WO2025085826A1 - Activation of orl1: a novel therapy to prevent heart failure progression - Google Patents

Abstract

Embodiments of the disclosure include methods treating a cardiac medical condition in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of one or more compositions or a pharmaceutically acceptable salt thereof, wherein the one or more compositions comprises at least one or more ORL1-activating agents.

Description

ACTIVATION OF ORL1: A NOVEL THERAPY TO PREVENT HEART FAILURE PROGRESSION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/591,680, filed October 19, 2023, which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

[0002] Embodiments of the disclosure include at least the fields of molecular biology, cell biology, cell therapy, and medicine, including cardiac medicine.

BACKGROUND

[0003] Despite medical and surgical innovations, heart failure (HF) is a major cardiovascular disease and remains the number one cause of death in the world. Although current main therapeutic strategies targeting HF -related G-protein-coupled receptors (GPCR), such as angiotensin receptor antagonists, markedly improve the outcome of HF, HF morbidity and mortality rates remain high. There is a critical need to explore new therapeutic approaches.

[0004] The present disclosure provides solutions to a long-felt need in the art for improving cardiac function.

BRIEF SUMMARY OF THE DISCLOSURE

[0005] Embodiments of the disclosure include methods and compositions for the treatment of any medical condition related to the mammalian heart.

[0006] Embodiments of the disclosure include a method of treating a cardiac medical condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more compositions, wherein the one or more compositions comprise one or more ORL1 (opioid related nociceptin receptor 1, also known as OPRLl)-activating agents or a pharmaceutically acceptable salt thereof. Embodiments of the disclosure include methods of inhibiting pathological hypertrophy in a subject in need thereof, inducing physiological hypertrophy in a subject in need thereof, suppressing fibrosis in a subject in need thereof, inducing angiogenesis in a subject in need thereof, preventing HF progression in a subject in need thereof, or attenuating or preventing hypertension progression in a subject in need thereof, the methods comprising providing to the subject a therapeutically effective amount of one or more compositions or a pharmaceutically acceptable salt thereof, wherein the one or more compositions comprises at least one or more ORL1 -activating agents. [0007] In certain embodiments, the one or more compositions are a small molecule, a peptide, a protein, a biologic, a carbohydrate, and/or a nucleic acid.

[0008] In certain embodiments, the at least one or more ORL1 -activating agents comprise (R)- Ro65-6570, [(pF)Phe4]N/OFQ-(l-13)-NH2, [3H]Leu-N/OFQ, [3H]N/OFQ, [3H]Tyrl4-N/OFQ, [Argl4Lysl5]N/OFQ, [F/G]N/OFQ-(1-13)-NH2, Ac-RYYRIK-NH2, Ac-RYYRWK-NH2, AT- 121, AT -403, BU08028, buprenorphine, buprenorphine hydrochloride, cebranopadol, etorphine, JNJ-19385899, MCOPPB (also known as l-[l-(l-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3- piperidinyl]-lH-benzimidazole), MCOPPB trihydrochloride hydrate, MT-7716, N/OFQ-(1-13)- NH2, N/OFQ-NH2, NNC 63-0532, nociceptin/orphanin FQ (N/OFQ), norbuprenorphine, PWT2- N/OFQ, Ro64-6198, Ro6570, SCH221510, SCH486757, SerlOO (ZP120), SR16835, SR16435, TH-030418, UFP-102, and/or UFP-112. In specific embodiments, the at least one or more ORL1- activating agents comprise MCOPPB.

[0009] In certain embodiments, the cardiac medical condition induces heart failure, and wherein the cardiac medical condition may be with aortic stenosis, arrhythmia, cerebrovascular accident, chronic obstructive pulmonary disease, cigarette smoking, congenital heart disease, diabetic cardiomyopathy, dilated cardiomyopathy, a genetic defect, heart failure, hypertension, ischemic coronary disease, kidney disease, low baseline left ventricular ejection fraction, low platelet count, male gender, mitral regurgitation, myocardial infarction, myocarditis, obstructive hypertrophic cardiomyopathy, obesity, old age, peripheral vascular disease, renal disease, rheumatic heart disease, valvular disease, and/or viral myocarditis. In specific embodiments, the cardiac medical condition is heart failure or is associated with heart failure.

[0010] In certain embodiments, the at least one or more ORL1 -activating agents are provided in a pharmaceutical composition. In certain embodiments, the at least one or more compositions are delivered systemically. In other embodiments, the at least one or more compositions are delivered locally. In certain embodiments, the at least one or more compositions are provided intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, locally, via inhalation, via injection, via infusion, via continuous infusion, via localized perfusion, via a catheter, and/or via a lavage. In certain embodiments, the at least one or more compositions are provided as a tablet, troche, lozenge, aqueous suspension, oily suspension, emulsion, hard capsule, soft capsule, and/or syrup.

