatment of heart failure with an elabela-derived conjugate results in improvement in one or more symptoms (e.g., dyspnea (worse on exertion), leg swelling, orthopnea, fatigue, and/or nocturnal urinary frequency), improvement of heart function (e.g., as assessed by chest x- ray, cardiac ultrasound (e.g., ejection fraction), stress test (e.g., exercise tolerance, blood oxygen level, and/or cardiac catheterization), and/or prolonged survival. In some embodiments, treatment of ischemia or reperfusion-induced renal fibrosis with an elabela-derived conjugate results in improvement in one or more symptoms (e.g., inflammation, fibrosis, cellular apoptosis, renal dysfunction, and renal tubular lesions), and/or prolonged survival.

Treating pulmonary arterial hypertension, heart failure, and/or ischemia or reperfusion-induced renal fibrosis can result in an increase in average survival time of a population of subjects treated according to the present invention in comparison to a population of untreated subjects. For example, the average survival time is increased by more than 30 days (more than 60 days, 90 days, or 120 days). An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with the elabela-derived conjugates described herein. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an elabela-derived conjugate described herein.

Treating pulmonary arterial hypertension, heart failure, and/or ischemia or reperfusion-induced renal fibrosis can also result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. For example, the mortality rate is decreased by more than 2% (e.g., more than 5%, 10%, or 25%). A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an elabela-derived conjugate described herein. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an elabela-derived conjugate as described herein.

Treating a subject identified as at risk for developing pulmonary arterial hypertension, heart failure, and/or ischemia or reperfusion-induced renal fibrosis can result in an increase in the time to onset of the disease of a population of subjects treated according to the present invention in comparison to a population of untreated subjects. For example, the delay in the onset to disease is increased by more than 30 days (more than 60 days, 90 days, 120 days, 1 year, 2 years, 5 years, or more). An increase in the time to onset of the disease may be measured, for example, by calculating for a population the average time to disease onset following initiation of treatment with the elabela-derived conjugates described herein. An increase in the time to onset of the disease of a population may also be measured, for example, by calculating for a population the average time to disease onset following completion of a first round of treatment with an elabela-derived conjugate described herein.

Selection of Subjects

Subjects that may be treated using the methods described herein are subjects having or at risk for developing ischemia or reperfusion-induced renal fibrosis, PAH, or HF (e.g., congestive heart failure (CHF) as a consequence of a myocardial infarction). Patients at risk for HF may show early symptoms of heart failure or may not yet be symptomatic when treatment is administered. Similarly, patients at risk for worsening ischemia or reperfusion-induced renal fibrosis or PAH may show early symptoms of ischemia or reperfusion-induced renal fibrosis or PAH or may not yet be symptomatic when treatment is administered. Subjects that may be treated include those that have HF diagnosed by the standard and routine diagnostic procedures known in the art (e.g., electrocardiography (ejection fraction), radionuclide imaging, magnetic resonance imaging, computed tomography imaging, cardiac catheterization with angiography, heart muscle biopsy, and/or assessment of atrial natriuretic peptide (ANP) and/or B-type natriuretic peptide levels in the blood (BNP)).

Subjects that may be treated also include those that have PAH diagnosed by the standard and routine diagnostic procedures known in the art (e.g., blood tests (e.g., arterial blood gas measurement), cardiac magnetic resonance imaging, chest radiography, computed tomography of chest, echocardiography (e.g., with an optional bubble study), electrocardiography, pulmonary function test, right heart catheterization with vasoreactivity testing, and/or six-minute walk test (e.g., to assess the severity of PAH)).

Subjects that may be treated also include those that have had ischemia or reperfusion-induced renal fibrosis diagnosed by the standard and routine diagnostic procedures known in the art (e.g., blood tests (e.g., BUN and/or Cr), urinalysis (e.g., red blood cells, white blood cells, cellular casts and/or cytokines), and/or kidney biopsy (e.g., to assess inflammation and/or fibrosis)).

