WO2025106145A1

WO2025106145A1 - Hybrid form of cardiac targeting peptide with increased serum stability and enhanced cardiomyocyte targeting

Info

Publication number: WO2025106145A1
Application number: PCT/US2024/045793
Authority: WO
Inventors: Maliha Zahid
Original assignee: Mayo Foundation for Medical Education and Research; Mayo Clinic in Florida
Current assignee: Mayo Foundation for Medical Education and Research; Mayo Clinic in Florida
Priority date: 2023-11-13
Filing date: 2024-09-09
Publication date: 2025-05-22
Anticipated expiration: 2026-05-13

Abstract

Cardiac targeting peptides and methods for their use are provided herein. For example, cardiac targeting peptides containing D-amino acids at particular positions and having increased cardiomyocyte targeting ability are provided herein, as are methods for using the cardiac targeting peptides to deliver agents (e.g., radioisotopes or drugs) to the heart.

Description

HYBRID FORM OF CARDIAC TARGETING PEPTIDE WITH INCREASED SERUM STABILITY AND ENHANCED CARDIOMYOCYTE TARGETING

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Serial No. 63/567,225, filed March 19, 2024, and from U.S. Provisional Application Serial No. 63/548,342, filed November 13, 2023. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named 07039-2277WOl_ST26_SL.XML.” The XML file, created on September 5, 2024, is 9,482 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This document relates to cardiac targeting peptides and methods for their use. For example, this document relates to cardiac targeting peptides with increased cardiomyocyte targeting ability, and to use of the peptides to deliver agents such as radioisotopes to the heart.

BACKGROUND

Despite several decades of advances, coronary artery disease remains the number one killer in the developed world, with numbers quickly catching up in the developing world. Cardiomyocytes are the cells responsible for the contractility of the heart and are the seat of common pathologies such as heart failure with reduced ejection fraction. Another type of heart failure, heart failure with preserved ejection fraction, results from a “stiff’ heart. This type of heart failure has been rising steadily within the population of heart failure patients, mainly due to aging of the population and increasing incidence of co-morbidities, such as obesity, hypertension, and diabetes mellitus. In contrast to heart failure with reduced ejection fraction (which is due to a “weak” heart), heart failure with preserved ejection fraction is a recalcitrant disease with very few beneficial therapies. Targeting the stiffened heart by changing the behavior of the hypertrophied cardiomyocyte, thus addressing the underlying pathophysiology, will require the ability to successfully and specifically target the cardiomyocyte.

Occlusive coronary artery disease, which can manifest as chest pain and/or shortness of breath, can be diagnosed by stress testing utilizing cardiac imaging technologies such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), stress echocardiography, stress MRI, or CT angiography. These methods have limitations, however, such as a high rate of false positives and false negatives (SPECT), short half-lives of seconds to a few minutes precluding exercise testing (PET), and requiring on-site cyclotrons or expensive generators, thereby limiting the use of PET to large, academic, research centers only. Of these imaging modalities, PET has been shown to have the highest sensitivity for diagnosing obstructive coronary artery disease, but its use remains confined to only large, academic centers that can support on-site cyclotrons.

SUMMARY

Targeted delivery of therapeutics to the heart is a long-sought goal, with strategies including direct intra-cardiac or intra-pericardial delivery, intra-coronary infusion, and the use of adenoviral, lentiviral, or adeno-associated viral vectors having preference, if not complete cardio-selectivity, for cardiac tissue. Another option is the use of cell penetrating peptides (CPPs), which are about 5-30 amino acids in length and can breach cell membrane barriers while carrying cargos up to several times their size, in an intact functional form. Several strategies can be used to identify cell- or tissue-specific CPPs, including phage display. Using this technique, a 12 amino acid, non-naturally occurring cardiomyocyte targeting peptide (CTP) was identified that targets cardiomyocytes selectively after an intravenous injection in as little as 5-30 minutes. This document is based, at least in part, on the development of variants of the synthetic CTP, resulting in peptides with enhanced serum stabilities, better ability to resist proteinase degradation, and significantly enhanced transduction of cardiomyocytes in vivo. The variant CTPs can be used, in some cases, to deliver radioisotopes (e.g., for imaging), drugs (e.g., small molecules), and oligonucleotides (e.g., in the form of microRNAs (miRNAs), small interfering RNAs (siRNAs), phosphorodiamidate morpholino oligomers, and/or peptide-nucleic acids) to the heart in order to, for example, alter cell behavior. For example, as described herein, variants of the CTP in which two amino acid residues in key positions were substituted with their D-enantiomers, or in which the N-terminal six amino acids were deleted, or in which the N-terminal six amino acids were deleted and two of the remaining amino acids in key positions were substituted with their D-enantiomers, demonstrated increased serum stability and enhanced cardiomyocyte targeting ability.

This document provides variant CTPs, compositions containing the variant CTPs, and methods for their use. For example, the variant CTPs can be radiolabeled and used as cardiac PET imaging agents, which may expand the use of PET beyond large academic centers. In some cases, the variant CTPs can be labeled (e.g., with Technetium-99m) and used as cardiac-specific SPECT imaging agents. In addition, the variant CTPs (e.g., a variant CTP having the amino acid sequence AP(d)WHLSSQYS(d)RT, which is SEQ ID NO:6 and also is referred to herein as “hybrid CTP” or “hCTP”) can be used as delivery vectors for therapeutic agents (e.g., small molecule drugs such as Amiodarone, or oligonucleotides such as siRNAs, miRNAs, phosphorodiamidate morpholino oligomers, and/or peptide-nucleic acids) for treating cardiac conditions such as atrial fibrillation, ventricular tachycardia, or heart failure (e.g., heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, or cardiomyopathy).

In a first aspect, this document features a polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, or having the amino acid sequence set forth in SEQ ID NO:6 but with one or two amino acid additions, subtractions, or substitutions, provided that the polypeptide does not have the amino acid sequence set forth in SEQ ID NO: 1 In another aspect, this document features a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, or having the amino acid sequence set forth in SEQ ID NO: 3 but with one amino acid addition, subtraction, or substitution.

In another aspect, this document features a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7, or having the amino acid sequence set forth in SEQ ID NO: 7 but with one amino acid addition, subtraction, or substitution.

In another aspect, this document features a composition containing a pharmaceutically acceptable carrier and a polypeptide having (a) the amino acid sequence set forth in SEQ ID NO:6, (b) the amino acid sequence set forth in SEQ ID NO:6 but with one or two amino acid additions, subtractions, or substitutions, provided that the polypeptide does not have the amino acid sequence set forth in SEQ ID NO:1, (c) the amino acid sequence set forth in SEQ ID NO:3, (d) the amino acid sequence set forth in SEQ ID NO:3 but with one amino acid addition, subtraction, or substitution, (e) the amino acid sequence set forth in SEQ ID NO: 7, or (f) the amino acid sequence set forth in SEQ ID NO:7 but with one amino acid addition, subtraction, or substitution.

In another aspect, this document features a conjugate containing (a) a polypeptide having (i) the amino acid sequence set forth in SEQ ID NO:6, (ii) the amino acid sequence set forth in SEQ ID NO: 6 but with one or two amino acid additions, subtractions, or substitutions, provided that the polypeptide does not have the amino acid sequence set forth in SEQ ID NO: 1, (iii) the amino acid sequence set forth in SEQ ID NO:3, (iv) the amino acid sequence set forth in SEQ ID NO:3 but with one amino acid addition, subtraction, or substitution, (v) the amino acid sequence set forth in SEQ ID NO:7, or (vi) the amino acid sequence set forth in SEQ ID NO:7 but with one amino acid addition, subtraction, or substitution, coupled to (b) a therapeutic agent or a detectable label. The polypeptide can be coupled to the therapeutic agent or the detectable label at its N-terminus or at its C-terminus. The polypeptide can be coupled to the therapeutic agent. The therapeutic agent can include an anti-arrhythmic agent (e.g., a small molecule drug that acts as an anti-arrhythmic agent), a siRNA, miRNA, phosphorodiamidate morpholino oligomer, peptide-nucleic acid, plasmid DNA, DNA origami, RNA origami, nucleic acid containing a sequence encoding a polypeptide, another therapeutic polypeptide, extracellular vesicles, liposomes, nanoparticles, or exosomes. The polypeptide can be coupled to the detectable label. The detectable label can be a fluorescent or radioisotope label. The detectable label can include a radioisotope (e.g., Technetium 99m, Gallium68, or F-18).

In another aspect, this document features a composition containing a pharmaceutically acceptable carrier and a conjugate containing (a) a polypeptide having (i) the amino acid sequence set forth in SEQ ID NO:6, (ii) the amino acid sequence set forth in SEQ ID NO:6 but with one or two amino acid additions, subtractions, or substitutions, provided that the polypeptide does not have the amino acid sequence set forth in SEQ ID NO: 1, (iii) the amino acid sequence set forth in SEQ ID NO:3, (iv) the amino acid sequence set forth in SEQ ID NO: 3 but with one amino acid addition, subtraction, or substitution, (v) the amino acid sequence set forth in SEQ ID NO: 7, or (vi) the amino acid sequence set forth in SEQ ID NO:7 but with one amino acid addition, subtraction, or substitution, coupled to (b) a therapeutic agent or a detectable label. The polypeptide can be coupled to the therapeutic agent or the detectable label at its N- terminus or at its C-terminus. The polypeptide can be coupled to the therapeutic agent. The therapeutic agent can include an anti-arrhythmic agent (e.g., a small molecule drug that acts as an anti -arrhythmic agent), a siRNA, miRNA, phosphorodiamidate morpholino oligomer, peptide-nucleic acid, plasmid DNA, DNA origami, RNA origami, nucleic acid containing a sequence encoding a polypeptide, another therapeutic polypeptide, extracellular vesicles, liposomes, nanoparticles, or exosomes. The polypeptide can be coupled to the detectable label. The detectable label can be a fluorescent or radioisotope label. The detectable label can include a radioisotope (e.g., Technetium 99m, or Gallium68, or F-18).

In another aspect, this document features a method for delivering an agent into a cardiac cell. The method can include, or consist essentially of, contacting a cardiac cell with a conjugate that includes (a) a polypeptide having (i) the amino acid sequence set forth in SEQ ID NO:6, (ii) the amino acid sequence set forth in SEQ ID NO:6 but with one or two amino acid additions, subtractions, or substitutions, provided that the polypeptide does not have the amino acid sequence set forth in SEQ ID NO: 1, (iii) the amino acid sequence set forth in SEQ ID NO:3, (iv) the amino acid sequence set forth in SEQ ID NO:3 but with one amino acid addition, subtraction, or substitution, (v) the amino acid sequence set forth in SEQ ID NO: 7, or (vi) the amino acid sequence set forth in SEQ ID NO:7 but with one amino acid addition, subtraction, or substitution, coupled to (b) the agent. The agent can be a therapeutic agent or a detectable label. The polypeptide can be coupled to the therapeutic agent or the detectable label at its N- terminus or at its C-terminus. The therapeutic agent can include an anti-arrhythmic agent (e.g., a small molecule drug that acts as an anti-arrhythmic agent), a siRNA, miRNA, phosphorodiamidate morpholino oligomer, peptide-nucleic acid, plasmid DNA, DNA origami, RNA origami, nucleic acid containing a sequence encoding a polypeptide, another therapeutic polypeptide, extracellular vesicles, liposomes, nanoparticles, or exosomes. The detectable label can be a fluorescent or radioisotope label. The detectable label can include a radioisotope (e.g., Technetium 99m, Gallium68, or F-18). The polypeptide can be coupled to the detectable label.

In another aspect, this document features a method that includes, or consists essentially of, administering, to a mammal, a conjugate containing (a) a polypeptide having (i) the amino acid sequence set forth in SEQ ID NO:6, (ii) the amino acid sequence set forth in SEQ ID NO: 6 but with one or two amino acid additions, subtractions, or substitutions, provided that the polypeptide does not have the amino acid sequence set forth in SEQ ID NO: 1, (iii) the amino acid sequence set forth in SEQ ID NO:3, (iv) the amino acid sequence set forth in SEQ ID NO:3 but with one amino acid addition, subtraction, or substitution, (v) the amino acid sequence set forth in SEQ ID NO:7, or (vi) the amino acid sequence set forth in SEQ ID NO:7 but with one amino acid addition, subtraction, or substitution, coupled to (b) a therapeutic agent or a detectable marker. The mammal can be a human. The mammal can have been identified as having a cardiovascular disorder. The cardiovascular disorder can include heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, pericarditis, recurrent pericarditis, or Friedreich’s ataxia associated cardiac hypertrophy. The cardiovascular disorder can include myocarditis (e.g., viral myocarditis, vaccine- associated myocarditis, lymphocytic myocarditis, giant-cell myocarditis, or eosinophilic myocarditis). The polypeptide can be coupled to the therapeutic agent or the detectable label at its N-terminus or at its C-terminus. The agent can be a therapeutic agent. The therapeutic agent can include an anti -arrhythmic agent (e.g., a small molecule drug that acts as an anti-arrhythmic agent), a siRNA, miRNA, phosphorodiamidate morpholino oligomer, peptide-nucleic acid, plasmid DNA, DNA origami, RNA origami, nucleic acid containing a sequence encoding a polypeptide, another therapeutic polypeptide, extracellular vesicles, liposomes, nanoparticles, or exosomes. The agent can be a detectable label. The detectable label can be a fluorescent or radioisotope label. The detectable label can include a radioisotope (e.g., Technetium 99m, Gallium68, or F-18). The polypeptide can be coupled to the detectable label.

In still another aspect, this document features a method for treating a mammal having a cardiovascular disorder. The method can include, or consist essentially of, administering, to the mammal, a conjugate containing (a) a polypeptide having (i) the amino acid sequence set forth in SEQ ID NO:6, (ii) the amino acid sequence set forth in SEQ ID NO:6 but with one or two amino acid additions, subtractions, or substitutions, provided that the polypeptide does not have the amino acid sequence set forth in SEQ ID NO: 1, (iii) the amino acid sequence set forth in SEQ ID NO:3, (iv) the amino acid sequence set forth in SEQ ID NO: 3 but with one amino acid addition, subtraction, or substitution, (v) the amino acid sequence set forth in SEQ ID NO:7, or (vi) the amino acid sequence set forth in SEQ ID NO:7 but with one amino acid addition, subtraction, or substitution, coupled to (b) a therapeutic agent. The mammal can be a human. The cardiovascular disorder can include heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, pericarditis, recurrent pericarditis, or Friedreich’s ataxia associated cardiac hypertrophy. The cardiovascular disorder can include myocarditis (e.g., viral myocarditis, vaccine-associated myocarditis, lymphocytic myocarditis, giant-cell myocarditis, or eosinophilic myocarditis). The polypeptide can be coupled to the therapeutic agent or the detectable label at its N- terminus or at its C-terminus. The polypeptide can be coupled to the therapeutic agent or the detectable label at its N-terminus or at its C-terminus. The agent can be a therapeutic agent. The therapeutic agent can include an anti-arrhythmic agent (e.g., a small molecule drug that acts as an anti-arrhythmic agent), a siRNA, a miRNA, phosphorodiamidate morpholino oligomer, peptide-nucleic acid, plasmid DNA, DNA origami, RNA origami, nucleic acid containing a sequence encoding a polypeptide, another therapeutic polypeptide, extracellular vesicles, liposomes, nanoparticles, or exosomes. The agent can include a detectable label. The detectable label can be a fluorescent or radioisotope label. The detectable label can include a radioisotope (e.g., Technetium 99m, Gallium68, or F- 18). The polypeptide can be coupled to the detectable label.