[0011] In certain embodiments, the methods of the disclosure further comprise the step of providing one or more additional therapeutic compositions to the subject in need thereof. [0012] In certain embodiments, the one or more compositions further comprise Gata4, fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); angiopoi etins, Angl and Ang2; matrix metalloproteinase (MMP); Delta-like ligand 4 (DIM); ITD-1; Mef2c, TBX5, Hand2, myocardin, Mesoderm posterior protein 1 (Mespl), miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, 5-azacytidine, Zebularine, and/or or peptides thereof, angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor plus neprilysin inhibitors (ARNIs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, diuretics, potassium, magnesium, potassium-sparing diuretics or aldosterone antagonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, Digoxin or Lanoxin or digitalis, hydralazine and isosorbide dinitrate (BiDil), Vericiguat or Verquvo or an oral soluble guanylate cyclase (sGC) stimulator, positive inotropes or inotropic therapy, vasodilators, anticoagulant medication, aspirin, nitrates, statins, and/or blood thinners.

[0013] In certain embodiments, administration of the at least one or more compositions occurs before administration of the at least one or more ORL-1 activating agents. In other embodiments, administration of the at least one or more compositions occurs simultaneously to administration of the at least one or more ORL-1 activating agents. In other embodiments, administration of the at least one or more compositions occurs after administration of the at least one or more ORL-1 activating agents. In certain embodiments, the at least one or more compositions are delivered via a same route of administration as the at least one or more ORL-1 activating agents. In other embodiments, the at least one or more compositions are delivered via a different route of administration as the at least one or more ORL-1 activating agents. In certain embodiments, the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in one formulation. In other embodiments, the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in separate formulations.

[0014] In certain embodiments, the methods of the disclosure further comprise treatment with at least one or more co-therapies. In specific embodiments, the at least one or more co-therapies further comprise drug therapy, surgery, coronary bypass, heart valve repair or replacement, an implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) or biventricular pacing, a ventricular assist device (VAD), VAT, percutaneous coronary intervention (PCI) and/or heart transplant. In certain embodiments, administration of the at least one or more co-therapies occurs before administration of the at least one or more ORL-1 activating agents. In other embodiments, administration of the at least one or more co-therapies occurs simultaneously to administration of the at least one or more ORL-1 activating agents. In other embodiments, administration of the at least one or more co-therapies occurs after administration of the at least one or more ORL-1 activating agents.

[0015] In certain embodiments, there is a kit comprising one or more compositions encompassed by the disclosure, said composition being housed in a suitable container. The kit may or may not one or more apparatuses for delivery of the composition, one or more reagents for dispersing the composition in, testing of the individual, or a combination thereof.

[0016] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter which form the subject of the claims of the disclosure. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present disclosure. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the disclosure as set forth in the appended claims. The novel features which are believed to be characteristic of the disclosure, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings.

[0018] FIG. 1. Depicts a graph showing Ca²⁺ intracellular levels (Y axis, as a ratio of MFIs for UV450-A and UV652LP-A) over time (X axis, as minutes) when cardiomyocytes were exposed to various conditions. Embryonic rat cardiomyocyte cells (e.g., H9C2) were treated for 48 hours with 50 MOI of lentivirus encoding Gata4 (“Gata4”) or mock treated (“Mock”), and then received lOOnM of a vasoconstrictor peptide (e.g., endothelin-1, e.g., ET-1) via addition to the culture medium. The cells were then bathed in Ca²⁺-free buffer with the sarcoplasmic reticulum Ca²⁺ ATPase inhibitor cyclopiazonic acid (CPA) for 30 minutes, before transfer to buffer containing ImM Ca²⁺ and CPA. Ca²⁺ levels were measured by indo-1 fluorescence via flow cytometry prior and after the cell transfer to buffer containing ImM Ca²⁺. The results showed Ca²⁺ influx was significantly diminished in the Gata4 condition, demonstrating that Gata4 inhibits calcium influx in cardiomyocytes.