In some embodiments, a subject has previously undergone treatments for HF, PAH, and/or ischemia or reperfusion-induced renal fibrosis (e.g., digoxin, ACE Inhibitors, diuretics, beta blockers, and/or anti-hypertensive compounds for HF, endothelin receptor antagonists, prostacyclin analogs, and/or enhancers of nitric oxide signaling for PAH, antibiotics for sepsis, diuretic/contractile agents for chronic heart failure, and/or steroids for vasculitis). In other embodiments, the subject has not previously undergone treatments for HF, PAH, and/or ischemia or reperfusion-induced renal fibrosis.

Pharmaceutical Compositions

Elabela-derived conjugates for use in the methods described herein may be placed into a pharmaceutically-acceptable suspension, solution, or emulsion. The composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage at 2-8°C (e.g., 4°C).

A composition including an elabela-derived conjugate can also be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). A composition including an elabela-derived conjugate can further be formulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 1 ½ years, or 2 years) at 2-8°C (e.g., 4°C).

In some embodiments, an elabela-derived conjugate is formulated in a phosphate-buffered saline. Additional active compounds can also be incorporated into the composition.

Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington’s Pharmaceutical Sciences (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (USP 41 NF36), published in 2018. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe. Routes of Administration

Pharmaceutical compositions including an elabela-derived conjugate described herein may be administered, for example, by subcutaneous, intravenous, via percutaneous coronary intervention (e.g., via cardiac catheter), aerosolized inhalation, or nasal application.

The most suitable route for administration in any given case will depend on the particular cell or composition administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the diseases being treated, the patient’s diet, and the patient’s excretion rate. Multiple routes of administration may be used to treat a single subject. Multiple routes of administration may be used to treat a single subject at one time, or the subject may receive treatment via one route of administration first, and receive treatment via another route of administration during a second appointment, e.g., 1 day later, 2 days later, 5 days later, 1 week later, 2 weeks later, 1 month later, 6 months later, or 1 year later. Compositions may be administered to a subject once, or compositions may be administered one or more times (e.g., 2-10 times) per day, week, month, or year to a subject for prevention or treatment for HF, PAH and renal fibrosis.

Dosing

The dosage of a pharmaceutical composition including an elabela-derived conjugate described herein, can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the subject to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. Pharmaceutical compositions including an elabela-derived conjugate described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response.

In some embodiments, suitable dosages of pharmaceutical compositions including an elabela- derived conjugate described herein include dosages of between 0.001 mg to about 1000mg per dose (e.g., from 0.001 mg to 1000 mg, from 0.05 mg to 500 mg, from 0.1 mg to 20 mg, from 5 mg to 500 mg, from 0.1 mg to 100 mg, from 10 mg to 100 mg, from 0.1 mg to 50 mg, from 0.5 mg to 25 mg, from 1.0 mg to 10 mg, from 1.5 mg to 5 mg, or from 2.0 mg to 3.0 mg) or from 1 mg to 1 ,000 mg (e.g., from 5 mg to 1 ,000 mg, from 1 mg to 750 mg, from 5 mg to 750 mg, from 10 mg to 750 mg, from 1 mg to 500 mg, from 5 mg to 500 mg, from 10 mg to 500 mg, from 1 mg to 100 mg, from 5 mg to 100 mg, from 10 mg to 100 mg, from 1 mg to 50 mg, from 5 mg to 50 mg, or from 10 mg to 50 mg). In some embodiments, the suitable dosage of the pharmaceutical compositions including an elabela-derived conjugate comprises 1 mg, 5 mg, or 10 mg per dose.

Combination Therapies

A pharmaceutical composition including an derived-elabela-derived conjugate as described herein, can be administered alone or in combination with an additional therapeutic agent. In combination treatments, the dosages of one or more of the therapeutic agents may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et al. , Neurology 65:S3-S6 (2005)). In this case, dosages of the agents or compounds when combined should provide a therapeutic effect.

For example, when used to prevent or treat PAH, the elabela-derived conjugates may be administered in combination with one or more additional therapeutic agents routinely used in the treatment of PAH (e.g., endothelin receptor antagonists, prostacyclin analogs, and/or enhancers of nitric oxide signaling).