In another aspect, this document features a method for treating a mammal having a cardiac disorder, where the method includes, or consists essentially of, administering to the mammal a polypeptide having the amino acid sequence set forth in SEQ ID NO:6. The mammal can be a human. The cardiovascular disorder can include myocarditis, pericarditis, recurrent pericarditis, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, or Friedreich’s ataxia associated cardiac hypertrophy. The myocarditis can be viral myocarditis, vaccine- associated myocarditis, lymphocytic myocarditis, giant-cell myocarditis, or eosinophilic myocarditis.

In another aspect, this document features a method for treating a mammal having a cardiac disorder, where the method includes, or consists essentially of, administering to the mammal a composition containing a pharmaceutically acceptable carrier and a polypeptide having the amino acid sequence set forth in SEQ ID NO:6. The mammal can be a human. The cardiovascular disorder can include myocarditis, pericarditis, recurrent pericarditis, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, or Friedreich’s ataxia associated cardiac hypertrophy. The myocarditis can be viral myocarditis, vaccine-associated myocarditis, lymphocytic myocarditis, giant-cell myocarditis, or eosinophilic myocarditis. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph plotting fluorescent activated cell sorting (FACS) results using H9C2 cells incubated with 10 pM of various CTP alanine mutants or the CTP-Aand CTP-B fragments, as indicated, in biological quadruplicates. Results were corrected for baseline differences in fluorescence labeling of the peptides.

FIG. 2 is a graph plotting fluorescence intensities of human left ventricular derived cardiomyocytes incubated with various CTP alanine mutants at a concentration of 10 pM. In particular, these cells showed increased uptake of CTP-B (SEQ ID NO:3) and hCTP (SEQ ID NO:6), in which amino acids at positions 3 and 11 were substituted with their D-enantiomers. *p<0.05, ***p<0.001.

FIG. 3 includes representative images and a graph showing confocal fluorescent intensities in hearts from mice injected with CTP, CTP-B, CTP-P2A or random (RAN) peptide (DARHYQGPWNIL; SEQ ID NO:8) at 10 mg/kg for 15 minutes. CTP-B and P2A showed significantly greater fluorescence than CTP. N=3 in each group. *p-value<0.01.

FIGS. 4A and 4B are graphs plotting serum stability of hCTP versus CTP in fetal bovine serum (FBS) (FIG. 4A) and in pooled human serum (FIG. 4B). The FBS and the pooled human serum both exhibited increased serum stability of hCTP compared to CTP over time.

FIG. 5 is a graph plotting the results of FACs in a human cardiomyocyte cell line incubated with the indicated linear and cyclic peptides at 10 pM concentrations for 30 minutes before washing, trypsinizing, fixing and sorting.

FIG. 6 shows representative PET images of [⁶⁸Ga]Ga-NOTA-CTP injected into a CD1 mouse with dynamic imaging for 30 minutes followed by imaging at 60 and 120 minutes.

FIG. 7 shows the general design of various cardiac targeting peptides as PET probes.

FIG. 8 includes a trio of graphs plotting average radiation efficiencies in the heart of mice injected with Cy5.5-labeled CTP or hCTP. In particular, mice were injected with vehicle (PBS), CTP (10 mg/kg), or hCTP (10 mg/kg) and euthanized at 5, 15, 30, 60 and 120 minutes, followed by harvesting of the hearts and IVIS imaging. N=3 for each peptide and time point. Data for each of the 3 mice are plotted individually.

FIG. 9 is a graph plotting the mean radiation efficiencies in the heart of mice injected with Cy5.5-labeled CTP or hCTP. N=3. Error bars represent 1 SD.

FIG. 10A includes 2D confocal images of representative hearts from mice that were injected with CTP (10 mg/kg) or hCTP (10 mg/kg) and euthanized at the indicated time points. 2D confocal quantification (FIG. 10B) showed that hCTP was about 3 times more efficient than CTP in transducing the mouse heart. The peak uptake for CTP occurred at 5 minutes as opposed to hCTP, where the peak uptake occurred at 30 minutes. **p<0.01; ***p<0.001.

FIG. 11A includes 3D confocal images of representative hearts from mice that were injected with CTP (10 mg/kg) or hCTP (10 mg/kg) and euthanized at the indicated time points. 3D confocal quantification (FIG. 11B) showed that hCTP was about 4 times more efficient than CTP in transducing the mouse heart. The peak uptake for CTP occurred at 5 minutes as opposed to hCTP, where the peak uptake occurred at 30 minutes. FIGS. 12A-12C are graphs plotting free cytosolic calcium levels in a human cardiomyocyte cell line that was treated daily with the indicated concentrations of CTP or hCTP daily for 4 days; FLIPR assays for free cytosolic calcium were performed on day 5. hCTP was about 10 times more efficacious at reducing cytosolic calcium levels than CTP. All experiments were done (represented by bars) in octuplicate, and biological triplicates were performed on three different days (FIGS. 12A, 12B, and 12C, respectively). This effect was not due to changes in cell viability (representative graphs plotted in FIG. 12D). *p<0.05; ***p<0.001; ****p<0.0001. FIG. 12D also includes a schematic illustrating the study design.

FIGS. 13A-13C are graphs plotting luminescence as a measure of NF-KB activation in a human cardiomyocyte cell line that was transfected with a reporter plasmid expressing the luciferase gene under an NF-KB promoter. 48 hours posttransfection, cells were treated once with varying concentrations of CTP or hCTP, and 24 hours later the cells were challenged with TNF-a for 4 hours and luminescence was read. hCTP was about 10 times more efficacious as an anti-inflammatory at reducing NF-KB activation than CTP. All experiments were done (represented by bars) in octuplicate, and biological triplicates performed on three different days (FIGS. 13A, 13B, and 13C, respectively). This effect was not due to changes in cell viability (plotted in FIG. 13D). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

FIG. 14A includes a series of representative confocal microscopy images showing hCTP accumulation in the heart, kidney, liver, and lungs of mice over time. FIG. 14B is a graph plotting fluorescence intensities in the indicated organs over time.

FIG. 15A is a graph plotting cytosolic calcium levels in human cardiomyocytes treated with the indicated concentrations of hCTP. Calcium levels were determined by FLIPR 6 assay. FIG. 15B includes representative images showing Western blots for DHPR and SERCA2a expression in human cardiomyocytes after treatment with the indicated concentrations of hCTP. GAPDH was used as a control. Graphs plotting the results also are shown. FIG. 15C is a graph plotting levels of free elemental calcium in solution after a 20 minute incubation of human cardiomyocytes with the indicated concentrations of hCTP. FIG. 16A includes graphs plotting luminescence caused by expression of an NF- KB promoter-driven luciferase reporter in human cardiomyocytes treated with TNF-a alone or in combination with the indicated concentrations of hCTP, to indicate NF-KB activation in response to TNF-a stimulation in the cells with hCTP. Each graph is from a separate experiment. FIG. 16B is a graph plotting IL-8 secretion by human cardiomyocytes treated with TNF-a and the indicated concentrations of hCTP. FIG. 16C is a graph plotting IL- 1 P production by human cardiomyocytes treated the indicated concentrations of hCTP in combination with angiotensin 2 (Ang2) and phenylephrine (PE). FIG. 16D includes representative graphs plotting luminescence caused by expression of an NF-KB promoter-driven luciferase reporter in human cardiomyocytes treated with the indicated concentrations of hCTP (left and center graphs) or CTP (right graph) in combination with Ang2 and PE. A schematic illustrating the study protocol is shown at the top of the figure.

FIG. 17A is a graph plotting exercise capacity (measured as running distance) for mice treated with vehicle or hCTP for five days/week. *, p = 0.03. FIG. 17B is a graph plotting weight as a correlation of running distance, showing that running distance was unrelated to weight and increased significantly with daily hCTP treatments. FIG. 17C is a graph plotting E/e’ ratios for mice, before and after treatment with vehicle or two difference concentrations of hCTP for 3 or 5 days per week. FIG. 17D is a graph plotting ejection fractions for each group of mice before and after treatment with hCTP. FIG. 17E includes a pair of graphs plotting growth rates of control mice or Heart Failure with preserved Ejection Fraction (HFpEF) mice, before and after treatment with hCTP.

FIG. 18A is a series of histograms showing uptake of hCTP by human cardiomyocytes, demonstrating inhibition of hCTP uptake in these cells by pre-treatment with varying concentrations of benzamil, a Na(+)/Ca(2+) exchanger (NCX) inhibitor. FIG. 18B includes a pair of graphs plotting uptake of hCTP (left panel) or CTP (right panel) by human cardiomyocytes treated with benzamil. FIG. 18C is a graph plotting cell viability, showing that inhibition of hCTP uptake was not secondary to changes in cell viability. FIGS. 19A and 19B include graphs plotting uptake of CTP or hCTP, either alone or in combination with the indicated NCX inhibitors by, human cardiomyocytes.

FIG. 20A shows IVIS imaging of hearts from mice that were injected with vehicle only (Control), Benzamil followed by Cy5.5-labeled hCTP (B+hCTP), or Cy5.5- labeled hCTP only (hCTP). FIG. 20B is a graph plotting fluorescence intensity (average radiant efficiency) for the mouse hearts shown in FIG. 20A.

DETAILED DESCRIPTION

This disclosure provides methods and materials related to cardiac cell targeting by cell penetrating peptides that can traverse the cardiomyocyte cell membrane either without a functional cargo or while carrying functional cargo (e.g., a therapeutic agent, a radiolabel, a siRNA, or a miRNA), thus introducing the cargo into the cardiomyocytes. For example, this document provides substantially pure variants of a CTP that has the amino acid sequence set forth in SEQ ID NO: 1 (APWHLSSQYSRT), where the variants have increased cardiomyocyte targeting ability and/or increased stability in vivo as compared to the CTP having the sequence of SEQ ID NO: 1. This document also provides compositions containing the variant CTPs provided herein, as well as methods for using the variant CTPs and compositions to treat mammals having cardiovascular disorders. In some cases, a variant CTP can be used to treat mammals having a cardiovascular disorder. In some cases, a variant CTP conjugated to a therapeutic agent can be used to deliver the agent into a cardiac cell and/or to treat mammals having cardiovascular disorders.

The term “isolated” as used herein with reference to a polypeptide means that the polypeptide (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source (e.g., free of human proteins), (3) is expressed by a cell from a different species, or (4) does not occur in nature. An isolated polypeptide can be, for example, encoded by DNA or RNA, including synthetic DNA or RNA, or some combination thereof.

The term “substantially pure” as used herein with reference to a polypeptide means the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. A substantially pure polypeptide can be any polypeptide that is removed from its natural environment and is at least 60 percent pure. A substantially pure polypeptide can be at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent pure, or about 65 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, or 95 to 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. In some cases, a substantially pure polypeptide can be a chemically synthesized polypeptide.

Any method can be used to obtain a substantially pure polypeptide. For example, polypeptide purification techniques, such as affinity chromatography and HPLC, as well as polypeptide synthesis techniques can be used. In addition, any material can be used as a source to obtain a substantially pure polypeptide. For example, tissue from wild-type or transgenic animals can be used as a source material. In addition, tissue culture cells engineered to over-express a particular polypeptide can be used to obtain substantially pure polypeptide. Further, a polypeptide can be engineered to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, or FLAG™ tag (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini, or in between. Other fusions that can be used include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.

The variant CTPs provided herein can be variants of the 12-amino acid CTP having the sequence set forth in SEQ ID NO: 1 (in which all amino acids are L-amino acids). In some cases, a variant CTP provided herein can contain the entire amino acid sequence set forth in SEQ ID NO: 1, except that the amino acid sequence contains one or more (e.g., two, three, four, five, six, or more than six) amino acid additions, subtractions, or substitutions (e.g., substitutions with other amino acid residues, or substitutions of one or more (e.g., two, three, four, five, six, or more than six) L-amino acid residues with their D-isoforms). For example, a variant CTP can contain the amino acid sequence set forth in SEQ ID NO: 1 but with one amino acid addition, subtraction, or substitution. In some cases, a variant CTP can contain the amino acid sequence set forth in SEQ ID NO: 1 but with two amino acid residue additions, subtractions, or substitutions. In some cases, a variant CTP can contain the amino acid sequence set forth in SEQ ID NO: 1 but with three amino acid residue additions, subtractions, or substitutions. In some cases, a variant CTP can contain the amino acid sequence set forth in SEQ ID NO: 1 but with four amino acid residue additions, subtractions, or substitutions. In some cases, a variant CTP can contain the amino acid sequence set forth in SEQ ID NO: 1 but with five amino acid residue additions, subtractions, or substitutions. In some cases, a variant CTP can contain the amino acid sequence set forth in SEQ ID NO:1 but with six amino acid residue additions, subtractions, or substitutions.

Any amino acid residue set forth in SEQ ID NO: 1 can be subtracted, and any amino acid residue (e.g., any of the 20 conventional amino acid residues, including D- isoforms of these amino acid residues) can be added to or substituted within the sequence set forth in SEQ ID NO: 1. The majority of naturally occurring amino acids are L-amino acids, and naturally occurring polypeptides are largely comprised of L-amino acids. D- amino acids are the enantiomers (or “mirror images”) of L-amino acids. In some cases, a variant CTP provided herein can include one or more D-amino acids substituted for the amino acids within SEQ ID NO: 1. For example, a variant CTP can have the amino acid sequence set forth in SEQ ID NO: 6 (AP(d)WHLSSQYS(d)RT; also referred to herein as “hCTP”), where the amino acid residues at positions 3 and 11 are D-amino acids.

In some cases, a variant CTP provided herein can have three, four, five, or six additions, subtractions, or substitutions relative to SEQ ID NO: 1. For example, a variant CTP can be a fragment or truncated version of the CTP having the sequence set forth in SEQ ID NO: 1, where the fragment or truncated version lacks three, four, five, or six amino acid residues from the N- or C-terminal end of SEQ ID NO: 1. For example, a variant CTP can have the amino acid sequence set forth in SEQ ID NO:3 (SQYSRT; also referred to herein as “CTP-B”), which corresponds to the C-terminal half of SEQ ID NO: 1 and contains only L-amino acids. In some cases, a variant CTP can have the amino acid sequence set forth in SEQ ID NO:7 (SQ(d)YS(d)RT; also referred to herein as “hCTP-B”), which corresponds to SEQ ID NO: 3 but includes D-amino acids at positions 3 and 5. In some cases, a variant CTP provided herein can have the sequence set forth in SEQ LD N0:6, SEQ ID N0:3, or SEQ ID NO:7, but with one or two subtractions, additions, or substitutions with respect to SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO: 7, provided that the variant CTP does not have the amino acid sequence set forth in SEQ ID NO: E

Variant CTPs having one or more amino acid additions, subtractions, or substitutions relative to SEQ ID NO: 1 (or SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7) can be prepared and modified as described herein. In some cases, amino acid substitutions can be made by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. For example, naturally occurring residues can be divided into groups based on side-chain properties: (1) hydrophobic amino acids (methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine); (5) amino acids that influence chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Examples of useful conservative substitutions can include, without limitation, substitution of valine, leucine, or isoleucine for alanine; lysine, glutamine, or asparagine for arginine; glutamine for asparagine; glutamic acid for aspartic acid; serine for cysteine; asparagine for glutamine; aspartic acid for glutamic acid; proline for glycine; arginine, glutamine, lysine, or asparagine for histidine; leucine, valine, methionine, alanine, or phenylalanine for isoleucine; isoleucine, valine, methionine, alanine, or phenylalanine for leucine; arginine, glutamine, or asparagine for lysine; leucine, phenylalanine, or isoleucine for methionine; leucine, valine, isoleucine, or alanine for phenylalanine; glycine for proline; threonine for serine; serine for threonine; tyrosine for tryptophan; phenylalanine, tryptophan, threonine, or serine for tyrosine; and leucine, isoleucine, methionine, phenylalanine, or alanine for valine.