[0019] FIG. 2. Depicts imaging of cell extracts produced by cardiomyocytes exposed to various conditions (X axis). Over 72 hours, embryonic rat cardiomyocyte cells (e.g., H9C2) were not treated (“Mock”); treated with lOOnM of a vasoconstrictor peptide (e.g., ET-1) at hour 48 (“Mock+ET-1”); treated with 50 MOI lentivirus encoding Gata4 at hour 24, or with 0.5uM MCOPPB at hours 24 and 48, and treated with ET-1 at hour 48 (“Gata4+ET-1” and “MCOPPB+ET-1”, respectively); treated with 50 MOI lentivirus encoding Gata4 at hour 24, or with 0.5uM ORL1 activator (e.g. MCOPPB) at hours 24 and 48, and treated with lOuM anti- ORL1 (e.g., [Nphel]Nociceptin(l-13)NH2) and ET-1 at hour 48 (“Gata4+anti-ORLl+ET-l” and “MCOPPB+anti-ORLl+ET-1”, respectively). Nuclear fractions of cell extracts were analyzed by Western blotting with NFATc4 (e.g., anti-NFATc4, e.g., NF AT c4 Santacruz Cat no sc-271597). Lamin b (Santa Cruz Biotechnology- SC 374015) was used a loading control. Addition of anti- ORL1 significantly increased NFATc4 expression, demonstrating that anti-ORLl diminished Gata4’s and MCOPPB’s downregulation of NFATc4 expression in the nuclei of cardiomyocytes. [0020] FIGs. 3A-3B. Show expression of NPPA and NPPB, which are downstream transcription genes of the NF AT signaling pathway and related to pathological hypertrophy, in cells exposed to various conditions. 3A) Depicts a chart showing NPPA and NPPB expression (Y axis, as relative mRNA expression as compared to mock cells) in cardiomyocytes exposed to various conditions (X axis). Embryonic rat cardiomyocyte cells (e.g., H9C2) were treated for 72 hours with 50 MOI of lentivirus encoding Gata4 (“Gata4” and “Gata4 + anti-ORLl”) via addition to the culture medium. All groups then received lOOnM of a vasoconstrictor peptide (e.g., ET-1) and the “Gata4 + anti-ORLl” group received lOuM anti-ORLl (e.g., [Nphel]Nociceptin(l- 13)NH2). NPPA and NPPB expression were then measured via a SYBR Green qPCR protocol. The results showed that both NPPA and NPPB were downregulated by Gata4, while anti-ORLl (e.g., [Nphel]Nociceptin(l-13)NH2) diminished Gata4’s downregulation of NPPA and NPPB in cardiomyocytes. 3B) Depicts a chart showing NPPA and NPPB expression (Y axis, as relative mRNA expression as compared to control cells) in cardiomyocytes exposed to various conditions (X axis). Embryonic rat cardiomyocyte cells (e.g., H9C2) were treated for 48 hours with 0.5 uM MCOPPB (“MCOPPB” and “MCOPPB + anti-ORLl”) via addition to the culture medium. All groups then received lOOnM of a vasoconstrictor peptide (e.g., ET-1) and the “MCOPPB + anti- 0RL1” group received lOuM anti-ORLl (e.g., [Nphel]Nociceptin(l-13)NH2). NPPA and NPPB expression were measured via a SYBR Green qPCR protocol. The results showed that both NPPA and NPPB were downregulated by the 0RL1 activator (e.g., MCOPPB), while anti-ORLl (e.g., [Nphel]Nociceptin(l-13)NH2) diminished MCOPPB’s downregulation of NPPA and NPPB in cardiomyocytes.

[0021] FIG. 4. Depicts a chart showing expression of Collal and Fnl (Y axis, as relative mRNA expression as compared to control cells), which are downstream transcription genes of the NF AT signaling pathway and related to fibrosis, in cardiac cells exposed to various conditions (X axis). Primary rat cardiac fibroblasts were isolated from harvested rat cardiac tissue and cultured in DMEM with 10% fetal bovine serum and 1% penicillin/ streptomycin for 3-5 days. The primary rat cardiac fibroblasts were then treated for 48 hours with saline (“Mock”) and 0.5uM MCOPPB (“MCOPPB” and “MCOPPB + anti-ORLl”) via addition to the culture medium. All groups then received lOOnM of a vasoconstrictor peptide (e.g., ET-1) and the “Gata4 + anti-ORLl” group received lOuM anti-ORLl (e.g., [Nphel]Nociceptin(l-13)NH2). Collal and Fnl expression were measured via a SYBR Green qPCR protocol. The results show that ORL1 activator (e.g., MCOPPB) downregulated Collal and Fnl expression, while anti-ORLl (e.g., [Nphel]Nociceptin(l-13)NH2) diminished MCOPPB’s downregulation of Collal and Fnl expression.

[0022] FIG. 5. Depicts imaging of western blotting for extracts produced by H9C2 cells exposed to various conditions. Cellular responses to increasing concentrations of Norepinephrine (NE) was measured by the extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation (P-ERK1/2). Cellular responses to increasing concentrations of MCOPPB was measured by the ERK1/2 phosphorylation. Cellular responses to increasing concentrations of MCOPPB in the presence of lOOnM NE was measured by phosphoERKl/2. The efficacy of NE to mediate ERK1/2 phosphorylation decreased when MCOPPB was co-applied. The antibodies anti-ERKl/2 (Cell Signaling, #4695) and phospho-ERKl/2 (Cell Signaling, #4370) were used.