In another example, for the prevention or treatment of HF, the elabela-derived conjugates may be administered in combination with one or more additional therapeutic agents routinely used in the treatment of HF (e.g., digoxin, ACE Inhibitors, diuretics, beta blockers, and/or anti-hypertensive compounds).

In another example, for the prevention or treatment of ischemia or reperfusion-induced renal fibrosis, the elabela-derived conjugates may be administered in combination with one or more additional therapeutic agents routinely used in the treatment of chronic kidney dysfunction (e.g., antibiotics for sepsis, diuretic/contractile agents, and/or steroids).

Evaluation of Efficacy

Assessment of the efficacy of a treatment including an elabela-derived conjugate of the invention may be assessed through any suitable clinical means as is known to those of skill in the art. For example, the development, progression, and or amelioration of HF in response to treatment may be assessed through electrocardiography (e.g., ejection fraction), radionuclide imaging, magnetic resonance imaging, computed tomography imaging, cardiac catheterization with angiography, heart muscle biopsy, atrial natriuretic peptide levels in the blood, and/or B-type natriuretic peptide levels in the blood.

In another example, the development, progression, and or amelioration of PAH in response to treatment may be assessed using blood tests (e.g., arterial blood gas measurement); one or more non- invasive tests such as cardiac magnetic resonance imaging, chest radiography, computed tomography of chest, echocardiography (e.g., optionally with a bubble study), electrocardiography, and/or pulmonary function test; or with one or more invasive tests (e.g., right heart catheterization with vasoreactivity testing and/or a six-minute walk test (e.g., to assess severity of PAH)).

In another example, the development, progression, and/or amelioration of ischemia or reperfusion-induced renal fibrosis in response to treatment may be assessed using blood tests (e.g., BUN and/or Cr), urinalysis (e.g., red blood cells, white blood cells, cellular casts and/or cytokines), and/or kidney biopsy (e.g., to assess inflammation and/or fibrosis).

Kits

The compositions described herein can be provided in a kit for use in in (i) the prevention or treatment of ischemia/reperfusion induced renal fibrosis, (ii) the prevention or treatment of heart failure (e.g., post myocardial infarction heart failure), and (iii) the prevention or treatment pulmonary arterial hypertension. Compositions may include elabela-derived conjugates described herein. The kit can include a package insert that instructs a user of the kit, such as a physician, to perform the methods described herein. The kit may optionally include a syringe or other device for administering the composition.

Examples

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 may be used, made, 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.

Example 1: Generation of elabela-derived conjugates

Elabela-derived conjugates were generated using standard established peptide synthesis methodologies. Briefly, in one example, an elabela-derived peptide having an amino acid sequence of SEQ ID NO: 1 (elabela-14) was made using a standard solid phase synthesis on Wang resin (0.45 meq/g). HPLC purification was performed using 0.1% TFA/water and 0.1% TFA/aceto nitrile buffers on a reversed-phase C-18 column. Following purification, a palmitate moiety linked to a b alanine-2xglycine linker was then coupled to the N-terminus of elabela-14 using DIC/Oxyma.

Example 2: Evaluation of activity of elabela-derived conjugates

The activity of elabela-derived conjugates was assessed using a cell culture luciferase-based reporter assay. Briefly, HEK293 cells were transiently co-transfected for 24 hours with cDNAs encoding the human apelin receptor, an SRE-luciferase reporter gene, Gq5i (to assess Gai mediated signaling), and a b-galactosidase control gene (as a transfection control). Apelin receptor agonists including Ela-14 conjugated to a fatty acid acyl (lipidated Ela-14) were added during the last four hours of the transfection protocol. Luciferase activity was assessed after a total of 24 hours. Notably, lipidated Ela-14 exhibited conserved agonist efficacy and potency (EC50=1.3nM) compared to unconjugated apelin-13 and unconjugated Ela-14 (Figure 1). Potencies similarto lipidated Ela-14 were also observed for two exemplary peptides of the invention: Pyr-Arg-Lys(beta-Ala-Palmitoyl)-Cys-Met-Pro-Leu-His-Ser-Arg-Val- Pro-Phe-Pro-OH (SEQ ID NO: 26) and Pyr-Arg-Arg-Cys-Met-Lys(beta-Ala-Palmitoyl)-Leu-His-Ser-Arg- Val-Pro-Phe-Pro-OH (SEQ ID NO: 27).