In some embodiments, a variant CTP provided herein can include one or more non-conservative substitutions. Non-conservative substitutions typically entail exchanging a member of one of the classes described above for a member of another class. Such production can be desirable to provide large quantities or alternative embodiments of such compounds. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the peptide variant using, for example, methods disclosed herein.

In some embodiments, a variant CTP provided herein can include an amino acid sequence with at least 83% (e.g., at least 83% or at least 91%) but less than 100% sequence identity to the reference CTP sequence set forth in SEQ ID NO: 1. Percent sequence identity is calculated by determining the number of matched positions in aligned amino acid sequences, dividing the number of matched positions by the total number of aligned amino acids, and multiplying by 100. A matched position refers to a position in which identical amino acids occur at the same position in aligned amino acid sequences. Percent sequence identity also can be determined for any nucleic acid sequence.

In particular, the percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows. First, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained online at fr.com/blast or at ncbi.nlm.nih.gov. Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seql.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. To compare two amino acid sequences, the options of B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence (e g., SEQ ID NO: 1), or by an articulated length (e.g., 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleotide sequence that has 11 matches when aligned with the sequence set forth in SEQ ID NO: 1 is 91.6 percent identical to the sequence set forth in SEQ ID NO:1 (i.e., 11/12 x 100 = 91.6). It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 7.17, 75.18, and 7.19 are rounded up to 7.2. It also is noted that the length value will always be an integer.

Isolated polypeptides can be produced using any suitable methods, including solid phase synthesis, and can be generated using manual techniques or automated techniques (e.g., using an Applied BioSystems (Foster City, CA) Peptide Synthesizer or a Biosearch Inc. (San Rafael, CA) automatic peptide synthesizer). In some cases, isolated polypeptides can be synthesized as described in the Examples herein, using solid phase peptide synthesis with fluorenylmethyloxy-carbonyl (FMOC) chemistry.

In general, the variant CTPs provided herein have increased cardiomyocyte targeting ability, as compared to the CTP having the amino acid sequence set forth in SEQ ID NO: 1. Variant CTPs provided herein (e.g., CTPs having the sequences set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7, or containing one or two subtractions, additions, or substitutions with respect to SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO: 7) can be screened for cardiomyocyte targeting ability using any appropriate assay. For example, the cardiomyocyte targeting ability of a variant CTP or of a variant CTP conjugate provided herein can be evaluated using methods such as fluorescence activated cell sorting (FACS) as described in the Examples herein. As used herein, an “increase” in cardiomyocyte targeting is an increase of at least 5% (e.g., at least 10%, at least 20%, at least 30%, at least 50%, or at least 100%) in labeling of a population of cardiomyocytes, as compared to a reference level of labeling of a corresponding population of cardiomyocytes. A “reference level” can be a control level of labeling of a population of cardiomyocytes by the CTP having the amino acid sequence of SEQ ID NO:1, coupled to the same label as the variant CTP being evaluated.

In some cases, the variant CTPs described herein (e.g., hCTP, CTP-B, and hCTP- B, having the amino acid sequences set forth in SEQ ID NOs:6, 3, and 7, respectively, or variants thereof) can be coupled to a therapeutic or diagnostic agent (e.g., a therapeutic agent for treating a cardiovascular disease, or a detectable label).

For example, a variant CTP provided herein can be coupled to a therapeutic agent for treating a cardiovascular disorder. In some cases, a variant CTP can be coupled to an anti-arrhythmic agent. Examples of anti-arrhythmic agents include, without limitation, sodium channel blockers that can prevent sodium from getting through cell membranes, which can slow electrical impulses in the heart muscle (e.g., disopyramide, flecainide, mexiletine, propafenone, and quinidine), beta blockers that can slow the heart rate, often by blocking hormones such as adrenaline (e.g., acebutolol, atenolol, bisoprolol, metoprolol, nadolol, and propranolol), potassium channel blockers that can prevent potassium from getting through cell membranes, which can slow electrical impulses in heart cells (e.g., amiodarone, bretylium, dofetilide, dronedarone, ibutilide, and sotalol), nondihydropyridine calcium channel blockers that can block calcium channels in the heart muscle, which can decrease heart rate and contractions (e.g., diltiazem and verapamil), as well as adenosine (which can block or slow electrical impulses at the atrioventricular node between the upper and lower chambers of the heart), digoxin (which can reduce the heart rate and increase contractility of the heart), and anticoagulants. Coupling a variant CTP to such a therapeutic agent can facilitate delivery of the agent to cardiomyocytes.

In some cases, a variant CTP provided herein can be coupled to a siRNA, a miRNA, phosphorodiamidate morpholino oligomer (also referred to as a “morpholino”), or a peptide-nucleic acid (PNA). For example, a variant CTP can be coupled to a miRNA such as miRNA106a (which targets CAMKIIdelta; see Lebek et al., Circulation, 148: 1490-1504, 2023) or RBM20 (a regulator of splicing of various cardiac genes). Coupling a variant CTP to such a siRNA, miRNA, morpholino, or PNA can decrease expression of the targeted gene in cardiomyocytes. In some cases, a variant CTP provided herein can be coupled to an anti-arrhythmic agent (e.g., a small molecule drug that acts as an anti-arrhythmic agent, such as amiodarone), a plasmid, DNA origami, RNA origami, a nucleic acid containing a sequence encoding a polypeptide, a therapeutic polypeptide (e g., heme-oxygenase 1 or HIF1 -alpha), extracellular vesicles, liposomes, nanoparticles, or exosomes.

Any appropriate method can be used to conjugate a variant CTP (e.g., hCTP) to a therapeutic agent. For example, a variant CTP (e.g., hCTP) can be conjugated to small molecule at its N-terminus or C-terminus via a disulfide bond, or via an ester linker. In some cases, a variant CTP (e.g., hCTP) can be synthesized directly with a therapeutic peptide of interest at its N-terminus or C-terminus, or via a disulfide bridge or ester linker. In some cases, a variant CTP (e.g., hCTP) can be used to label DNA origami by conjugating it to the “staples” used to make DNA or RNA origami. In some cases, a variant CTP (e.g., hCTP) can be attached to nanoparticles or exosomes via a pegylated linker. In some cases, a variant CTP provided herein (e.g., hCTP) can be coupled to a detectable label. In some cases, the detectable label can be a fluorescent label. For example, a variant CTP provided herein can be coupled to Cyanine 5.5, Sulfo-Cyanine7, Fluorescein, or Rhodamine. In some cases, the detectable label can be a radiolabel. For example, a variant CTP can be labeled with Technetium 99m using HYNIC as a chelator, or with Gallium68 using 1,4,7- triazacyclononane- 1, 4, 7-triacetic acid (NOTA), 1,4,7,10- tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (DOTA), l,4,7-triazacyclononane,l- glutaric acid-4, 7-acetic acid (NODAGA), or any other appropriate chelator. The radiolabeled variant CTP can be used in imaging technologies such as, without limitation, PET or SPECT scanning.

Any appropriate method can be used to couple a variant CTP provided herein to a therapeutic agent or a radiolabel. For example, a method can include steps described in the Examples herein for attaching a radiolabel to CTP, where a bifunctional chelator (e.g., 2-S-(4-isothiocyanatobenzyl)-l, 4, 7-triazacyclononane-l, 4, 7-triacetic acid) is first coupled to the CTP and then the resulting conjugate is labeled with a detectable label (e.g., a radiolabel such as Gallium68, Technetium 99m, or F-18, or a fluorescent label).

In some cases, a siRNA, miRNA, PNA, morpholino, DNA origami, peptidenucleic acid, RNA origami, or another nucleic acid or plasmid DNA can be coupled to a variant CTP provided herein (e g., hCTP) using a method that includes one or more of the following steps. First, the peptide can be synthesized on a resin (e.g., a THR-2- chlorotrityl resin) using FMOC chemistry and ethyl-(2Z)-2-cyano-2- hydroxyiniinoacetateVVA' diisopropylcarbodiimide (Oxyma/DIC) activation. In some cases, FMOC-D-Trp(Boc)-OH and FMOC-D-Arg(Pbf)-OH can be incorporated at amino acid positions 3 and 11, respectively, to generate a peptide having the sequence set forth in SEQ ID NO:6. After the peptide chain is assembled, the free N-terminal amino group of the fully protected hybrid-CTP peptide resin can be manually conjugated with 3- (tritylthio)propionic acid using N,N, A^f'A''-tetramethyl-O-(benzotriazol- l -yl) uroniumtetrafluorob orate/ 1 -hy droxyb enzotri azol e hydrate/ di i sopropy 1 ethyl ami ne/ dimethylformamide (TBTU/HObt/DIEA/DMF). The fully-protected Trt-SH-hCTP-CCh peptide can be cleaved from the solid support (e.g., under mildly acidic conditions), followed by C-terminal activation using DIPCD1/DMF and, in some cases, reaction with Cy5.5-amine. Final cleavage with trifluoroacetic acid (TFA):thioanisole:anisole: ethanedithiol (90:5:2:3) can be followed by precipitation in diethyl ether (EtCh). The resulting crude product can be purified (e.g., by semi -preparative C-5 RP-HPLC on an appropriate chromatography system, such as the Waters Delta Prep 4000 chromatography system using standard acetonitrile/0.1%TFA gradient conditions). Separately, C6 protected siRNA oligomers can be reduced to their free thiol form (e.g., using DL- dithiothreitol (DTT) in 0.1M triethylammonium bicarbonate at pH 8.5) and then reacted with dithio-bis-maleimidoethane (DTME). Purification of the siRNA-DTME intermediate can be followed by reaction with the purified SH-hCTP peptide Purification of the resulting siRNA-DTME-hCTP conjugate can be accomplished using a chromatography system (e.g., with a trimethylamine acetate (TEAA)/acetonitrile gradient), followed by lyophilization and re-lyophilization from nuclease free water if desired. MALDI-Tof analysis of the purified conjugates can be carried out to confirm the expected mass and identity of the final siRNA-DTME-hCTP product.

In some cases, a siRNA can be coupled to a cyclic peptide. In some cases, for example, solid phase peptide synthesis of an N-terminal lysine-modified peptide can be carried out using FMOC chemistry, with stepwise addition of each FMOC-protected amino acid performed on a resin (e.g., a THR-2-chlorotrityl resin) using ethyl-(2Z)-2- Oxyma/DlC activation chemistry. Cleavage of the fully side-chain protected linear peptide from the resin can be accomplished under mildly acidic conditions, and the peptide can be head-to-tail cyclized using 3-(diethoxyphosphoryloxy)-l,2,3-benzotriazin- 4(3H)-one (DEPBT) in dimethylformamide/dichloromethane. The peptide can be deprotected (e.g., using trifluoroacetic acid:triisopropylsilane:H2O and precipitated, and the epsilon amino group of the N-terminal lysine can be thiolated (e.g., with 2- iminothiolane; Traut’s reagent). Separately, C6 protected siRNA oligomers can be reduced to their free thiol form using (e.g., using DL-dithiothreitol in 0.1 M triethylammonium bicarbonate, pH 8.5) and then reacted with dithio-bis- maleimidoethane (DTME). After purification of the siRNA-DTME intermediate, it can be reacted with the purified cyclic peptide (e.g., in 300mM NaOAc/acetonitrile at pH 5.2) to yield the conjugate. The siRNA-DTME-cyclic-peptide conjugate can be purified (e.g., using a chromatography system), lyophilized and then re-lyophilization if desired. MALDI-Tof analysis of the purified conjugates can be carried out to confirm the expected mass and identity of the final siRNA-DTME-cyclic peptide product.

In some cases, a variant CTP described herein (e.g., hCTP, CTP-B, and hCTP-B, having the amino acid sequence set forth in any of SEQ ID NOs:6, 3, and 7, respectively, or variants thereof) can be incorporated into a composition for administration to a mammal (e.g., a mammal having or at risk for having or developing a cardiovascular disease). In some cases, a variant CTP can be admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds such as, for example, liposomes, receptor or cell targeted molecules, or oral, topical, or other formulations for assisting in uptake, distribution and/or absorption.

In some cases, a variant CTP described herein (e.g., hCTP, CTP-B, and hCTP-B, having the amino acid sequence set forth in any of SEQ ID NOs:6, 3, and 7, respectively, or variants thereof) can be coupled to a therapeutic or diagnostic agent (e.g., a therapeutic agent for treating a cardiovascular disease, or a radiolabel) and incorporated into a composition for administration to a mammal (e.g., a mammal having or at risk for having or developing a cardiovascular disease). In some cases, the variant CTP can be admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds such as, for example, liposomes, receptor or cell targeted molecules, or oral, topical, or other formulations for assisting in uptake, distribution and/or absorption.

In some cases, a composition can contain a variant CTP provided herein (e.g., a variant CTP provided herein that is or is not coupled to a therapeutic or diagnostic agent) in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert vehicles for delivering antibodies to a subject. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties, when combined with one or more therapeutic compounds and any other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, without limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methyl cellulose); fdlers (e g., lactose or dextrose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

Pharmaceutical compositions containing molecules described herein can be administered by a number of methods, depending upon whether local or systemic treatment is desired. Administration can be, for example, parenteral (e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous (i.v.) drip); oral; topical (e.g., transdermal, sublingual, ophthalmic, or intranasal); or pulmonary (e.g., by inhalation or insufflation of powders or aerosols), or can occur by a combination of such methods. Administration can be rapid (e.g., by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow-release formulations).

In some cases, pharmaceutical compositions can include, without limitation, solutions, emulsions, aqueous suspensions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, for example, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Emulsion formulations can be particularly useful for oral delivery of therapeutic compositions due to their ease of formulation and efficacy of solubilization, absorption, and bioavailability. Liposomes can be particularly useful due to their specificity and the duration of action they offer from the standpoint of drug delivery.

Compositions provided herein can contain any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to a subject, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof for the relevant compound (e.g., variant CTP coupled to a therapeutic agent or a radiolabel). Accordingly, for example, this document provides pharmaceutically acceptable salts of the variant CTP conjugates, prodrugs and pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. A prodrug is a therapeutic agent that is prepared in an inactive form and is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the peptides useful in methods provided herein (i.e., salts that retain the desired biological activity of the parent variant CTP conjugate without imparting undesired toxicological effects). Examples of pharmaceutically acceptable salts include, but are not limited to, salts formed with cations (e.g., sodium, potassium, calcium, or poly amines such as spermine); acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or nitric acid); salts formed with organic acids (e.g., acetic acid, citric acid, oxalic acid, palmitic acid, or fumaric acid); and salts formed with elemental anions (e.g., bromine, iodine, or chlorine).