[0023] FIG. 6. Depicts imaging of western blotting for extracts produced by H9C2 cells exposed to various conditions. H9C2 cells were untreated or treated with VEGF (lOOnM) or MCOPPB (0.5pM) in the presence or absence of anti-ORLl (10pM). Cellular material was solubilized and immunoblotted with anti-phospho-tyrosine VEGF receptor antibody (Sigma- Aldrich, #SAB4504567). MCOPPB induced tyrosine phosphorylation in VEGF receptors, which was reduced by anti-ORLl. [0024] FIGs. 7A-7J. Show the effects of treatment with ORL1 activator over time on various cardiac characteristics in rats after myocardial infarction (MI). Three weeks after MI was induced via LAD coronary artery ligation, twelve Sprague Dawley rats (6 male and 6 female) began intraperitoneal treatment with either an ORL1 activator (e.g., 2.5 mg/kg of MCOPPB) or saline (6 rats per treatment condition). Treatment continued 5 days per week for 3 months. Data points were collected at various time points for each test condition: day 0 (e.g., before MI), day 3 (e.g., three days after MI), day 21 (e.g., three weeks after MI and before initial treatment), day 49 (e.g., one month after initial treatment), day 77 (e.g., two months after initial treatment), and day 105 (e.g., three months after initial treatment).

[0025] FIGs. 7A-7B. Show the effects of ORL1 activator (e.g., MCOPPB) treatment over time on ejection fraction (EF) the rats after myocardial infarction (MI). 7A) Depicts a chart showing average ejection fraction (Y axis, in percent) in the rats at various time points for each test condition (X axis). Individual ejection fractions were determined via echocardiography at days 0, 3, 21, 49, 77, and 105, and then averaged for each test condition. Then, the significance of the difference between the average EF for each test condition at day 105 was determined via a two- tailed ANOVA analysis with a Bonferroni post hoc test. 7B) Depicts a chart showing averaged individual relative percent changes in EF (Y axis, in percent) for the rats at various time points for each test condition (X axis). Individual EFs were determined via echocardiography at days 0, 21, 49, 77, and 105. Then, individual percent changes in EF for each time point were determined relative to EF at day 21, and then the significance of the difference between the averaged relative individual percent changes in EF for each test condition at day 105 was determined via a two- tailed ANOVA analysis with a Bonferroni post hoc test. The results showed that ORL1 activator (e.g., MCOPPB) administration increased ejection fraction after MI, and thus improved post-MI cardiac function in rats.

[0026] FIGs. 7C-7D. Show the effects of ORL1 activator (e.g., MCOPPB) treatment over time on systolic wall thickness in the rats after MI. 7C) Depicts a chart showing average systolic interventricular septum (IVS) thickness (Y axis, in millimeters) in the rats at various time points for each test condition (X axis). Systolic IVS thickness in each rat was determined via echocardiography at days 0, 3, 21, 49, 77, and 105, and then the significance of the difference between the average systolic IVS thickness for each test condition at day 105 was determined via two-tailed ANOVA analysis with a Bonferroni post hoc test. 7D) Depicts a chart showing average systolic left ventricular (LV) posterior wall thickness (Y axis, in millimeters) in the rats at various time points for each test condition (X axis). Systolic LV posterior wall thickness in each rat was determined via echocardiography at days 0, 3, 21, 49, 77, and 105, and then the significance of the difference between the average systolic LV posterior wall thickness for each test condition at day 105 was determined via a two-tailed ANOVA analysis with a Bonferroni post hoc test. The results showed that ORL1 activator (e.g., MCOPPB) administration increased both systolic IVS thickness and systolic LV posterior wall thickness after MI, and thus induced cardiac hypertrophy. Physiological hypertrophy maintains cardiac function over time, whereas pathological hypertrophy is accompanied by cardiac dysfunction and eventually develops heart failure. Since cardiac function was demonstrated to improve in FIGs. 7A-7B, the hypertrophy demonstrated in FIGs. 7C-7D was demonstrated to be physiological, not pathological.

[0027] FIG. 7E. Shows the effects of ORL1 activator (e.g., MCOPPB) treatment over time on end-systolic LV volume in the rats after MI, by depicting a chart showing average end-systolic LV volume (Y axis, in microliters) in the rats at various time points for each test condition (X axis). End-systolic LV volume in each rat was determined via echocardiography at days 0, 3, 21, 49, 77, and 105, and then the significance of the difference between the average end-systolic LV volume for each test condition at day 105 was determined via a two-tailed ANOVA analysis with a Bonferroni post hoc test. The results showed that ORL1 activator (e.g., MCOPPB) administration decreased end-systolic LV volume after MI, and thus improved post-MI cardiac contractility, which resulted in improved cardiac function, in rats.