Example 3: Evaluation of stability of elabela-derived conjugates

In vitro stability of unconjugated Ela-14 and lipidated Ela-14 was assessed by incubation of the peptides with varying concentrations of trypsin. Briefly, peptides were pre-incubated with trypsin at various dilutions for 1.5 hours at 37°C. Following incubation, bioactivity was assessed using the cell culture luciferase-based reporter assay described in Example 2, above. Unconjugated Ela-14 exhibited an 11- and >800-fold potency loss, respectively after preincubation with a 2.5x10⁵ and 2.5x10⁴ dilutions of trypsin, indicating that 91 and 99.9% of the peptide had been degraded (Figure 2, left panel). In contrast, no trypsin-induced potency loss was observed with lipidated Ela-14 (Figure 2, right panel). Protease resistance for two exemplary peptides of the invention, Pyr-Arg-Lys(beta-Ala-Palmitoyl)-Cys- Met-Pro-Leu-His-Ser-Arg-Val-Pro-Phe-Pro-OH (SEQ ID NO: 26) and Pyr-Arg-Arg-Cys-Met-Lys(beta-Ala- Palmitoyl)-Leu-His-Ser-Arg-Val-Pro-Phe-Pro-OH (SEQ ID NO: 27), was also improved relative to that of lipidated elabela.

Example 4: In vivo pharmacodynamics and pharmacokinetics evaluation of elabela-derived conjugates

Elabela-derived conjugates can be injected percutaneously, subcutaneously, or intravenously into a vertebrate animal (e.g., a rat, mouse, or pig). For pharmacokinetic analysis radiolabeled elabela- derived conjugates can be injected to characterize absorption, half-life, metabolism, distribution, and excretion of the peptide. In one example, changes in blood pressure can be used to determine whether an elabela-derived conjugate is bioactive and capable of activating the apelin receptor system.

Example 5: Evaluation of efficacy of elabela-derived conjugates in an animal model of congestive heart failure

Mouse models may be used to evaluate elabela-derived conjugates in vivo for the treatment of heart failure. In one particular example, mice are implanted with telemetric monitors seven days prior to the administration of elabela-derived conjugates. Prior to administration of the elabela-derived conjugates, baseline blood pressure is recorded. Elabela-derived conjugates or control compositions are then administered subcutaneously to mice in increasing doses, each separated by a three-day washout/recovery phase. Sequential drug doses may be administered as follows: 1 , 5, 25, 125, and 625 pg conjugate/kg body weight. Following each dose, systolic, diastolic, and mean blood pressures are monitored for 24 hours and quantified.

Following a seven-day washout period, mice are further assessed by echocardiography. LV systolic function (e.g., LV fractional shortening and ejection fraction) and diastolic function (e.g., isovolumic relaxation time and mitral inflow velocities) are recorded prior to administration of the elabela- derived conjugates to establish baseline measurements. The following day, mice receive subcutaneous administration of the elabela-derived conjugates. The dose and time point for echocardiography can be selected based on the maximum observed effect on blood pressure. An additional echocardiographic measurement is recorded after twenty-four hours to evaluate the duration of observed effects.

In another example, the elabela-derived conjugates can be further assessed in a mouse myocardial infarction model. After baseline ECHO, a myocardial infarction is surgically induced by ligation of the left anterior descending artery. Immediately thereafter, the animals receive daily subcutaneous injections of either an elabela-derived conjugate or vehicle at the previously determined optimal dose. Infarct size and LV contractile function is assessed by echocardiography at two and seven days post-infarct. At day seven, hearts are removed for quantification of infarct size, and organs weighed to measure cardiac chamber hypertrophy as well as lung mass (an index of pulmonary edema, a sequela of heart failure). Elabela-derived conjugate-induced improvement in one or more of these parameters is indicative of therapeutic efficacy. Example 6: Evaluation of efficacy of elabela-derived conjugates in an animal model of Pulmonary Arterial Hypertension (PAH)

The monocrotaline (MCT)-induced PAH model, a well-established preclinical disease model, can be used to evaluate effectiveness of the elabela-derived conjugates. Both early and late treatment paradigms can be assessed.