Compositions additionally can contain other adjunct components conventionally found in pharmaceutical compositions. Thus, the compositions also can include compatible, pharmaceutically active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or additional materials useful in physically formulating various dosage forms of the compositions, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents, and stabilizers. Furthermore, the composition can be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings, penetration enhancers, and aromatic substances. When added, however, such materials should not unduly interfere with the biological activities of the other components within the compositions.

In some cases, a variant CTP provided herein (e.g., a variant CTP provided herein that is or is not coupled to a therapeutic or diagnostic agent) can be formulated as a sustained release dosage form. For example, a variant CTP can be formulated into a controlled release formulation. In some cases, coatings, envelopes, or protective matrices can be formulated to contain one or more of the polypeptides provided herein. Such coatings, envelopes, and protective matrices can be used to coat indwelling devices such as stents, catheters, and peritoneal dialysis tubing. In some cases, a polypeptide provided herein can incorporated into a polymeric substance, liposomes, microemulsions, microparticles, nanoparticles, or waxes.

Pharmaceutical formulations as disclosed herein, which can be presented conveniently in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients (i.e., the antibodies) with the desired pharmaceutical carrier(s). Typically, the formulations can be prepared by uniformly and intimately bringing the active ingredients into association with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Formulations can be sterilized if desired, provided that the method of sterilization does not interfere with the effectiveness of the molecules(s) contained in the formulation.

Pharmaceutical compositions also can include auxiliary agents or excipients, such as glidants, dissolution agents, surfactants, diluents, binders, disintegrants, and/or lubricants. For example, dissolution agents can increase the dissolution rate of the polypeptide from the dosage formulation, and can include, for example, organic acids and/or salts of organic acids (e.g., sodium citrate with citric acid). Other examples of excipients useful in such formulations include synthetic, semi-synthetic, modified, and natural polymers (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol, starches, gum acacia, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, PEG, cyclodextrin, alkoxy- modified cyclodextrins, hydroxyethylcellulose, hydroxypropylcellulose, microcrystalline cellulose, albumin, dextran, maltitol, xylitol, kaolin, and methyl cellulose). The polypeptide also can be mixed with a lubricating agent (e.g., talc, magnesium stearate, stearic acid, or mineral oil, calcium stearate, hydrogenated vegetable oils, sodium benzoate, sodium chloride, leucine carbowax, magnesium lauryl sulfate, or glyceryl monostearate), a wetting agent, an emulsifying and suspending agent, or a preserving agent (e.g., methyl or propyl hydroxybenzoate).

Other agents that can be added to a pharmaceutical composition can alter the pH of the microenvironment on dissolution and establishment of a therapeutically effective plasma concentration profile of the polypeptide. Such agents include salts of inorganic acids and magnesium hydroxide. Other agents that can be used include surfactants and other solubilizing materials.

Useful diluents include, for example, pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, sucrose, fructose, glucose dextrose, or other sugars, dibasic calcium phosphate, calcium sulfate, cellulose, ethylcellulose, cellulose derivatives, kaolin, mannitol, lactitol, maltitol, xylitol, sorbitol, or other sugar alcohols, dry starch, saccharides, dextrin, maltodextrin or other polysaccharides, inositol, or combinations thereof. Water-soluble diluents can be particularly useful.

Glidants can be used to improve the flow and compressibility of composition ingredients during processing. Useful glidants include, for example, colloidal silicon dioxide (also referred to as colloidal silica, fumed silica, light anhydrous silicic acid, silicic anhydride, and silicon dioxide fumed).

Surfactants that are suitable for use in the pharmaceutical compositions provided herein include, without limitation, sodium lauryl sulphate, polyethylene stearates, polyethylene sorbitan fatty acid esters, polyoxyethylene castor oil derivatives, polyoxyethylene alkyl ethers, benzyl benzoate, cetrimide, cetyl alcohol, docusate sodium,, glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate, lecithin, medium chain triglycerides, monoethanolamine, oleic acid, poloxarners, polyvinyl alcohol and sorbitan fatty acid esters.

Suitable disintegrants include, for example, starches, sodium starch glycolate, crospovidone, croscarmellose, microcrystalline cellulose, low substituted hydroxypropyl cellulose, pectins, potassium methacrylate- divinylbenzene copolymer, polyvinyl alcohol), thylamide, sodium bicarbonate, sodium carbonate, starch derivatives, dextrin, beta cyclodextrin, dextrin derivatives, magnesium oxide, clays, bentonite, and combinations thereof.

In some embodiments, a pharmaceutical composition for controlled release delivery of a variant CTP in a subject can include (a) a complex of the polypeptide (where the polypeptide has at least one basic functional group) and a polyanion derived from hexahydroxycyclohexane (where the polyanion has at least two negatively charged functional groups); and (b) a pharmaceutically acceptable carrier containing a biodegradable, water-insoluble polymer. Such compositions are described in, for example, PCT Publication No. WO 2006/017852, and can be prepared in the form of solutions, suspensions, dispersions, emulsions, drops, aerosols, creams, semisolids, pastes, capsules, tablets, solid implants, or microparticles, for example. The term “controlled release delivery,” as used herein, refers to continual delivery of a pharmaceutical agent in vivo over a period of time (e.g., several days to weeks or months) following administration. Sustained controlled release delivery of a variant CTP conjugate can be demonstrated by, for example, continued therapeutic effects of the polypeptide over time (e.g., continued reductions in symptoms over time). Sustained delivery of the polypeptide also can be demonstrated by detecting the presence of the polypeptide in vivo over time. The compositions can provide a low initial burst delivery, followed by stable, controlled release of the polypeptide in vivo for prolonged periods of time (e.g., from days to months).

Also provided herein are articles of manufacture containing one or more variant CTPs, variant CTP conjugates, or pharmaceutical compositions as described herein in a bottle, vial, syringe, or other vessel. The article of manufacture also can include a transfer set and/or a water-based vehicle in a separate vessel, or the polypeptide/composition and vehicle can be separated in a double chamber syringe.

In some cases, an isolated variant CTP provided herein (e.g., CTP variant having the amino acid sequence set froth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7) can be used to treat cardiovascular disease in a mammal (e.g., a human, a non-human primate, a mouse, a rat, a sheep, a dog, a pig, a horse, or a cow). For example, a variant CTP provided herein can be used to treat cardiovascular disorders such as, without limitation, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, pericarditis, recurrent pericarditis, Friedreich’s ataxia associated cardiac hypertrophy, and myocarditis (including, without limitation, giant cell myocarditis, eosinophilic myocarditis, viral myocarditis, vaccine-associated myocarditis, and/or lymphocytic myocarditis). In some cases, a variant CTP provided herein can reduce one or more symptoms of cardiovascular disease (e.g., heart failure), including clinical parameters such as edema, shortness of breath, and fatigue, as well as cardiac unloading (i.e., reduced pressure in the heart), increased glomerular filtration rate (GFR), decreased PRA, decreased levels of angiotensin II, decreased proliferation of cardiac fibroblasts, decreased left ventricular (LV) hypertrophy, decreased LV mass (indicative of reduced fibrosis and hypertrophy), decreased PWCP (an indirect measure of left atrial pressure), decreased right atrial pressure, decreased mean arterial pressure, decreased levels of aldosterone (indicative of an anti-fibrotic effect), decreased ventricular fibrosis, increased ejection fraction, and decreased LV end systolic diameter. To determine whether a variant CTP is capable of inhibiting or reducing a symptom of heart failure, one or more of these parameters can be evaluated (e.g., before and after treatment with the variant CTP), using any appropriate method.

In some cases, an isolated variant CTP conjugate provided herein (e.g., a conjugate containing a variant CTP coupled to a therapeutic agent) can be used to treat cardiovascular disease in a mammal (e.g., a human, a non-human primate, a mouse, a rat, a sheep, a dog, a pig, a horse, or a cow). For example, a variant CTP conjugate provided herein can be used to treat cardiovascular disorders such as, without limitation, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, pericarditis, recurrent pericarditis, Friedreich’s ataxia associated cardiac hypertrophy, and myocarditis (including, without limitation, giant cell myocarditis, eosinophilic myocarditis, viral myocarditis, vaccine-associated myocarditis, and/or lymphocytic myocarditis). In some cases, a variant CTP conjugate provided herein can reduce one or more symptoms of cardiovascular disease (e.g., heart failure), including clinical parameters such as edema, shortness of breath, and fatigue, as well as cardiac unloading (i.e., reduced pressure in the heart), increased glomerular filtration rate (GFR), decreased PRA, decreased levels of angiotensin II, decreased proliferation of cardiac fibroblasts, decreased left ventricular (LV) hypertrophy, decreased LV mass (indicative of reduced fibrosis and hypertrophy), decreased PWCP (an indirect measure of left atrial pressure), decreased right atrial pressure, decreased mean arterial pressure, decreased levels of aldosterone (indicative of an anti-fibrotic effect), decreased ventricular fibrosis, increased ejection fraction, and decreased LV end systolic diameter. To determine whether a variant CTP conjugate is capable of inhibiting or reducing a symptom of heart failure, one or more of these parameters can be evaluated (e.g., before and after treatment with the variant CTP conjugate), using any appropriate method.

Any suitable method can be used to assess the presence or extent of cardiovascular disease in a mammal, including, without limitation, general clinical examination to evaluate blood pressure, heart rate, heart rhythm, arterial oxygen, and hemoglobin levels; echocardiography to measure ejection fraction, LV and left atrium (LA) diameter, LV wall motion, LV filling pressure, and diastolic function by pulse and tissue Doppler; use of a Swan-Ganz catheter to measure cardiac output, pulmonary wedge capillary pressure, pulmonary arterial pressure, right ventricle pressure, right atrium pressure, and systemic and pulmonary vascular resistance; assessment of kidney function by determination of glomerular filtration rate, serum creatinine, and blood urea nitrogen; and measurement of biomarkers such as BNP, amino-terminal proBNP (NT- proBNP), troponin-T, troponin-I, C-reactive protein (CRP), and creatine-kinase, serum cystatin-C, albuminuria, neutrophil gelatinize associated lopocalin (NGAL), N-acetyL beta-D-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1), angiotensin-II, renin, aldosterone, and inflammatory cytokines (e.g., interleukin (IL)-6, IL- 18, etc.).

Methods for selectively labeling cardiomyocytes in vitro or in a mammal also are provided herein. The methods can include administering a variant CTP provided herein to cells in culture or to a mammal (e.g., intravenously), where the variant CTP is coupled to a detectable label (e.g., a fluorescent label or a radio label such as Technetium 99m or Gallium68). Any appropriate method can be used to detect the presence and or measure the level of the label in the cardiomyocytes in vitro or in a mammal. For example, FACS can be used to assess cardiomyocyte labeling of cells in culture, and positron emission tomography (PET) imaging can be used to assess cardiomyocyte labeling in a mammal.

This document also provides methods for treating cardiovascular disorders (e.g., heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, pericarditis, recurrent pericarditis, Friedreich’s ataxia associated cardiac hypertrophy, and myocarditis including, without limitation, giant cell myocarditis, eosinophilic myocarditis, viral myocarditis, vaccine-associated myocarditis, and/or lymphocytic myocarditis) in a mammal. In some cases, the methods can include administering a variant CTP provided herein to a mammal having a cardiovascular disorder. In some cases, the methods can include administering a variant CTP provided herein conjugated to a therapeutic agent for the cardiovascular disorder. The terms “treat” and “treatment” as used herein refer to prescribing, administering, or providing a medication to beneficially affect or alleviate one or more symptoms associated with a disease or disorder, or one or more underlying causes of a disease or disorder.

Before administering a variant CTP or a variant CTP conjugate provided herein to a mammal, the mammal can be assessed to determine whether or not the mammal has a need for treatment of a cardiovascular disorder. After identifying a mammal as having a need for such treatment, the mammal can be treated with a variant CTP, a variant CTP conjugate, or a composition provided herein. For example, a composition containing a variant CTP coupled to a therapeutic agent can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to reduce one or more symptoms of a cardiovascular disorder, or to prevent or delay worsening of one or more such symptoms). In some cases, a composition containing a variant CTP can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to reduce one or more symptoms of a cardiovascular disorder, or to prevent or delay worsening of one or more such symptoms). Dosages typically are dependent on the responsiveness of the subject to the variant CTP or the conjugate, with the course of treatment lasting from several days to several months, or until a suitable response is achieved. Persons of ordinary skill in the art can readily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of an antibody, and generally can be estimated based on the EC50 found to be effective in in vitro and/or in vivo animal models. Compositions containing the variant CTPs or variant CTP conjugates provided herein may be given once or more daily, weekly, monthly, or even less often, or can be administered continuously for a period of time (e.g., hours, days, or weeks). In some cases, composition containing a variant CTP or a variant CTP conjugate provided herein (e.g., a variant CTP having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7 coupled to a therapeutic agent for treating cardiovascular disease) can be administered at a dose of at least about 0.01 ng conjugate/kg to about 100 mg conjugate/kg of body mass (e.g., about 10 ng conjugate/kg to about 50 mg conjugate/kg, about 20 ng conjugate/kg to about 10 mg conjugate/kg, about 0.1 ng conjugate/kg to about 20 ng conjugate/kg, about 3 ng conjugate/kg to about 10 ng conjugate/kg, or about 50 ng conjugate/kg to about 100 pg conjugate/kg) of body mass, although other dosages also may provide beneficial results. A composition can be administered at a dose of, for example, about 0.1 ng conjugate/kg/minute to about 500 ng conjugate/kg/minute (e.g., about 0.5 ng conjugate/kg/minute, about 1 ng conjugate/kg/minute, about 2 ng conjugate/kg/minute, about 3 ng conjugate/kg/minute, about 5 ng conjugate/kg/minute, about 7.5 ng conjugate/kg/minute, about 10 ng conjugate/kg/minute, about 12.5 ng conjugate/kg/minute, about 15 ng conjugate/kg/minute, about 20 ng conjugate/kg/minute, about 25 ng conjugate/kg/minute, about 30 ng conjugate/kg/minute, about 50 ng conjugate/kg/minute, about 100 ng conjugate/kg/minute, or about 300 ng conjugate/kg/minute). In some cases, composition containing a variant CTP provided herein (e.g., a variant CTP having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7) can be administered at a dose of at least about 0.01 ng variant CTP/kg to about 100 mg variant CTP/kg of body mass (e.g., about 10 ng variant CTP/kg to about 50 mg variant CTP/kg, about 20 ng variant CTP/kg to about 10 mg variant CTP/kg, about 0.1 ng variant CTP/kg to about 20 ng variant CTP/kg, about 3 ng variant CTP/kg to about 10 ng variant CTP/kg, or about 50 ng variant CTP/kg to about 100 pg variant CTP/kg) of body mass, although other dosages also may provide beneficial results. A composition can be administered at a dose of, for example, about 0.1 ng variant CTP/kg/minute to about 500 ng variant CTP/kg/minute (e.g., about 0.5 ng variant CTP/kg/minute, about 1 ng variant CTP/kg/minute, about 2 ng variant CTP/kg/minute, about 3 ng variant CTP/kg/minute, about 5 ng variant CTP/kg/minute, about 7.5 ng variant CTP/kg/minute, about 10 ng variant CTP/kg/minute, about 12.5 ng variant CTP/kg/minute, about 15 ng variant CTP/kg/minute, about 20 ng variant CTP/kg/minute, about 25 ng variant CTP/kg/minute, about 30 ng variant CTP/kg/minute, about 50 ng variant CTP/kg/minute, about 100 ng variant CTP/kg/minute, or about 300 ng variant CTP/kg/minute).