[0028] FIG. 7F. Shows the effects of ORL1 activator (e.g., MCOPPB) treatment over time on LV myocardial wall fibrosis in the rats after MI, by depicting a chart showing average LV myocardial wall fibrosis (Y axis, in microliters) in the rats for each test condition (X axis). LV myocardial wall fibrosis in each rat was determined via Masson-Trichrome staining of collagen in selected myocardial tissue samples harvested at day 105. The heart was then cut transversally and sectioned with 2 (2- to 3 -mm) slices obtained, 1 immediately cephalad and 1 immediately caudad to the transverse centerline of the infarct region, which was readily identifiable by gross inspection. After paraffin embedding of these slices, 22 sections per animal (at a 120-pm interval between each section) obtained as described earlier were stained with Masson's tri chrome. The fibrotic area (blue) and the nonfibrotic region (red) were outlined using Adobe Photoshop CS5 software, and then quantified with MATLAB and Simulink software (MathWorks, Inc). The total area of fibrosis was calculated as: (total of blue pixels from all sections/total of blue plus red pixels from all sections). Then, the significance of the difference between the averaged LV myocardial wall fibrosis results for each test condition was determined via a two-tailed t-test analysis. The results showed that ORL1 activator (e.g., MCOPPB) administration decreased the extent of LV myocardial wall fibrosis after MI; since fibrosis induces pathological changes in the heart, 0RL1 activator administration thus reduced the risk of heart failure in rats.

[0029] FIGs. 7G-7H. Show the effects of ORL1 activator (e.g., MCOPPB) treatment over time on cardiomyocyte diameters in the rats after MI. Cardiomyocyte diameters from each rat were determined via Masson-Trichrome staining of collagen in the same myocardial tissue samples as FIG. 7F. 20 cardiomyocytes were counted per sample region at x400 magnification. 7G) Depicts a chart showing average cardiomyocyte diameter (Y axis, in micrometers) in the rats from each sample region for each test condition (X axis). The significance of the difference between the average cardiomyocyte diameter for each test condition was determined via a two-tailed t-test analysis. 7H) Shows an image depicting the three sample regions. The results showed that ORL1 activator (e.g., MCOPPB) administration increased cardiomyocyte diameter after MI. Physiological hypertrophy maintains cardiac function over time, whereas pathological hypertrophy is accompanied by cardiac dysfunction and eventually develops heart failure. Since cardiac function was demonstrated to improve in FIGs. 7A-7B, the cardiomyocyte hypertrophy demonstrated in FIG. 7H was physiological, not pathological.

[0030] FIGs. 7I-7J. Show the effects of ORL1 activator (e.g., MCOPPB) treatment over time on myocardial angiogenesis in the rats after MI. Myocardial angiogenesis in each rat was determined via CD31 immunohistochemical staining (e.g., with R&D Systems AF3628) of the same myocardial tissue samples as FIG. 7F. After staining, the number of capillary vessels were counted per sample region at x200 magnification. 71) Depicts a chart showing the average number of vessels (Y axis) per counting field for each test condition (X axis). The significance of the difference between the averaged CD31 positivity for each test condition was determined via a two- tailed t-test analysis. 7J) Shows representative counting field images for each test condition. The results showed that the most CD31 -positive cells were found in the peri -infarct regions, as well as in the rats treated with MCOPPB. The results further showed that ORL1 activator (e.g., MCOPPB) administration increased angiogenesis after MI, and thus improved post-MI cardiac function in rats.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0031] As used herein, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method. [0032] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0033] The phrase “and/or” means “and” or “or”. To illustrate, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and/or” operates as an inclusive or.

[0034] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0035] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the disclosure.

[0036] Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and/or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0037] The term “cardiac medical condition” as used herein refers to any medical condition that affects heart tissue, including that affects heart function.

[0038] As used herein, the term “therapeutically effective amount” is synonymous with “effective amount”, “therapeutically effective dose”, and/or “effective dose” and refers to the amount of compound that will elicit the biological, cosmetic or clinical response being sought by the practitioner in an individual in need thereof. The appropriate effective amount to be administered for a particular application of the disclosed methods can be determined by those skilled in the art, using the guidance provided herein. For example, an effective amount can be extrapolated from in vitro and in vivo assays as described in the present specification. One skilled in the art will recognize that the condition of the individual can be monitored throughout the course of therapy and that the effective amount of a compound or composition disclosed herein that is administered can be adjusted accordingly. [0039] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0040] As used herein, the term “individual” or “subject” generally refers to an individual in need of a therapy for a liver condition. The individual can be a mammal, such as a human, dog, cat, horse, pig or rodent. The individual can be one that has or is suspected of having or at risk for having a liver disease or liver medical condition. The individual may have a disease or be suspected of having the disease. The individual may be asymptomatic. The individual may be of any gender or biological sex.