Hemodynamic measurements:

In one example, groups of eight rats, 8-10 weeks of age, with equal numbers of male and female animals, are administered a single subcutaneous injection of 60 mg/kg MCT or vehicle control. MCT causes moderate PAH at 21 days (early disease), while at 35 days, animals have severe PAH (late disease). The animals are then treated with daily intraperitoneal injections of unmodified synthetic elabela (ELA, 450 pg/kg), a dose shown to partially attenuate MCT-induced PAH, or with a stable elabela- derived conjugate at the same dose, and compared with corresponding saline-injected controls. At the end of treatments, RV and systemic pressures as well as cardiac outputs are obtained via jugular venous, carotid artery, and femoral artery cannulation, respectively. After lung inflation for histological preparation, lungs and hearts are removed and processed as described below.

Drug-induced attenuation of pulmonary arterial remodeling:

To assess treatment effects of the stable elabela-derived conjugate on the pulmonary vasculature, paraffin-embedded lung sections will be stained with Verhoeff-VanGieson for elastin, followed by morphometric analysis of the vessels by light microscopy (Zeiss, Thornwood, NY). In each animal, 80-100 intra-acinar arteries (20-80 pm diameter) will be categorized as fully muscular (>75% of the circumference of the vessel), partially muscular (25-75%), or nonmuscular (<25%).

Drug-induced attenuation of pathologic cardiac remodeling:

In the setting of PAH, RV undergoes hypertrophy and fibrosis, resulting in right heart failure and death. Therefore, an effective treatment with a stable elabela-derived conjugate will result in a reduction of pathological RV hypertrophy and/or RV fibrosis. RV hypertrophy can be quantified by the Fulton index: RV/(Left Ventricle+Septum) = weight ratio, and by analysis of RV myocyte size (hematoxylin/eosin staining). RV fibrosis can be quantified after RV collagen staining (mason trichrome) and by hydroxyproline assay.

Example 7: Prevention or Treatment of Heart Failure

A subject diagnosed as having or at risk for developing heart failure (e.g., congestive heart failure (CHF) as a consequence of a myocardial infarction) can be treated with an elabela-derived conjugate or pharmaceutical composition thereof as described herein. The elabela-derived conjugate can be administered at an effective dose to treat the subject diagnosed with or at risk for developing HF. When effectively treated, the subject diagnosed as at risk for developing HF will have a decreased risk of post myocardial infarction-induced CHF with attendant symptoms and signs. For example, the subject may experience an increase in the time to disease onset as compared to a subject not treated with an elabela- derived conjugate or pharmaceutical composition according to the methods described herein. For a subject having HF, following treatment, fibrosis will be minimized and the ejection fraction will be maintained. Treatment may also result in an improvement in one or more symptoms (e.g., dyspnea (worse on exertion), leg swelling, orthopnea, fatigue, and/or nocturnal urinary frequency), improvement of heart function (e.g., as assessed by chest x-ray, cardiac ultrasound (e.g., ejection fraction), stress test (e.g., exercise tolerance, blood oxygen level, and/or cardiac catheterization), and/or prolonged survival.

The subject can be diagnosed prior to treatment by a variety of diagnostic methods known in the art. For example, a subject presenting with indicators of myocardial necrosis following acute myocardial infarction (as evidenced by the appearance of cardiac enzymes (e.g., creatine kinase, myocardial specific enzyme CK-MB, myocardial cell protein troponin I, and/or myocardial cell protein troponin T) in the circulation) may be identified as at risk for developing HF. In another example, a subject can be diagnosed as having HF using electrocardiography (ejection fraction), radionuclide imaging, magnetic resonance imaging, computed tomography imaging, cardiac catheterization with angiography, heart muscle biopsy, atrial natriuretic peptide levels in the blood and/or assessment of B-type natriuretic peptide levels in the blood.

Example 8. Prevention or Treatment of Pulmonary Arterial Hypertension

A subject diagnosed as having or at risk for developing pulmonary arterial hypertension can be treated with an elabela-derived conjugate or pharmaceutical composition thereof as described herein.