The methods provided herein can include administering to a mammal an effective amount of a variant CTP or a variant CTP conjugate provided herein (e.g., a variant CTP having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO: 7, or a variant CTP having the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO:3, or SEQ ID NO:7 coupled to a therapeutic agent for treating cardiovascular disease), or an effective amount of a composition containing a variant CTP or a variant CTP conjugate provided herein. As used herein, the term “effective amount” is an amount of a variant CTP or a variant CTP conjugate that is sufficient to reduce the occurrence of a symptom of cardiovascular disease by at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%). In some cases, for example, an “effective amount” of a variant CTP or a variant CTP conjugate provided herein is an amount that reduces a symptom of cardiovascular disease in a treated mammal by at least 10% as compared to the level of the symptom in the mammal prior to administration of the variant CTP or the conjugate, or as compared to the level of the symptom in a control, untreated mammal. The presence or extent of such symptoms can be evaluated using any appropriate method.

In some embodiments, the amount and frequency of administering to a mammal a variant CTP or a variant CTP conjugate provided herein (e.g., a variant CTP having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7, or a variant CTP having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7 coupled to a therapeutic agent for treating cardiovascular disease), or a pharmaceutical composition containing the variant CTP or the conjugate, can be titrated in order to, for example, identify a dosage that is most effective to treat a cardiovascular disease in the mammal, while having the least amount of adverse effects. For example, an effective amount of a composition can be any amount that reduces arrhythmia within a mammal without having significant toxicity in the mammal. If a particular mammal fails to respond to a particular amount, then the amount can be increased by, for example, twofold, three-fold, five-fold, or ten-fold. After receiving this higher concentration, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments in the dosage can be made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment.

In some cases, an effective frequency of administration of a variant CTP or a variant CTP conjugate provided herein (e.g., a variant CTP having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7, or a variant CTP having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7 coupled to a therapeutic agent for treating cardiovascular disease), or a pharmaceutical composition containing the variant CTP or the conjugate, can be a frequency that reduces one or more symptoms associated with a cardiovascular disorder (e.g., heart failure) in the mammal, without producing significant toxicity to the mammal. In some cases, an effective frequency of administration of a variant CTP or a variant CTP conjugate provided herein, or a pharmaceutical composition containing the variant CTP or the conjugate can be a frequency that reduces one or more symptoms associated with a cardiovascular disorder (e.g., heart failure) in a mammal as compared to a control mammal having a comparable disorder and not treated with the variant CTP, the variant CTP conjugate, or the composition. For example, an effective frequency of administration of a variant CTP or a variant CTP conjugate provided herein, or a pharmaceutical composition containing the variant CTP or the conjugate, can be from about four times a day to about once every other month, or from about once a day to about once a month, or from about once every other day to about once a week. The frequency of administration of a variant CTP or a variant CTP conjugate provided herein or a pharmaceutical composition containing the variant CTP or the conjugate can remain constant or can be variable during the duration of treatment. Various factors can influence the actual effective frequency used for a particular application. For example, the effective amount, the severity of the disorder when treating a mammal, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments, and the judgment of the treating physician may require an increase or decrease in the actual effective frequency of administration.

In some cases, an effective duration of administration of a variant CTP or a variant CTP conjugate provided herein (e.g., a variant CTP having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7, or a variant CTP having the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:3, or SEQ ID NO:7 coupled to a therapeutic agent for treating cardiovascular disease) or a pharmaceutical composition containing the variant CTP or the conjugate can be a duration that reduces one or more symptoms associated with a cardiovascular disorder (e g., heart failure) in a mammal, without producing significant toxicity to the mammal. In some cases, an effective duration of administration of a variant CTP or a variant CTP conjugate provided herein or a pharmaceutical composition containing the variant CTP or the conjugate can be a duration that reduces one or more symptoms associated with a cardiovascular disorder (e.g., heart failure) in a mammal as compared to a control mammal having a comparable disorder and not treated with the variant CTP, the conjugate, or the composition. For example, an effective duration of administration of a variant CTP or a variant CTP conjugate provided herein, or a pharmaceutical composition containing the variant CTP or the conjugate, can range from one to several days, to several weeks, months, or years. In general, the effective duration can range in duration from several days to several months. For example, an effective duration can range from about one to two weeks to about 36 months. Prophylactic treatments can be typically longer in duration and may last throughout an individual mammal’s lifetime. Multiple factors can influence the actual effective duration used for a particular application. For example, the severity of the disorder, the effective frequency, the effective amount, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments, and the judgment of the treating physician may require an increase or decrease in the actual effective duration of administration of a compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound or the pharmaceutically acceptable salt as described herein.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1 - Identification of variant CTPs with increased cardiac targeting ability

As described elsewhere (Zahid et al., PLoS One 5 :e 12252, 2010; and Zahid, M. & Robbins, Methods Mol Biol 683:277-289, 2011), combinatorial in vitro and in vivo M13 phage display methodology was used to identify a synthetic 12-amino acid, mildly basic, non-naturally occurring peptide having the amino acid sequence APWHLSSQYSRT (SEQ ID NO: 1). The peptide was capable of specifically targeting cardiomyocytes in vivo following intravenous injection into mice, and therefore was referred to as Cardiac Targeting Peptide, or CTP. Biodistribution studies showed that CTP was able to transduce heart tissue in as little as 15 minutes, with complete disappearance of fluorescence by 6 hours (Zahid et al., Biomolecules 8, 2018). The targeting was not species limited, as incubating explanted heart tissue from patients undergoing heart transplants showed robust uptake of CTP by the normal cardiomyocytes with sparing of fibroblasts present in scar tissue (Zahid et al., Adv Genet 69:83-95, 2010). Further, human fibroblast-derived induced pluripotent stem cells differentiated into beating cardiomyocytes were also targeted by CTP when the cells were incubated with a fluorescently dual -labeled CTP and then subjected to live cell confocal imaging, which showed uptake occurring within 5-10 minutes and accumulation of the Rhodamine label into the cardiomyocytes (Zahid 2018, supra). CTP was successfully labelled with technetium-99m (a radioisotope commonly used in clinical SPECT imaging) using hydrazino-ni cotinamide as the chelator, and the labelled CTP was used to image normal mouse heart without uptake by other organs over time (Zahid 2018, supra).

To obtain insight into the mechanism of transduction of CTP, smaller 6-amino acid N-terminal (CTP-A; APWHLS; SEQ ID NO:2) and C-terminal (CTP-B: SQYSRT; SEQ ID NO:3) versions of the full-length peptide. An alanine scan also was performed in which each amino acid in CTP was sequentially replaced with alanine. The transduction abilities of these peptides were tested in a rat cardiomyoblast cell line (H9C2 cells; ATCC CRL1446) using fluorescence activated cell sorting (FACs). Of all the tested peptides, CTP-B, CTP-P2A (AAWHLSSQYSRT, with alanine substituted for the proline at position 2; SEQ ID NO:4) and H4A (APWALSSQYSRT, with alanine substituted for the histidine at position 4; SEQ ID NO:5) had substantial and statistically significant enhanced transduction abilities as compared to CTP in vitro (FIG. 1). hCTP and CTP-B also had higher uptake by human cardiomyocytes as compared to the parent CTP (FIG. 2). CTP-B and P2A showed higher, though similar, transduction capabilities in vivo as well (FIG. 3). Interestingly, S7A and R11 A, though with highest uptake in H9C2 cells, targeted the lungs instead of the heart in vivo in mice.

Studies were conducted to determine whether serum stability and transduction abilities of the CTPs would be increased by replacing select amino acids with their D- isoforms, particularly at positions that are most frequently the site of peptidase/proteinase breakdown. Using a peptide cutter website (expassy.or ) that tested for degradation sites for various proteinase and peptidase enzymes, positions 3 and 11 were identified as likely being the most frequent sites of enzymatic lysis (TABLE 1). A “hybrid CTP” (hCTP) in which amino acid residues 3 and 11 of CTP were altered to contain D-amino acids (AP(d)WHLSSQYS(d)RT; SEQ ID N0:6) while the other amino acids remained L- amino acids, and a “hybrid CTP-B” (hCTP-B) in which positions 3 and 5 of CTP-B were altered to contain D-amino acids (SQ(d)YS(d)RT; SEQ ID N0:7) while the other amino acids remained L-amino acids were synthesized. Serum stability studies of CTP versus hCTP using LC-MS/MS showed that replacing residues 3 and 11 with their D- enantiomers significantly increased serum stability of the peptide (FIGS. 4A and 4B).

Transduction efficiencies of the linear peptides (CTP, CTP-B, P2A, and H4A) and cyclic peptides (CTP, P2A, H4A), as well as hybrid CTP (hCTP) and hybrid CTP-B (hCTP-B) were tested in a human cardiomyocyte cell line in a head-to-head fashion in quadruplicate using FACs (FIG. 2). CTP-B was not able to be cyclized due to stearic hinderance posed by its short length. Live-dead co-staining and gating on the live cell population showed >90% cell viability with all peptide incubations. CTP-B and hCTP had significantly enhanced uptake in cells compared to CTP. Hybridization of CTP-B with D-amino acids at positions 3 and 5 did not enhance uptake. These experiments identified CTP-B and hCTP as peptides with significantly improved transduction abilities compared to CTP, and much greater than those of P2A and H4A (FIG. 5). Additionally, cyclization of peptides did not improve their transfection efficiencies (FIG. 2).

TABLE 1 : Sites in CTP vulnerable to enzymatic degradation

The parent CTP was then synthesized using solid state synthesis with NOTA (a chelator) conjugated to its N-terminus, and the CTP was labeled with ⁶⁸GA. The labeling of CTP -NOTA with ⁶⁸GA was optimized, with a yield of 98% radiolabeling efficiency at room temperature in ten minutes, and with >97% radiochemical purity, without the need of a heating block. The conjugate was injected into wild-type adult CD1 mice (n=6) with dynamic PET imaging performed for first 30 minutes, followed by imaging at 60 and 120 minutes. The results indicated that [⁶⁸Ga]Ga-NOTA-CTP was taken up from the vascular compartment and by the heart tissue within 5 minutes of injection as evidenced by the “donut” appearance of the heart on transverse and coronal images (FIG. 6). Example 2 - Identification of a CTP candidate as a cardiac PET imaging agent CTP and hCTP were labeled at their N-termini with a fluorophore (Cy5.5) and injected into wild-type mice. The mice were sacrificed at various time-points, and heart, liver, lung, kidneys, and brain fluorescence was measured using ex vivo TVIS imaging, followed by confocal microscopy to assess which cellular compartment exhibits uptake. A scrambled, random peptide served as a negative control.

Peptides were synthesized using solid phase peptide synthesis on a Liberty CEM microwave synthesizer using fluorenylmethyloxycarbonyl (FMOC) chemistry. Stepwise addition of each FMOC protected amino acid was performed on THR-2-Chlorotrityl resin using ethyl-(2Z)-2-cyano-2-hydroxyiminoacetate/N,N-diisopropylcarbodiimide activation chemistry. The N-termini of the peptides were fluorescently labeled with Cyanine 5.5 (Cy5.5), and the labeled peptides were purified using preparative C-18 RP-HPLC on a Waters Delta Prep 4000 chromatography system using standard Acetonitrile/0.1%TFA gradient conditions followed by lyophilization.

To develop and validate the various PET probes based on CTP, a bifunctional chelator 2-S-(4-isothiocyanatobenzyl)-l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA-Bn-SCN) was conjugated to CTP, with subsequent radiolabeling with a PET isotope ⁶⁸GA (FIG. 7). Conjugating the peptides with NOTA-chelator and radiolabeling them with ⁶⁸GA ensured that structural variability lay only in the CTPs, such that any variation in uptake profile was the result of variation in the chemical structure of the peptides only. Further, ⁶⁸GA was selected due to its superior imaging characteristics (E_Pmax 1.8 MeV, Ep_mean 0.89 MeV, p⁺ 89%,), half-life (ti/₂, 67.7 min), and wide availability from ⁶⁸Ge/⁶⁸GA generators using either liquid or solid targets. Similar conjugation and radiolabeling condition were applied to each PET probe. For quality control analysis, both rad-TLC and HPLC were utilized to measure radiolabeling efficiency, radiochemical purity, and chemical purity. The stability of the PET probes in their final formulation also was determined in fetal bovine serum as well as in pooled human serum over time. Developed quality control methods were employed after every production of radiotracer and before any studies were undertaken. For in vivo studies, adult wild-type CD1 mice were injected intravenously with radiolabeled CTP or hCTP at a dose of 10 mg/kg. For each peptide, 3 mice were injected intravenously with each peptide, and the peptides were allowed to circulate for 5, 15, 30, 60, and 120 minutes. The mice were then euthanized, and heart, lungs, liver, kidneys, spleen, brain, small bowel, and large bowel were dissected out and weighed, and ex vivo IVIS imaging of the organs performed. The average radiant efficiencies of each organ were calculated by drawing ROIs around the organs, as well as total radiant efficiencies normalized to organ weights. The average radiant efficiencies in the heart of each mouse are plotted in the graph shown in FIG. 8, and the mean radiation efficiencies are plotted in the graph shown in FIG. 9. Confocal micrographs as 2D and 3D with heart uptake quantified by fluorescence are shown in FIGS. 10A and 11A, and demonstrate that hCTP had 3- to 4-fold greater uptake efficiency than the parent CTP (see, FIGS. 10B and 11B)

Example 3 - Micro PET imaging and in vivo biodistribution of CTPs in normal and infarcted murine heart

CTP, hCTP, CTP-B, hCTP-B, and a random peptide (RAN; TABLE 2) are labeled with ⁶⁸Ga and used for head-to-head imaging of normal and infarcted murine hearts using dual-modality dynamic and time restricted PET/CT studies. Adult wild-type mice with myocardial infarction undergo PET/CT imaging performed 4-6 weeks post infarction (allowing conversion of infarcted myocardium to scar tissue). Competition studies with “cold” conjugates are carried out at 10- and 20-times excess peptide. Comparison to N-13 ammonia (current standard), and F-18 Flupiridaz are carried out.

TABLE 2: Peptide amino acid sequences

In vivo PET studies: In vivo mouse studies are performed with the NOTA conjugated versions of the four peptides labeled with ⁶⁸GA. Adult wild-type CD1 mice (n=6; 3 male, 3 female mice for each peptide) are injected intravenously via tail vein injections with the [⁶⁸Ga]Ga-NOTA-CTPs, immediately followed by a 30 minute dynamic PET scan and then static PET imaging at 60 minutes and 120 minutes. For PET imaging, anesthetized animals are imaged using a small animal micro-PET/X-ray system, Genesys4 (Sofie BioSciences, Dulles, VA, USA). PET images are reconstructed using imaging software of the small animal micro-PET/X-ray system, Genesys4 (Sofie BioSciences, Dulles, VA, USA). The reconstructed images are visualized, analyzed, and scaled to SUV using image analysis software (a Medical Image Data Examiner or “AMIDE;” Pandey et al., Am J Nucl Med Mol Imaging 4:303-310, 2014). The maximum intensity projection (MIP) PET images are generated in the coronal and sagittal planes using image analysis software, MIM 7.2.7 software (MIM Software Inc.; Cleveland, OH), showing uptake of radiolabeled [⁶⁸Ga]Ga-NOTA-CTP or [⁶⁸Ga]Ga-NOTA-hCTP in different organs. After imaging at 120 minutes post-injection, animals are euthanized via thoracotomy under anesthesia (2% isoflurane). Organs and tissues are collected, and radioactivity is counted using a gamma counter for ex vivo biodistribution to calculate standardized uptake value (SUV) in different organs. SUV is calculated using the following formula:

SUV = radioactivity concentration in tissue (pCi/g)/injected dose(pCi)/body weight(g).