[0041] As used herein, the terms “treatment,” “treat,” or “treating” refers to intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of pathology of a liver disease or liver medical condition. Treatment may serve to accomplish one or more of various desired outcomes, including, for example, preventing occurrence or recurrence of disease, alleviation of symptoms, and diminishment of any direct or indirect pathological consequences of the disease, lowering the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0042] Embodiments of the disclosure include methods and compositions for the therapy or prevention of any cardiac medical condition.

I. Embodiments of Methods

[0043] Embodiments of the present disclosure are directed to methods and/or compositions related to therapy and/or prevention of one or more cardiac-related medical conditions. Certain embodiments relate to reversal of a cardiac medical condition (or improvement of at least one symptom thereof), including at least aortic stenosis, arrhythmia, cardiac disease, cardiomyopathy, cardiotoxicity, cerebrovascular accident, chronic obstructive pulmonary disease, heart failure, congestive heart failure, ischemic heart disease, myocardial infarction, coronary artery disease, coronary pulmonale, inflammatory heart disease, inflammatory cardiomegaly, myocarditis, congenital heart disease, rheumatic heart disease, cardiac systolic dysfunction, cardiac diastolic dysfunction, angina, dilated cardiomyopathy, idiopathic cardiomyopathy, diabetic cardiomyopathy, a genetic defect, hypertension, kidney disease, low baseline left ventricular ejection fraction, low platelet count, male gender, mitral regurgitation, obstructive hypertrophic cardiomyopathy, obesity, old age, peripheral vascular disease, valvular disease, viral myocarditis, or other conditions resulting in cardiac fibrosis, for example.

[0044] In some embodiments, the cardiac-related medical condition is the result of one or more genetic defects in the individual. Genetic defects may be caused by an atrial septal defect, atrioventricular septal defect, coarctation of the aorta, complete atrioventricular canal defect (CAVC), double-outlet right ventricle, D-transposition of the great arteries, Ebstein’s anomaly, hypoplastic left heart syndrome, I-transposition of the great arteries, interrupted aortic arch, patent ductus arteriosis (PDA), pulmonary atresia, pulmonary valve stenosis, single ventricle, tetralogy of Fallot, total anomalous pulmonary venous return, tricuspid atresia, truncus arteriosus, or ventricular septal defect, for example.

[0045] In particular aspects of the disclosure, any cardiac medical condition that induces heart failure may be the cardiac medical condition to be treated. The cardiac medical condition may be caused by one or more of a variety of characteristics, including, for example, long-term high blood pressure; excess cholesterol, partial or full blockage of a heart vessel, heart valve problems; heart tissue damage (such as from one or more previous heart attack(s) or chronic or acute and/or recurrent episodes or sequelae of ischemic heart disease); chronic rapid heart rate; metabolic disorders, such as thyroid disease or diabetes; nutritional deficiencies of essential vitamins or minerals, such as thiamin (vitamin B-l), selenium, calcium and/or magnesium; pregnancy; alcohol abuse; drug abuse, including of narcotics or prescription drugs, such as cocaine or antidepressant medications, such as tricyclic antidepressants; use of some chemotherapy drugs to treat cancer (including Adriamycin); certain viral infections; side effects of a drug and/or vaccine, hemochromatosis and/or an unknown cause or undetected cause, i.e. idiopathic cardiomyopathy.

[0046] In some cases, methods and compositions of the present disclosure are employed for treatment or prevention of one or more cardiac medical conditions or delay of onset of one or more cardiac medical conditions and/or reduction of extent and/or delay of onset of one or more symptoms of one or more cardiac medical conditions. In particular cases, such prevention, delay or onset, or reduction of extent of one or more symptoms, occurs in an individual that is at risk for a cardiac medical condition. Exemplary risk factors include one or more of the following: age, gender (male, although it occurs in females), high blood pressure, high serum cholesterol levels, tobacco use (including smoking), excessive alcohol consumption, sugar consumption, family and/or personal history, obesity, lack of physical activity, psychosocial factors, diabetes mellitus, overweight, genetic predisposition, and/or exposure to air pollution.