The elabela-derived conjugate can be administered at an effective dose to treat the subject diagnosed with PAH. When effectively treated, the subject diagnosed as having or at risk for developing PAH will exhibit a slowed or arrested disease progression. Treatment may also result in an improvement in one or more symptoms (e.g., decreased dyspnea, fatigue, chest pain, and palpitations), enhanced exercise tolerance, improvement in pulmonary function (e.g., as assessed by pulmonary function tests, arterial blood gas measurement, echocardiography, cardiac MRI, right heart catheterization with vasoreactivity testing, and six-minute walk test (to assess severity of PAH)), and/or prolonged survival

The subject can be diagnosed prior to treatment by a variety of diagnostic methods known in the art. For example, a subject can be diagnosed as having or at risk for developing PAH by blood tests (e.g., arterial blood gas measurement), cardiac magnetic resonance imaging, chest radiography, computed tomography of chest, echocardiography (e.g., with an optional bubble study), electrocardiography, pulmonary function test, right heart catheterization with vasoreactivity testing, and/or six-minute walk test (e.g., to assess the severity of PAH).

Example 9. Prevention or Treatment of Ongoing Renal Fibrosis

A subject diagnosed as having or at risk for developing renal fibrosis associated with kidney disease characterized by ischemia and/or reperfusion injury resulting from vascular compromise secondary to heart failure, sepsis, or vascular disease can be treated with an elabela-derived conjugate or pharmaceutical composition thereof as described herein. Additionally, a subject diagnosed as having renal fibrosis associated with a kidney disease resulting from a drug and/or toxin-induced kidney damage (e.g., cyclosporin) can be treated with an elabela-derived conjugate or pharmaceutical composition thereof as described herein. The elabela-derived conjugate can be administered at an effective dose to treat the subject diagnosed with renal fibrosis. When effectively treated, the subject diagnosed having or at risk for developing renal fibrosis will exhibit a slowed or arrested disease progression. Treatment may also result in an improvement in one or more symptoms (e.g., inflammation, fibrosis, cellular apoptosis, renal dysfunction, and renal tubular lesions) and/or prolonged survival. The subject can be diagnosed prior to treatment by a variety of diagnostic methods known in the art. For example, a subject can be diagnosed as having renal fibrosis by blood tests (e.g., BUN and/or Cr), urinalysis (e.g., red blood cells, white blood cells, cellular casts and/or cytokines), and/or kidney biopsy (e.g., to assess inflammation and/or fibrosis). Abnormalities in one or more of these indices would provide evidence for a diagnosis.

Other Embodiments

Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

Other embodiments are in the claims.

Claims

CLAIMS What is claimed is:

1. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a fatty acid acyl covalently linked through a linker to an elabela peptide or a fragment thereof comprising at least 11 contiguous amino acid residues.

2. The conjugate of claim 1 , wherein the elabela peptide or fragment thereof comprises at least 14 contiguous amino acid residues.

3. The conjugate of claim 1 or 2, wherein the elabela peptide or fragment thereof comprises an amino acid sequence from N-terminus to C-terminus of formula (I):

Glnoi-Argo2-Proo3-Valo4-Asno5-Leuo6-Thro7-Meto8-Argo9-Argio-Lysii-Leui2-Argi3-Lysi4-Hisi5-Asni6- Cysi7-Leui8-Glni9-Arg20-Arg2i-Cys22-Met23-Pro24-Leu25-HiS26-Ser27-Arg28-Val29-Pro30-Phe3i-Pro32 (I) (SEQ ID NO: 25).

4. The conjugate of claim 3, wherein one or more of Glnig to Pro3₂ is substituted with Lys, Ala, Val, pyroglutamic acid (Pyr), or Gly.