Competition Inhibition Study: To evaluate the selectivity and specificity of a selected peptide, a blocking study is conducted in which lOx and 20x excess of a nonradioactive version of the peptide is injected along with the radiotracer ([⁶⁸Ga]Ga- NOTA-peptide) via tail vein. The cold mass of the lOx and 20x excess of nonradioactive conjugate is determined based on its molar specific activity. Three different groups of animals are treated: (i) control group treated with just radiotracer, (ii) test group treated with lOx of nonradioactive version of the conjugate along with the radiotracer, and (iii) test group treated with 20x of the nonradioactive conjugate along with the radiotracer. The obtained results are analyzed by comparing SUV of all three groups in heart and other organs. A significant drop in uptake of radiotracer in heart is observed on coinjection with lOx and 20x of nonradioactive versions of the conjugates containing CTP, hCTP, and CTP-B, but not RAN.

In vivo infarct study: A PET probe selected from the in vivo imaging studies is evaluated with whole body PET/CT imaging for biodistribution in normal mice, and uptake patterns are assessed in infarcted mouse hearts. An equal number of male and female infarcted, adult mice are obtained from Inotiv Co. (West Lafayette, IN). Mice undergo infarction by a surgeon and are allowed to recover and kept under observation until they no longer require medications for post-operative pain management (a minimum of 72 hours). After a least 4-weeks post infarct (to allow conversion of myocytes to established fibroblast scar tissue), mice undergo imaging with the selected peptide, as described above (“Zn vivo PET studies’ f Standard statistical analysis is performed to compare the results between control and infarcted heart groups at various timepoints. Student’ s t-test showing a two-tailed p-value of<0.05 is considered a statistically significant difference.

Comparison to Current Tracers: Head-to-head comparisons of the selected [⁶⁸Ga]Ga-NOTA-peptide with other established radiotracers in the field are conducted to compare the clinical value of the selected [⁶⁸Ga]Ga-NOTA-peptide. For example, [⁶⁸Ga]Ga-NOTA-hCTP uptake into the heart is compared with uptake of N-13 ammonia (and, if available, with uptake of F-18 Flurpiridaz) in a 30-minute dynamic PET scan followed by biodistribution. Since the half-life of N-13 ammonia is only 10 minutes, longer studies are not feasible.

Example 4 - Automated cGMP synthesis and toxicity studies of the selected [⁶⁸Ga]Ga-NQTA-peptide

The selected PET probe from Example 3 is synthesized under cGMP environment using an automated radiosynthesis module. Toxicity studies by a certified research organization under GLP conditions are performed. Briefly, Sprague-Dawley rats are injected once intravenously with lOOx the proposed human dose based on body surface area, and well-being is monitored for up to 14 days with blood chemistries, necropsy, and histopathology on days 2 and 14. cGMP radiosynthesis: To prepare for clinical translation, the selected PET probe from Example 3 is synthesized under clinical good manufacturing practice (cGMP) using an automated radiosynthesis module. For automated synthesis, a Trasis all-in-one module is programed and tested for automated radiosynthesis. For cGMP synthesis, a batch record, method of standard operating procedures for reagents, chemicals, radiosynthesis, purification, quality control, and analysis are prepared. The final quality of the drug is tested by performing standard cGMP analyses, including evaluation of endotoxin, halflife, pH, filter integrity, sterility, radiochemical purity, radionuclide purity, chemical purity, and molar activity. Three validation runs and stability analyses are performed at the highest radioactivity concentration to ensure stability of the drug over time.

Toxicological studies: As described above, a selected cardiac targeting peptide is developed into a cardiac imaging PET probe. The [⁶⁸Ga]Ga- NOTA-peptide is synthesized under cGMP conditions, and toxicity studies are performed. The final imaging tracer undergoes limited toxicity studies following good laboratory process (GLP) in a single vertebrate, mammalian species for an imaging, microdose, exploratory IND application for a Phase I human limited imaging study, as per the FDA position paper (Guidance for Industry; fda.gov), which states that for a Phase I clinical trial of an imaging agent that leads to 100 pg or less of a peptide being injected as a single dose in a 60 kg human, limited toxicity studies in a single mammalian species lasting 14 days are acceptable. The dose tested in the animal species is 100-fold the human equivalent dose based on the body surface area conversion factor as outlined by the FDA (TABLE 3).

Per these recommendations/guidelines, the maximum human dose for a [⁶⁸Ga]Ga- NOTA-hCTP imaging agent, for example, is 1.67 pg/Kg (100 pg in a 60 Kg human), with a rat equivalent dose of 10.35 pg/Kg (1.67*6.2=10.35), 6.2 being the conversion from human to rat equivalent dose.

Toxicity is tested in rats at 100-fold the human dose. A single injection of the [⁶⁸Ga]Ga- NOTA-peptide is administered and rats are observed for up to 14 days, with a group undergoing necropsy followed by blood chemistries and histopathology at day 2, and another group undergoing the same necropsy and blood chemistries at day 14. Specifically, an equal number of male and female 6-week-old (adult) Sprague-Dawley rats are injected once intravenously via tail vein with the selected [⁶⁸Ga]Ga-NOTA- peptide conjugate (N=16). Rats are monitored for 2 hours continuously post-injection, followed by daily checks of one cohort for up to 2 days and a second cohort for up to 14 days. Rats are weighed prior to injection and daily thereafter. Eight age- and sex-matched rats (4 males, 4 females) serve as controls, with half euthanized at day 2 and half on day 14. Half of the treated rats (an equal number of males and females) are euthanized at day 2 post-injection with inhalational CO2, their chests are opened, and 1-2 mL of blood is drawn via left ventricular puncture and sent for complete blood count, blood chemistries including kidney and liver function studies, Troponin-I levels, high-sensitivity C-reactive protein (hs-CRP), and brain natriuretic peptide (BNP) levels. Troponin-I is a marker of cardiac injury, hs-CRP is a marker of inflammation, and BNP is a marker of congestive heart failure. After the blood draw, the heart, liver, lung, kidney, brain, and spleen are dissected out, fixed, paraffin embedded, and sectioned, and slides are prepared for H&E staining in a blinded fashion. The other half of the cohort is observed for up to 14 days, and then similarly euthanized with blood and organ collection at the end of the 14-day observation period. Prior to fixation for histochemistry, a small piece of the heart is snap frozen in liquid nitrogen and stored at -80°C for later mRNA extraction and RNA sequencing, if desired.

TABLE 3: Conversion of animal doses to human equivalent doses based on body surface area

HED can be calculated from the following formula: HED = animal dose in mg/kg x (animal weight in kg/human weight in kg)^{0 33} ^bThis km value is provided for reference only since healthy children will rarely be volunteers for phase 1 trials.

Tor example, cynomolgus, rhesus, and stumptail monkeys. Example 5 - Effects of CTP and hCTP on free cytosolic calcium levels

Human left ventricular myocyte cells were plated on Day 0, and were treated (in octuplet) with varying concentrations of CTP or hCTP on Days 1, 2, 3, and 4. On Day 5, a Fluorometric Imaging Plate Reader (FLIPR®) calcium assay was run. The experiments were repeated on 3 separate days as biological triplicates. In addition, cell viability was tested after 4 days of treatment using live-dead stain followed by FACS instead of the FLIPR® assay.

These studies demonstrated that both CTP and hCTP decreased free calcium levels, with hCTP having a greater effect than CTP. The maximum effect was observed at the 2.5 to 5 pM concentration (FIGS. 12A-12C). Live-dead staining revealed that this effect was not due to changes in cell viability (FIG. 12D).

Example 6 - Anti-inflammatory effects of CTP treatments effects, and effects on TNF-q mediated activation of NF-KB

Studies using CTP conjugated to amiodarone via a disulfide bond in guinea pigs revealed that daily injections of CTP along had salutary effects on calcium handling (by increasing expression of the calcium genes SERCA2a and DHPR) in the heart, and also had anti-inflammatory effects by decreasing expression of NF-KB genes. See, Zahid et al., Pharmaceutics, 15:2107, 2023 (doi.org/ 10.3390/ pharmaceuticsl5082107); and Zahid et al., Front Chem. 11 :1220573, 2023 (doi: 10.3389/fchem.2023.1220573).

This effect was further studied in a human cardiomyocyte cell line that was treated daily for 4 days with varying concentrations of CTP or hCTP. A FLIPR assay for free cytosolic calcium was performed on Day 5. hCTP was about 10 times more efficacious at reducing cytosolic calcium levels than CTP. All experiments were conducted in octuplicate, and biological triplicates were performed on different days (FIGS. 12A, 12B, and 12C, respectively). Cell viability was also tested after the experiments using live-dead staining followed by FACS instead of the luciferin assay, demonstrating that the effect on calcium levels was not due to changes in cell viability (FIG. 12D)

In further studies, the human cardiomyocyte cell line was transfected with a reporter plasmid expressing luciferase gene under an NF-KB promoter. After 48 hours, cells were treated once with varying concentrations of CTP or hCTP (ranging from 10 pM to 100 pM), and 24 hours later the cells were challenged with TNF-a for 4 hours and luminescence was read. hCTP was about 10 times more efficacious as an anti- inflammatory at reducing NF-KB activation than CTP. All experiments were done in octuplicate, and biological triplicates were performed on different days (FIGS. 13A, 13B, and 13C, respectively). Cell viability was also tested after the experiments using live- dead stain followed by FACS instead of the luciferin assay, demonstrating that the effect of hCTP on NF-KB activation was not due to changes in cell viability (FIG. 13D).

Thus, hCTP significantly decreased free calcium levels in the cardiomyocyte line, and it also acted as an anti-inflammatory by significantly decreasing TNF-a mediated activation of NF-KB. Neither the calcium handling effects that required daily treatments or the anti-inflammatory effects were due to decrease in cell viability (FIGS. 12D and 13D). Although CTP showed similar effects, they were marginal and seen at almost 10- fold greater concentrations, making hCTP a 10-fold more potent at salutary calcium handling effects and as an anti-inflammatory agent. Thus, hCTP and even CTP along may help to ameliorate the pathology associated with heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, pericarditis, recurrent pericarditis, as well as myocarditis (e g., giant cell myocarditis, eosinophilic myocarditis, viral myocarditis, vaccine-induced myocarditis, and/or lymphocytic myocarditis), both of which are conditions in which calcium handling and inflammation pay a major role with dire consequences and mortality in humans as high as 50% (giant cell myocarditis).

Example 7 - Further studies of hybrid CTPs having D-amino acids substituted for L-amino acids, which increased serum stability and transduction efficiency The results in this Example re-present and expand on at least some of the results provided in other Examples.

The CTP sequence (APWHLSSQYSRT; SEQ ID NO: 1) includes sites that are vulnerable to enzymatic degradation by enzymes as indicated in TABLE 1. As discussed above, the amino acids at two of those sites (positions 3 and 11) were substituted with d- amino acids, yielding the “hCTP” peptide set forth in SEQ ID NO:6. Uptake of hCTP into human monocytes was increased as compared to CTP (FIG. 2). In addition, hCTP was found to have higher serum stability than CTP in fetal bovine serum (FIG. 4A) and pooled human serum (FIG. 4B). Biodistribution studies in mice demonstrated that hCTP was about 3 times more efficient in transducing mouse hearts than CTP (FIGS. 8 and 9). This was confirmed via confocal micrographs as 2D and 3D with heart uptake quantified by fluorescence as shown in FIGS. 10A and 11A and plotted in FIGS. 10B and 11B, which also show that hCTP had 3- to 4-fold greater uptake efficiency than the parent CTP. Uptake of hCTP into mouse hearts was also significantly higher than uptake into kidney, liver, and lungs (FIGS. 14A and 14B). When human left ventricular myocytes (also referred to as human cardiomyocytes) were treated with varying concentrations of CTP or hCTP for 4 days and then subjected to FLIPR® calcium assays, both CTP and hCTP decreased free calcium levels, but hCTP had a greater effect than CTP (FIGS. 12A-12C). This effect was not due to changes in cell viability (FIG. 12D).

Intracellular calcium handling plays a central role in cardiac contractility and relaxation. Genes such as the Ca²⁺-ATPase pump (SERCA2a) and the Dihydropteridine reductase (DHPR) enzyme are involved in the cycling of intracellular calcium. To test hCTP’s calcium handling abilities, human cardiomyocytes were plated on day 1 and allowed to settle for 24 hours, after which they received daily treatment with various concentrations (l-25pM) of hCTP for three days. On day 5, cells either underwent intracellular calcium measurement using the FLIPR 6 Calcium assay, or were prepared for western blotting. Western blotting was performed to determine the protein concentrations of SERCA2a and DHPR, as compared to a GAPDH control. To test hCTP’s intrinsic calcium handling abilities, various concentrations of hCTP were mixed with a constant amount of elemental calcium dissolved in water. After 20 minutes at 37°C, the concentration of free Ca²⁺ was measured with an ABL90 Flex Plus radiometer. These studies demonstrated that treatment of cardiomyocytes with hCTP led to a decrease in cytosolic calcium in a dose dependent fashion; and that concentrations as low as 1 pM led to a significant decrease in cytosolic calcium (FIG. 15A; p< 0.0001 for all groups). Western blotting performed on the treated cardiomyocytes revealed an increase in expression of both DHPR and SERCA2a, normalized to GAPDH control (FIG. 15B). Outside of the cellular environment, hCTP did not appear to display inherent calcium sequestering properties (FIG. 15C), suggesting that improvements in calcium handling were due to upregulation of gene expression. Thus, hCTP appeared to lower cytosolic calcium and improve calcium handling through upregulation of SERCA2a and DHPR. These data indicated that hCTP is useful as a stand-alone, cardio- selective therapy for cardiomyopathies involving calcium mishandling.