[0047] In some embodiments, the subject in need of treatment is an adult. In some cases, the adult is at least 65 years of age. In other cases, the subject is at least 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0048] In some cases, the subject in need of treatment is 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-25, or 20-25 years old. In other cases, the subject in need of treatment is 25-100, 25-90, 25-80, 25-70, 25-60, 25-50, 25-40, or 25-30 years old. In other cases, the subject in need of treatment is 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, or 30-35 years old. In other cases, the subject in need of treatment is 35-100, 35-90, 35-80, 35-70, 35-60, 35-50, or 35-40 years old. In other cases, the subject in need of treatment is 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, or 40-45 years old. In other cases, the subject in need of treatment is 45-100, 45-90, 45-80, 45-70, 45-60, or 45-50 years old. In other cases, the subject in need of treatment is 50-100, 50-90, 50-80, 50-70, 50-60, or 50-55 years old. In other cases, the subject in need of treatment is 55-100, 55-90, 55-80, 55-70, or 55-60 years old. In other cases, the subject in need of treatment is 60-100, 60-90, 60-80, 60-70, or 60-65 years old. In other cases, the subject in need of treatment is 65-100, 65-90, 65-80, or 65-70 years old. In other cases, the subject in need of treatment is 70-100, 70-90, 70-80, or 70-75 years old. In other cases, the subject in need of treatment is 75-100, 75-90, or 75-80 years old. In other cases, the subject in need of treatment is 80-100, 80-90, or 80-85 years old. In other cases, the subject in need of treatment is 85-100, or 85-90 years old. In other cases, the subject in need of treatment is 90-100, 90-95, or 95-100 years old.

[0049] In some embodiments, the subject in need of treatment is an adolescent (i.e. 13-19 years old) that may or may not have a cardiac genetic defect. In other embodiments, the subject in need of treatment is a child (i.e. 1-12 years old) that may or may not have a cardiac genetic defect. In other embodiments, the subject in need of treatment is an infant (i.e. younger than 1 year old) that may or may not have a cardiac genetic defect. In cases wherein the subject is less than about 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1 year old, the subject may have a cardiac genetic defect.

[0050] Particular aspects of the disclosure concern delivery of at least one polynucleotide (including a gene), small molecule, peptide, polypeptide, shRNA polynucleotide, siRNApolynucleotide, and so forth to cardiac tissue for treatment of certain cells in the tissue. In specific embodiments, a nucleic acid is the active agent, whereas in some embodiments a polypeptide produced from the nucleic acid is the active agent. The tissue may be of any kind, but in specific cases it is cardiac muscle and/or scar tissue, including scar tissue in and/or on the heart. [0051] Particular aspects for such embodiments result in reversal of one or more cardiac medical conditions. Certain aspects for such embodiments result in improvement of at least one symptom of a cardiac medical condition. In exemplary embodiments, the cardiac medical condition is heart failure. The heart failure may be the result of one or more causes, including coronary artery disease and heart attack, high blood pressure, faulty heart valves, cardiomyopathy (such as caused by disease, infection, alcohol abuse and the toxic effect of drugs, such as cocaine or some drugs used for chemotherapy), idiopathic cardiomyopathy and/or genetic factors.

[0052] Particular but exemplary indications of embodiments of the disclosure include at least applications for 1) heart failure, including congestive heart failure; 2) prevention of ventricular remodeling; and/or 3) cardiomyopathy. Other indications may also include coronary artery disease, ischemic heart disease, valvular heart disease, etc.

[0053] In cases where the individual has cardiomyopathy, the cardiomyopathy may be ischemic or non-ischemic cardiomyopathy. The cardiomyopathy may be caused by long-term high blood pressure, heart valve problems, heart tissue damage from a previous heart attack, chronic rapid heart rate, metabolic disorders, nutritional deficiencies, pregnancy, alcohol abuse, drug abuse, chemotherapy drugs, viral infection, hemochromatosis, genetic condition, elevated cholesterol levels, or a combination thereof, as examples only. The cardiomyopathy may also have no identified cause, i.e. idiopathic cardiomyopathy.

[0054] In cases where the individual has hypertrophy, the hypertrophy may be pathological or physiological. In cases where the individual has pathological hypertrophy, the method of treatment may inhibit the pathological hypertrophy. In cases where the individual has pathological hypertrophy, the method of treatment may induce the physiological hypertrophy.

[0055] In certain embodiments, the method of treatment may suppress fibrosis in the individual. The fibrosis may be of the lung (i.e., pulmonary fibrosis), liver (i.e., cirrhosis), heart (e.g., ventricular fibrosis), lymph nodes (i.e., mediastinal fibrosis), soft tissue in the retroperitoneum (i.e., retroperitoneal cavity fibrosis), bone marrow (i.e., myelofibrosis), skin (i.e., keloid), scleroderma, and/or systemic sclerosis.

[0056] In certain embodiments, the method of treatment may induce angiogenesis, prevent heart failure progression, and/or attenuate or prevent hypertension progression in the individual.