5. The conjugate of claim 4, wherein one or more of Glnig to Pro3₂ is substituted with Lys.

6. The conjugate of claim 5, wherein Arg_åi or Pro_å4 is substituted with Lys.

7. The conjugate of claim 4 or 5, wherein one or more of Glnig to Pro3₂ is substituted with Ala.

8. The conjugate of claim 7, wherein Arg_åi is substituted with Ala.

9. The conjugate of claim 7 or 8, wherein Arg_åo is substituted with Ala

10. The conjugate of any one of claims 6-9, wherein Arg_ås is substituted with Ala.

11. The conjugate of any one of claims 4-10, wherein Glnig is substituted with Pyr.

12. The conjugate of any one of claims 3-11 , wherein one or more of Glnoi to Leuis is substituted with

Lys, Ala, Val, Pyr, or Gly, or is absent.

13. The conjugate of claim 12 wherein Glnoi to Leuis is absent.

14. The conjugate of claim 12, wherein Glnoi to Lysn is absent.

15. The conjugate of claim 12, wherein Glnoi is substituted with Pyr.

16. The conjugate of any one of claims 13-15, wherein the elabela peptide or fragment thereof comprises at least one lysine residue.

17. The conjugate of claim 16, wherein the at least one lysine residue is at least two lysine residues.

18. The conjugate of claim 17, wherein the at least two lysine residues is at least three lysine residues.

19. The conjugate of claim 18, wherein the at least three lysine residues is at least four lysine residues.

20. The conjugate of claim 1 or 2, wherein the elabela peptide or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 7-24.

21 . The conjugate of claim 20, wherein the elabela peptide or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 8, 9, 11 , 12, 14, 15, 16, or 20.

22. The conjugate of any one of claims 1 -21 , wherein the linker comprises the structure:

X1-L-X2; wherein Xi is one or more amino acids selected from Gly, Ala, Asn, Cys, and combinations thereof, or is absent;

L is a linker selected from the group consisting of a b alanine linker, a b alanine-2x glycine linker, a y-glutamyl linker, a bis-y-glutamyl linker, an animo-3,6-dioxaoctanoic acid (OEG) linker, a y-glutamyl-2x oligo ethylene glycol linker, 2-(2-(2-AminoEthoxy)Ethoxy)Acetic acid, Aminoethylethanolamine, (PEG)s, glutamine, glutamic acid, benzophenone-4-lsothiocyanate, bis-((N- lodoacetyl)Piperazinyl)Sulfonerhodamine, succinimidyl 2-(2-Pyridyldithio)Propionate, 4-Azido-2, 3,5,6-

Tetrafluorobenzoic acid, (N-((2-Pyridyldithio)ethyl)-4- Azidosalicylamide), succinimidyl trans-4-

(maleimidylmethyl)cyclohexane-l-carboxylate, and N-(t-BOC)-aminooxyacetic acid; and

X2 is one or more amino acids selected from Gly, Ala, Asn, Cys, and combinations thereof, or is absent.

23. The conjugate of claim 22, wherein L is a b alanine linker.

24. The conjugate of claim 22, wherein L is a b alanine-2x glycine linker.

25. The conjugate of claim 22, wherein L is a g-glutamyl linker.

26. The conjugate of claim 22, wherein L is a y-glutamyl-2x oligo ethylene glycol linker.

27. The conjugate of claim 22, wherein L is a 2-(2-(2-AminoEthoxy)Ethoxy)Acetic acid linker.

28. The conjugate of claim 22, wherein L is a (PEG)s linker.

29. The conjugate of any one of claims 22-28, wherein (a) Xi is absent, (b) X2 is absent, or (c) both Xi and X2 are absent.

30. The conjugate of any one of claims 22-29, wherein Xi and/or X2 comprises Ala-Ala-Ala.

31 . The conjugate of any one of claims 22-30, wherein Xi and/or X2 comprises Ala-Gly.

32. The conjugate of any one of claims 22-30, wherein Xi and/or X2 comprises Asn-Gly-Asn-Gly.

33. The conjugate of any one of claims 3-32, wherein the elabela peptide or fragment thereof comprises a lysine residue having a side chain covalently linked by the linker to the fatty acid acyl.

34. The conjugate of any one of claims 1-33, wherein the fatty acid acyl is covalently linked through a linker to the N-terminus of the elabela peptide or fragment thereof.