In further studies, the human cardiomyocyte cell line also was transfected with a reporter plasmid expressing luciferase gene under an NF-KB promoter, treated with concentrations of CTP or hCTP ranging from 10 pM to 100 pM, and 24 hours later challenged with TNF-a for 4 hours. Luminescence readings demonstrated that hCTP was about 10 times more efficacious as an anti-inflammatory at reducing NF-KB activation than CTP. All experiments were done in octuplicate, and biological triplicates were performed on different days (FIGS. 13A, 13B, and 13C, respectively). Cell viability was tested after the experiments using live-dead stain followed by FACS, demonstrating that the effect of hCTP on NF-KB activation was not due to changes in cell viability (FIG. 13D)

To further determine the effects of hCTP on TNF-a mediated NF-KB activation and on expression of NF-KB’ s downstream inflammatory markers IL-13 and IL-8, human cardiomyocytes were transfected using LIPOFECT AMINE™ 3000 (Thermo Fisher Scientific) with a reporter luciferase plasmid carrying an NF-KB promoter site upstream of the luciferase gene. Cells were treated with lower concentrations of hCTP (ranging from 1 pM to 10 pM) for 24 hours, followed by a TNF-a challenge (20 ng/mL), addition of luciferin as substrate, and measurement of luminescence. Cell viability in response to these manipulations was assessed by FACS using a live-dead stain. In addition, cell culture supernatants of the cardiomyocytes were analyzed for IL-8 levels using a human ELISA kit. In other experiments, human cardiomyocytes were transfected with the NF- KB reporter plasmid, and after treatment for 24 hours with various concentrations of hCTP, were incubated with 10 nM angiotensin 2 (Ang2) and 100 nM phenylephrine (PE) for 3 hours to assess levels of IL-10 in the cell culture media using an ELISA assay. Further, human cardiomyocytes transfected with the luciferase reporter plasmid and treated with hCTP or CTP for 24 hours were incubated in Ang2/PE for 3 hours, and NF- KB activation was then measured based on luminescence. These studies demonstrated that hCTP inhibited TNF-a mediated NF-KB activation with a dose-response curve, with maximum inhibition seen at 10 pM concentrations of hCTP (p<0.001) (FIG. 16A). Treatment with hCTP inhibited TNF-a mediated IL-8 secretion, with significant decreases seen even at the 1 pM dose (FIG. 16B). Similarly, secretion of IL- 10 by the cardiomyocytes in response to Ang2/PE stimulation was suppressed by hCTP even at 1 pM, the lowest tested dose (FIG. 16C; p<0.5). In addition, treating human cardiomyocytes with Ang2/PE resulted in a significant increase in NF-kB activation, which was prevented by hCTP/CTP pre-treatment (FIG. 16D). These data suggested that hCTP inhibited the NF-KB pathway transcriptional activity in response to TNF-a stimulation in a dose-dependent fashion in human cardiomyocytes. Further, treatment with hCTP inhibited TNF-a induced IL-8 and IL-10 secretion even at doses as low as 1 pM. The ability of hCTP to efficiently targeting this inflammatory pathway suggested its therapeutic utility in the treatment of heart failure.

Additional studies were conducted to determine the effect of hCTP on the Heart Failure with preserved Ejection Fraction (HFpEF) phenotype in mice. HFpEF is a form of heart failure that is rapidly rising in incidence, but no therapies with a mortality benefit exist for this syndrome. To establish a HFpEF model, 6-week-old, C57BL/6NJ mice were placed on a high-fat diet (HFD), 60% kCal from lard, and water with 0.5 g/L of an iNOS inhibitor (N-Nitro-L-arginine methylester) to induce hypertension. After 5 weeks on the diet, HFD mice and their littermate controls underwent baseline exercise testing and echocardiograms. Exercise capacity was measured after 2 days of exercise training with 2 days of rest in between; mice were placed on a treadmill at a 10-degree angle and made to run at increasingly fast speeds (5-30 m/min) until exhaustion. Echocardiograms were performed under 1.5% inhalational isoflurane. After baseline testing, the mice were treated with vehicle, 2.5 mg/Kg hCTP, or 5 mg/Kg hCTP, either 3 days a week or 5 days a week (five groups, n = 3 per group). After four weeks of treatment, the mice were again subjected to the same battery of tests and then euthanized. Data were compared using two-way ANOVA with Sidak’s test to compare measurements pre- and post-treatment. These studies demonstrated that treatment with hCTP at either concentration for five days/week led to an increase in running distance, from 39.8 m to 116.3 m (p = 0.03, n = 6), compared the change in control distance, which decreased from 208.4 m to 157.8 m (p = 0.399) (FIG. 17A). Running distance did not correlate with weight (R² < 0.001) (FIG. 17B). Treatment with 5 mg/Kg hCTP for 5 days/week also led to a normalization of the E/e’ ratio, from 43.3 to 26.9 (p = 0.004, n = 3) compared to a change in the control of 34.7 to 26.1 (p = 0.312) (FIG. 17C). Ejection fractions remained consistent (>55%) across all groups (FIG. 17D). In addition, treatment with hCTP normalized the growth rate of treated mice (FIG. 17E). Taken together, these data suggested that hCTP is useful as a treatment for ameliorating at least some of the physiological effects in a murine model of HFpEF.

Further studies demonstrated that benzamil, an inhibitor of the Na(⁺)/Ca(2⁺) exchanger (NCX), inhibited transduction of human cardiomyocytes by CTP and hCTP (FIGS. 18A and 18B), and that cell viability was not a factor (FIG. 18C). Other NCX inhibitors, including amiloride and SEA0400, also inhibited transduction of human cardiomyocytes by CTP and hCTP (FIGS. 19A and 19B). In addition, Alphafold 3 was used to predict binding of CTP to a particular NCX, SCL8A1. Taken together, these data indicated that CTP and hCTP bind to a NCX that is unique to the cardiomyocyte plasma membrane to gain entry into cardiomyocytes. The SCL8A1 transporter also is present in neurons in brain tissue, but is irrelevant to these cardiac targeting peptides since they do not cross the blood brain barrier due to their size.

In an in vivo study, a single, adult, wild-type B6 mouse was intraperitoneally injected with 1.5 mg/Kg Benzamil daily for 3 days. On Day 3, the mouse was injected with Cy5.5-labeled hCTP 10 mg/Kg. The peptide allowed to circulate for 30 minutes before the mouse was euthanized and its heart was dissected out for IVIS imaging. A negative control mouse was injected with vehicle for the peptide alone, and a positive control mouse (untreated with Benzamil) was injected with hCTP 10 mg/Kg only. This study demonstrated that pre-treating a mouse with Benzamil before administering hCTP markedly decreased the uptake of hCTP by the heart in vivo (FIGS. 20A and 20B). Example 8 - CTP derivatives for delivery of amiodarone

Conjugates of amiodarone attached to the N-terminus of CTP, CTP-B, hCTP, and a random peptide (RAN) are formulated, and the serum stability of each conjugate is tested.

The conjugate synthesis strategy is based on introducing a disulfide covalent bond between CTP, CTP-B, hCTP, or RAN and its cargo, amiodarone. The covalent linking makes this conjugate stable in serum, but once internalized into a cardiomyocyte, the reducing intracellular environment leads to reduction of the bond with subsequent release of amiodarone. Thus, the cardiomyocyte acts as a “sink” with intracellular accumulation of amiodarone, which is then free to diffuse out of the cell into the inter-cellular matrix, where it can act on various receptors (e.g., the beta-adrenergic receptor, calcium-sodium exchange channels, etc.). To achieve conjugation, thiolated versions of the various peptides and amiodarone are generated prior to linking the two together as described elsewhere (Yurko et al., Front Chem. 11:1220573, 2023). A 12-amino acid scrambled random peptide is generated using a web-based random peptide generator (Expasy- RandSeq) to serve as a negative control in all subsequent studies.

Synthesis of thiolated peptide. Solid phase peptide synthesis of peptides is accomplished on a microwave synthesizer using standard Fmoc/tBu chemistry and Oxyma pure coupling protocols at a 1 mM scale on Rink amide MB HA resin (10 reactors at 0.1 mM each). After completion of the peptide chain assembly, all reactor products are pooled, and the N-terminal amino group is manually conjugated on-resin with 3- (Tritylthio) propionic acid (5eq.) using TBTU (5eq.)/HOBt (5eq.)/DIPEA (lOeq.) in DMF. The resulting thiolated peptides are cleaved from the resin and side chains are deprotected using TFA:TIPS:H2O:EDT (94: 1:2.5:2.5) for 2 hours at room temperature, followed by precipitation of the resultant crude peptide-thiol product in ice cold Diethyl Ether (Et2O), which is centrifuged to a pellet at 2,500 rpm for 5 minutes. This process is repeated 2 additional times and the pellet is allowed to air dry in a chemical fume hood for about 30 minutes to remove residual ether from the sample. The crude peptide-thiol is dissolved in 50% TFE/0.1%TFA (aq.) in 20 mg batches and purified by preparative C-18 RP-HPLC. All fractions representing the desired product are pooled, shell frozen in dry ice/ethanol, and lyophilized to a dry powder using an SP Scientific SP VirTis BenchTop Pro freeze dryer. The yield of the final, purified CTP -thiol is expected to be about 60% based on initial reactor synthesis scale with determination of purity accomplished using analytical C-4 RP-HPLC. Therefore, production of peptides is scaled up, to about twice as much as needed for a molar 1 : 1 conjugation to thiolated amiodarone. MALDI analysis is performed of each of the peptide-thiol products, and the expected molecular weight of each of thiolated peptide is confirmed prior to progressing to the next step. Purified peptide-thiol intermediates are then modified with 2,2'-dithiobis (5-nitropyridine) (DTNP) in 80% trifluoroacetic acid (TFA) (aq.) for 15 minutes at room temperature with gentle mixing, and samples are dried to a film using a stream of nitrogen under mild heating conditions. The resulting NPys-S-peptide intermediate film is dissolved in 50%TFE/0.1% TFA and directly purified by preparative C-18 RP-HPLC on a Waters Delta Prep 4,000 chromatography system followed by lyophilization to a dry powder. Determination of final purity is accomplished using analytical C-4 RP-HPLC on a Waters Alliance chromatography system and processed with Empower software. MALDI analysis is performed after this step to confirm the correct mass of NPys-S-peptides.

Synthesis of thiolated amiodarone. Amiodarone hydrochloride is chloroalkylated at the tertiary nitrogen with a 10% solution of 3 -chloro- 1 -propanethiol in acetonitrile and a catalytic amount of sodium iodide and 1,2,2,6,6-pentamethylpiperidine, with gentle mixing overnight at 37°C to form the thiolated quaternary ammonium intermediate. The crude reaction mix is dissolved in 50% TFE/0.1%TFA and then directly purified by preparative C-18 RP-HPLC, followed by lyophilization to a clear film. Determination of purity is accomplished using analytical C-18 RP-HPLC on a Waters Alliance chromatography system along with Empower software. MALDI analysis is performed to confirm identity of the final amiodarone-thiol product.

Peptide-amiodarone conjugate synthesis'. Pure amiodarone-thiol is conjugated to the NPys-S-peptides in 20% 1 M ammonium acetate buffer (pH 4.0)/DMF at room temperature for 2 hours with gentle mixing. The crude amiodarone-SS-peptide conjugate is then purified using preparative C-18 RP-HPLC and the resulting fractions are pooled into 50 mL polypropylene conical tubes and shell frozen in dry ice/ethanol, followed by overnight lyophilization. The lyophilized conjugate is stored at -20 degrees until use.

Conjugate stability studies. Prior to embarking upon animal studies, all peptide- amiodarone conjugates are tested for serum stability at 37°C over time. To determine the stability, the peptide-amiodraone conjugates undergo HPLC characterization at baseline (time 0) before being placed at 37°C for various time points (24 hours, 48 hours, 72 hours, 7 days, and 21 days) and HPLC is performed at those times and compared to baseline to assess the stability of the conjugate over time. All conjugates and time points are tested in triplicate.

Tissue levels of amiodarone and its major metabolite (desethylamiodarone) are determined, and related toxicides are evaluated in mice treated chronically with amiodarone, CTP-amiodarone, CTP-B-amiodarone, hCTP -amiodarone, and RAN - amiodarone.

After successful completion of conjugation and HPLCs of the various conjugate studies at 37°C show acceptable stability (for up to 48 hours minimum), the conjugates are ready for animal testing. Adult (6-week old) wild-type, CD-I male and female mice (1: 1 ratio; 10 mice/group to allow for n=5 for each sex in all groups) are injected intraperitoneally with vehicle only, amiodarone only (MW 682; 25mg/Kg or 36.7pM/gm body weight), or the peptide-amiodarone conjugates at 1/15^th the molar dose of amiodarone at 2.4 pM/gm. All groups are injected 5 days/week for 4 weeks. The dose of amiodarone (25 mg/Kg) is based on literature available on toxicity studies of amiodarone in mice (Roomi et al., Exp Ther Med. 7:987-989, 2014; and Takai et al., J Appl Toxicol. 36:35-47, 2016), where investigators used higher doses of amiodarone (100 mg/Kg and 50 mg/Kg) but for much shorter durations of treatment that lasted 1 and 4 days, respectively. Mice are weighed on Day 1 of the treatments, and weekly thereafter as well as immediately prior to euthanasia. At the end of the treatments, mice undergo echocardiography for left ventricular size, mass, systolic (left ventricular ejection fraction, cardiac output) and diastolic function (mitral pulse-wave doppler inflow and mitral annular tissue doppler velocities) assessment, followed by euthanasia with inhalational CO2, opening of the thoracoabdominal cavity for obtaining blood through cardiac puncture, and then harvesting of the heart, lung, liver, kidney, thyroid, abdominal fat, skin, and ocular tissue. Tissues are rinsed in phosphate buffered saline (PBS), dabbed dry, weighed, and divided into two with one piece of each snap frozen in liquid nitrogen, and the other piece is put into formalin for later fixation and cryosectioning.

Amiodarone tissue accumulation studies'. Complete amiodarone accumulation studies are conducted by assessment of tissue as well as serum (collected at time of euthanasia) levels in mice treated with amiodarone alone versus the peptide-amiodarone conjugates. Tissues are snap frozen in liquid nitrogen and stored at -80°C until all animals have been euthanized for later extraction and quantification of amiodarone and its major metabolite (desethylamiodarone), using liquid chromatograph-tandem mass spectrometry (LC-MS) (Garcia et al., Circ Arrhythm Electrophysiol. l l:e006408, 2018; Kuhn et al., J Pharm Biomed Anal. 51 :210-216, 2010; and Rodrigues et al., J Chromatogr Sci. 51 :361-370, 2013). All collected organs, including heart, lung, liver, abdominal fat, skin, and retinal tissue (where amiodarone typically accumulates) are evaluated for levels of amiodarone/desethylamiodarone using a stable isotope dilution LC-MS protocol as reported elsewhere (Kuhn et al., supra), with a few modifications. Briefly, tissue is made into a homogenate by the addition of lx phosphate-buffered saline at 10 pL/mg prior to adding 20 pL of d4 isotope solution as an internal standard. Proteins are precipitated from the sample mixture by adding 1 mL of cold acetonitrile:methanol solution (50:50, v:v), and the supernatant is transferred to a 1-dram vial and dried in a speed vac without heat prior to being resuspended in running buffers for analysis. A 10- point calibration curve is constructed using amiodarone/desethylamiodarone obtained from Sigma-Aldrich (St. Louis, MO). Both standards and samples are analyzed on a Thermo Quantiva triple quadrupole mass spectrometer coupled with a Waters Acquity liquid chromatography system. Data acquisition is done using select ion monitor, m/z 646>201, 650>201, 618>547, 622>547 for amiodarone, d4 -amiodarone, desethylamiodarone and d4-desethylamiodarone, respectively.

Amiodarone toxicity studies'. Potential lung, liver, and thyroid toxicity signals are assessed at the end of the above experiments with chronic administration of amiodarone versus peptide-amiodarone conjugates. The organs saved from animals subjected to prolonged treatment with amiodarone and peptide-amiodarone conjugate are leveraged to study markers of lung toxicities. Lung sections are stained for fibrosis by Masson trichrome staining, and quantitative RT-PCR is performed for hydroxyproline, TGF-pi, and collagen type I, which are upregulated with amiodarone-induced lung toxicity (Nasri et al., Med Prine Pract. 25: 150-154, 2016; Card et al., Toxicol Sci. 75: 169-180, 2003; and Sharaf et al., Basic Clin Pharmacol Toxicol. 119:58-67, 2016). Serum procured at time of euthanasia is used for measurement of the level of high-sensitivity Troponin I (a marker of cardiac damage), liver function studies (alanine aminotransferase, aspartate transferase, alkaline phosphatase, bilirubin, etc.), and thyroid function studies (triiodothyronine, thyroxine, and TSH levels). Cardiac mass, end-diastolic dimensions, and function using echocardiography are analyzed by a technician blinded to the treatments. Liver, heart, and kidney tissues are embedded, cryosectioned, and stained for markers of apoptosis and compared to tissues from the control (vehicle only) animals. Light microscopy of at least three sections/tissue is assessed in 6 non-overlapping 20x high-power fields with number of apoptotic cells normalized by the number of DAPL stained nuclei in the field.

Efficacy of the lead conjugate against amiodarone for treating stable ventricular tachycardia (VT) is tested in a pig model.

Adult (2-3 month old) Yorkshire pigs in 1: 1 male to female ratio are utilized, with 10 animals with stable VT in the test group (peptide-amiodarone) and 10 animals in the control group (amiodarone only) in keeping with studies described elsewhere (Haasio et al., RegAnesth. 15: 174-179, 1990; Piktel et al., J Am Heart Assoc. 10:e016676, 2021; and Verrier et al., Heart Rhythm. 10:121-127, 2013). To achieve this number, 33 animals with myocardial infarction (MI) are generated. It is expected that up to 70-80% of the animals survive the MI procedure, yielding 25 infarcted animals. Post-infarct, animals are allowed to recover for 21 days prior to a pacing procedure, to achieve a target of 20 animals with sustained VT, defined as VT lasting > 1 minute. The 70-80% success rate of pacing induction is based on the work described elsewhere (Tschabrunn et al., Heart Rhythm. 13:262-273, 2016; and Lubberding et al., Am J Physiol Heart Circ Physiol. 318:H391-H400, 2020). Generating a pig model of M Animals are fasted for at least 12 hours prior to the procedure, after which they are prepped for aseptic surgery (including recording the general condition of each animal and its vital signs, application of eye lubricant, and clipping, cleaning and aseptically scrubbing the incision sites with antiseptic solution). The animals are treated with prophylactic antibiotics, Cefazolin (16-25 mg/kg IV) and Ceftiofur (3-5 mg/kg IV), followed by sedation with Tiletamine/Zolazepam (3.5-5.5 mg/kg)/Xylazine 1.5-3.5 mg/kg IM. Induction is done using propofol (2-8 mg/kg IV) and anesthesia is maintained with Isoflurane 0-5%. An endotracheal tube is placed in the mouth and manipulated past the arytenoids to achieve endo-tracheal intubation, and the cuff is inflated until no leak is audible while administering manual breaths. Lung intubation is confirmed by auscultation of both lungs. Once confirmed, the endotracheal tube is secured in place with tape, and the animal is hooked to the ventilator. The animals receive pre-operative pain medication (Ketoprofen 2-3.5 mg/kg) and intraoperative pain medication (Buprenorphine ER 0.12-0.24 mg/kg SQ). Bupivacaine (0.5%, 0.2-0.5ml) is administered along the incision site for local block at the thoracotomy site. Animals are maintained in stage III anesthesia while monitoring vital signs including heart rate, respiratory rate, end tidal CO2, pH, pCCh, SpCh, mean blood pressure, and body temperature. Animals receive appropriate maintenance isotonic fluids during the procedure at a rate of 5-10 mL/kg/hr. After an adequate depth of anesthesia is achieved, pigs are placed in a slight right lateral position, skin is prepared for surgery over the left rib cage and is draped, and an incision is made along the upper border of the 4^th or 5^th intercostal space. The rib space is opened with rib separators and pleura is cut into using electrocautery. The lungs are gently retracted, the pericardial space is opened, and The anterior wall/apex of the left ventricle is exposed. A short segment of the left anterior descending coronary artery is dissected free immediately distal to the takeoff of the first diagonal branch. A 2-0 silk suture is passed underneath and the artery is ligated. The pericardium is closed loosely. All catheters and sheaths following the procedure are removed and any bleeding vessel is ligated or cauterized. A chest tube is placed prior to closing the incision using standard surgical techniques, and bandages are placed over the incision prior to recovering the animal. Post-operative pain control is achieved with daily subcutaneous injection of Buprenorphine at 0.12-0.24 mg/Kg for the first three days, and with oral Tramadol (1-4 mg/Kg twice daily) after day 3 if needed. Animals are monitored daily for 21 days for any sign of infection, heart failure, or distress.

Generating ci pig model of stable VT After post-operative day 21 , an appropriately sized sheath is placed in the external jugular vein to allow for introduction of the pacing leads into the heart. A pacing lead is introduced into the right ventricle and connected to the external simulator. Brief bursts of rapid ventricular pacing from the right ventricle at cycle lengths of 150-250 msec are delivered for initiation of VT. Additional cycle lengths and stimulation protocols may be attempted to induce VT based on work described elsewhere (Garan et al., Circulation. 62:980-987, 1980; Reek et al., Pacing Clin Electrophysiol. 22:605-614, 1999; and Krejcy et al., Naunyn Schmiedebergs Arch Pharmacol. 346:213-218, 1992). Animals have their heart rhythm monitored continuously during this protocol, with sustained VT defined as a wide-complex tachyarrhythmia lasting > 1 minute.

Testing peptide-amiodarone conjugate efficacy compared to amiodarone'.

Baseline heart rate prior to induction of VT is recorded. Following induction of sustained VT, amiodarone is administered at a dose of 1.5 mg/Kg as a continuous intravenous infusion over 10 minutes. Amiodarone infusion is repeated once at the same dose/rate if normal sinus rhythm is not established during first infusion. If VT persists after the second infusion, external defibrillation shocks are applied starting at 150J energy, and are increased up to a maximum of 300J in 50J increments. Hemodynamics are monitored closely to ensure the animal’s physiologic stability. The total amiodarone dose, time to resolution of VT, need for defibrillatory shocks, number of shocks, energy of shocks, and highest energy of the shock required to revert to normal sinus rhythm are recorded. Following VT resolution, VT induction is repeated as described above for a total of 3 times, with pacing thresholds required for VT induction recorded. After the procedure, animals are recovered, and the experiment is repeated 3 times at weekly intervals. The exact same experiment is repeated and animals are treated with 1/15^th the molar dose of the chosen peptide-amiodarone conjugate in the treatment cohort. The efficacy outcome parameters outlined above are compared between the amiodarone (n=10) and peptide- amiodarone conjugate (n=10). At the end of three weeks of testing, animals are euthanized, and serum and tissue samples (heart, lung, liver, thyroid, skin, retina, abdominal fat) are collected and frozen for additional testing that includes thyroid and liver function tests, tissue embedding for cryosectioning for later histology and staining for fibrosis, as well as determining serum and tissue levels of amiodarone and desethylamiodarone. Parts of the tissues also are snap-frozen in liquid nitrogen for later RNA extraction and quantitative RT-PCR for hydroxyproline, TGF-pi, and collagen type I, which have been shown to be upregulated in animal studies of amiodarone lung toxicity described elsewhere (Nasri et al., supra, Card et al., supra, and Sharaf El-Din et al., supra).

Formulation and validation of a long-acting injectable form of optimized CTP- amiodarone.

A sustained-release formulation of the CTP-amiodarone is generated to achieve one-month steady levels, in order to improve the clinical potential/applicability of the CTP-amio conjugate.

Experimental Design & Methods'. Consegna Pharma Inc., a specialty pharma company, reformulates proven drugs into long-acting injectable (LAI) medications. Consegna Pharma’s ADSR™ software (Rothstein et al., J Mater Chem. 18: 1873-1880, 2008; and Rothstein et al., Biomaterials. 30:1657-1664, 2009) and microencapsulation techniques are used to achieve one-month sustained delivery of CTP-amiodarone. This software provides initial reformulation design and optimization in silico, and in vitro testing is performed to validate model predictions. ADSR™ is then used to convert the optimized dissolution profile into predicted pharmacokinetic (PK) behavior in Sprague- Dawley rats to inform preclinical testing, and amiodarone rat serum concentration is measured over 60 days to confirm the in vivo release. Consegna formulates the optimized CTP-amiodarone using FDA approved microencapsulation technology such as poly- lactic-co-glycolic acid (PLGA) microparticles into a LAI. Results from the ADSR™ software allows validation of the accuracy of formulation designs leading to de-risking of in vivo translation. Microparticle production and drug loading. CTP-amiodarone-loaded PLGA microparticles are produced by a double emulsion process using the Micropore Technologies AXF-1 system. The Micropore AXF-1 is a high throughput aseptic device scaling up Micropore’s advanced crossflow mixing technology to enable the continuous manufacturing of high-quality formulations in quantity. It retains a tightly controlled particle size distribution and is designed for commercial-scale production. Following complete solvent evaporation and subsequent microparticle hardening, the CTP- amiodarone loaded microparticle product is purified, dried, and stored until use.

In vitro dissolution testing'. CTP-amiodarone loaded microparticles are tested with a standard in vitro release assay. Briefly, rehydrated CTP-amiodarone loaded microparticles are incubated at 37°C in phosphate buffered saline at a 1: 10 mass-to- volume ratio (a near-perfect sink), and gently agitated. The accumulation of CTP- amiodarone in samples of supernatant is measured by injecting samples into a LC- MS/MS instrument equipped with a Cl 8 column. Release rate is determined by generating and employing a CTP-amiodarone calibration curve.

In vivo (rat) PK study. Consegna partners with a CRO that performs preclinical testing of novel therapeutics in an accredited vivarium. Highly trained staff perform in vivo PK study activities. In particular, CTP-amiodarone dosing is set based at least in part on literature reports, allometric scaling, and estimated oral-to-CTP scaling. Sprague Dawley rats (4 male and 4 female) are dosed with a subcutaneous injection of LAI CTP- amiodarone reconstituted at 200 mg/mL in 3% carboxymethyl cellulose sodium, (diluent approved in PLGA microparticle LAI products).

Data analysis and interpretation. Plasma sample collection captures the immediate release at 13 time points over the full course (one month) of sustained release and 30 days beyond. Hearts, livers, lungs, and thyroid tissue are harvested at the end of the study and tissue levels of amiodarone/desethylamiodarone are measured using LC- MS/MS. Heart tissue samples from the rats are analyzed to confirm targeting of the long- acting formulation of CTP. Additional tissue samples are analyzed to confirm nonaccumulation of amiodarone/desethylamiodarone in abdominal fat, liver, lungs, and thyroid. Plasma amiodarone/desethylamiodarone concentrations are measured by LC- MS/MS as detailed above.

OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide having the amino acid sequence set forth in SEQ ID NO:6.

2. A composition comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO:6 and a pharmaceutically acceptable carrier.

3. A conjugate comprising (a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:6 coupled to (b) a therapeutic agent or a detectable label.

4. The conjugate of claim 3, wherein said polypeptide is coupled to said therapeutic agent.

5. The conjugate of claim 4, wherein said therapeutic agent comprises an anti- arrhythmic agent, a siRNA, or a miRNA.

6. The conjugate of claim 3, wherein said polypeptide is coupled to said detectable label.

7. The conjugate of claim 6, wherein said detectable label comprises a radioisotope.

8. The conjugate of claim 7, wherein said radioisotope comprises Technetium 99m or Gallium68.

9. A composition comprising the conjugate of any one of claims 3 to 8 and a pharmaceutically acceptable carrier.

10. A method for delivering an agent into a cardiac cell, wherein said method comprises contacting said cardiac cell with a conjugate that comprises (a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:6 coupled to (b) said agent.

11. The method of claim 10, wherein said agent is a therapeutic agent or a detectable label.

12. The method of claim 11, wherein said agent is said therapeutic agent.

13. The method of claim 12, wherein said therapeutic agent comprises an anti- arrhythmic agent, a siRNA, or a miRNA.

14. The method of claim 11, wherein said agent is said detectable label.

15. The method of claim 14, wherein said detectable label comprises a radioisotope.

16. The method of claim 15, wherein said radioisotope comprises Technetium 99m or

Gallium68.

17. A method comprising administering, to a mammal, a conjugate comprising (a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:6 coupled to (b) a therapeutic agent or a detectable marker.

18. The method of claim 17, wherein said mammal is a human.

19. The method of claim 17 or claim 18, wherein said mammal has been identified as having a cardiovascular disorder.

20. The method of claim 19, wherein said cardiovascular disorder comprises myocarditis, pericarditis, recurrent pericarditis, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, or Friedreich’s ataxia associated cardiac hypertrophy.

21. The method of claim 20, wherein said myocarditis comprises viral myocarditis, vaccine-associated myocarditis, lymphocytic myocarditis, giant-cell myocarditis, or eosinophilic myocarditis.

22. The method of any one of claims 17 to 21, wherein said agent is said therapeutic agent.

23. The method of claim 22, wherein said therapeutic agent comprises an anti- arrhythmic agent, a siRNA, or a miRNA.

24. The method of claim 17, wherein said agent is said detectable label.

25. The method of claim 24, wherein said detectable label comprises a radioisotope.

26. The method of claim 25, wherein said radioisotope comprises Technetium 99m or

Gallium68.

27. A method for treating a mammal having a cardiovascular disorder, said method comprising administering, to said mammal, a conjugate comprising (a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:6 coupled to (b) a therapeutic agent.

28. The method of claim 27, wherein said mammal is a human.

29. The method of claim 27 or claim 28, wherein said cardiovascular disorder comprises myocarditis, pericarditis, recurrent pericarditis, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, or Friedreich’s ataxia associated cardiac hypertrophy.

30. The method of claim 29, wherein said myocarditis comprises viral myocarditis, vaccine-associated myocarditis, lymphocytic myocarditis, giant-cell myocarditis, or eosinophilic myocarditis.

31. The method of any one of claims 27 to 30, wherein said agent is said therapeutic agent.

32. The method of claim 31, wherein said therapeutic agent comprises an anti- arrhythmic agent, a siRNA, or a miRNA.

33. The method of any one of claims 27 to 30, wherein said agent is said detectable label.

34. The method of claim 33, wherein said detectable label comprises a radioisotope.

35. The method of claim 34, wherein said radioisotope comprises Technetium 99m or Gallium68.

36. A method for treating a mammal having a cardiac disorder, wherein said method comprises administering to said mammal a polypeptide having the amino acid sequence set forth in SEQ ID NO:6.

37. The method of claim 36, wherein said mammal is a human.

38. The method of claim 36 or claim 37, wherein said cardiovascular disorder comprises myocarditis, pericarditis, recurrent pericarditis, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, or Friedreich’s ataxia associated cardiac hypertrophy.

39. The method of claim 38, wherein said myocarditis comprises viral myocarditis, vaccine-associated myocarditis, lymphocytic myocarditis, giant-cell myocarditis, or eosinophilic myocarditis.

40. A method for treating a mammal having a cardiac disorder, wherein said method comprises administering to said mammal a composition comprising a pharmaceutically acceptable carrier and a polypeptide having the amino acid sequence set forth in SEQ ID NO:6.

41. The method of claim 40, wherein said mammal is a human.

42. The method of claim 40 or claim 41, wherein said cardiovascular disorder comprises myocarditis, pericarditis, recurrent pericarditis, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, Duchenne muscular dystrophy, cardiac hypertrophy, hypertrophic cardiomyopathy, cardiomyopathy, or Friedreich’s ataxia associated cardiac hypertrophy.

43. The method of claim 42, wherein said myocarditis comprises viral myocarditis, vaccine-associated myocarditis, lymphocytic myocarditis, giant-cell myocarditis, or eosinophilic myocarditis.