35. The conjugate of any one of claims 1-34, wherein the fatty acid acyl comprises 8 to 26 carbon atoms.

36. The conjugate of claim 35, wherein the fatty acid acyl is derived from a fatty acid selected from the group consisting of palmitic acid, myristic acid, stearic acid, palmitoleic acid, oleic acid, cholesterol, DPPE, GM1 , GM2, GM3, a-Linolenic acid, EPA, DHA, DPPC, DOPS, and DOPC.

37. The conjugate of claim 36, wherein the fatty acid acyl is palmitate.

38. The conjugate of claim 36, wherein the fatty acid acyl is octadecandioate.

39. The conjugate of any one of claims 1-38, wherein the elabela peptide or fragment thereof further comprises a C-terminal amidation modification.

40. The conjugate of any one of claims 1-39, wherein the elabela peptide or fragment thereof further comprises an N-terminal acylation modification.

41. A pharmaceutical composition comprising the conjugate of any one of claims 1-40 and one or more pharmaceutically acceptable carriers or excipients.

42. A method of agonizing the apelin receptor in a cell, the method comprising contacting the cell with an effective amount of the conjugate of any one of claims 1-40, or the pharmaceutical composition of claim 41 .

43. The method of claim 42, wherein the cell is a cell within the vasculature.

44. The method of claim 42, wherein the cell is a cardiomyocyte.

45. The method of claim 42, wherein the cell is a renal cell.

46. The method of any one of claims 42-45, wherein the cell is a human cell.

47. A method of preventing pulmonary arterial hypertension (PAH) in a subject identified as at risk for developing PAH, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1-40 or the composition of claim 41.

48. A method of treating PAH in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1-40 or the composition of claim 41 .

49. A method of preventing heart failure in a subject identified as at risk for developing heart failure, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1-40 or the composition of claim 41.

50. A method of treating heart failure in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1-40 or the composition of claim 41.

51 . The method of claim 49 or 50, wherein prior to administration, the subject has had an acute myocardial infarction.

52. A method of preventing ischemia or reperfusion-induced renal fibrosis in a subject identified as at risk for developing ischemia or reperfusion-induced renal fibrosis, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1-40 or the composition of claim 41.

53. A method of treating ischemia or reperfusion-induced renal fibrosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1 -40 or the composition of claim 41 .

54. The method of any one of claims 47-53, wherein the administering comprises subcutaneous, intravenous, percutaneous coronary intervention via cardiac catheter, aerosolized inhalation, or nasal administration of the conjugate or the pharmaceutical composition to the subject.

55. The conjugate of claim 1 , wherein the conjugate is Pyr-Arg-Lys(beta-Ala-Palmitoyl)-Cys-Met-Pro- Leu-His-Ser-Arg-Val-Pro-Phe-Pro-OH (SEQ ID NO: 26).

56. The conjugate of claim 1 , wherein the conjugate is Pyr-Arg-Arg-Cys-Met-Lys(beta-Ala-Palmitoyl)- Leu-His-Ser-Arg-Val-Pro-Phe-Pro-OH (SEQ ID NO: 27).

57. The pharmaceutical composition of claim 41 , wherein the conjugate is Pyr-Arg-Lys(beta-Ala- Palmitoyl)-Cys-Met-Pro-Leu-His-Ser-Arg-Val-Pro-Phe-Pro-OH (SEQ ID NO: 26).

58. The pharmaceutical composition of claim 41 , wherein the conjugate is Pyr-Arg-Arg-Cys-Met- Lys(beta-Ala-Palmitoyl)-Leu-His-Ser-Arg-Val-Pro-Phe-Pro-OH (SEQ ID NO: 27).

59. The method of any one of claims 47-54, wherein the conjugate is Pyr-Arg-Lys(beta-Ala- Palmitoyl)-Cys-Met-Pro-Leu-His-Ser-Arg-Val-Pro-Phe-Pro-OH (SEQ ID NO: 26).

60. The method of any one of claims 47-54, wherein the conjugate is Pyr-Arg-Arg-Cys-Met-Lys(beta- Ala-Palmitoyl)-Leu-His-Ser-Arg-Val-Pro-Phe-Pro-OH (SEQ ID NO: 27).