[0057] Although in particular embodiments an individual is treated in an in vivo or in situ manner, in alternative embodiments the individual is treated with compositions encompassed by the disclosure in an ex vivo manner. In such embodiments, cells that are to be subjected to nucleic acid composition(s) of the disclosure are either obtained from the individual or are obtained from another individual. Such cells may be subjected in vitro to the nucleic acid compositions such that they are uptaken by the cells, and the cells are then delivered to the individual to be treated.

[0058] In particular aspects, an individual is provided with an additional cardiac medical condition therapy.

A. ORL1 and Related Compositions

Opioid receptor like-1 encodes the nociception opioid peptide receptor (NOP) protein. NOP belongs to the opioid receptor family of G-protein coupled receptors (GPCRs). The neuropeptide N/OFQ, otherwise known as nociception or orphanin FQ, is an endogenous ligand for NOP. MCOPPB is an agonist for NOP and thus acts as an ORL1 -activating agent (Table 1).

TABLE 1

B. Masson Trichrome Staining

[0059] Fixed tissues may be paraffin embedded, sectioned (5pm), and stained with Masson Trichrome. Masson Trichrome is commonly used to differentiate collagen (stained blue-green) from surrounding muscle cells (stained red). Briefly, slides were stained with Weigert's iron hematoxylin followed by Ponceau acid fuchsin. After treatment with phosphomolybdic- phosphotungstic acid slides were stained with Light Green in acetic acid. Whole slides were scanned at 20* (Aperio Scanscope, Leica Biosystems Inc., Buffalo Grove, Ill.). Aperio ImageScope and Adobe Photoshop (Adobe Systems Inc., San Jose, Calif.) CS6 software was used to analyze the entire section on each slide. The quantity of blue-green pixels as a proportion of total pixels was used to determine percent (%) collagen as previously described (Jayes et al. 2016, Brunengraber et al. 2014).

C. Indo-1 Fluorescence Staining

[0060] Indo-1 is a fluorescent calcium indicator that enables the measurement of intracellular calcium levels. Cells can be loaded with the Indo-1, and then calcium levels can be measured via flow cytometry.

II. Administration of Therapeutic Compositions

[0061] The therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first cardiac medical condition therapy and a second cardiac medical condition therapy. The therapies may be administered in any suitable manner known in the art. For example, the first and second cardiac medical condition treatment may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the first and second cardiac medical condition treatments are administered in a separate composition. In some embodiments, the first and second cardiac medical condition treatments are in the same composition. [0062] In some embodiments, the first cardiac medical condition therapy and the second cardiac medical condition therapy are administered substantially simultaneously. In some embodiments, the first cardiac medical condition therapy and the second cardiac medical condition therapy are administered sequentially. In some embodiments, the first cardiac medical condition therapy and the second cardiac medical condition therapy, and a third therapy are administered sequentially. In some embodiments, the first cardiac medical condition therapy is administered before administering the second cardiac medical condition therapy. In some embodiments, the first cardiac medical condition therapy is administered after administering the second cardiac medical condition therapy.

[0063] Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0064] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the cardiac medical condition therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0065] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0066] In some embodiments, the cardiac medical condition therapy may comprise a therapeutically effective amount of one or more ORL1 -activating agents or a pharmaceutically acceptable salt thereof, a vector comprising the nucleic acid encoding for the one or more ORL1- activating agents, or a cell comprising the one or more ORL1 -activating agents, a nucleic acid encoding for the one or more ORL1 -activating agents. In some embodiments, a single dose of the one or more OR 1 -activating agents is administered. In some embodiments, multiple doses of the one or more OR 1 -activating agents are administered. In some embodiments, the one or more ORL1 -activating agents is administered at a dose of between 1 mg/kg and 5000 mg/kg. In some embodiments, the one or more OR 1 -activating agents is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17. 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,

52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,

78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, , 96, 97, 98, 99, 100, 101, 102,

103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,

122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,

141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,

160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,

179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,

198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,

217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,

236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,

255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,

274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,

293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,

312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,

331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,

350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,

369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,

388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,

407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,

426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,

445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,

464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,

483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501,

502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,

521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539,

540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558,

559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 1100, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg/kg, or any range derivable therein.

[0067] In some embodiments, a single dose of the one or more ORL1 -activating agents is administered. In some embodiments, multiple doses of the one or more ORL1 -activating agents are administered. In some embodiments, the one or more ORL1 -activating agents is administered at a dose of between 1 mg/kg and 100 mg/kg. In some embodiments, the one or more ORL1- activating agents is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg/kg, or any range derivable therein.

[0068] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.

[0069] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM, or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0070] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0071] It will be understood by those skilled in the art and made aware that dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of pg/ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0072] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.

[0073] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0074] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0075] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0076] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0077] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0078] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile inj ectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0079] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0080] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

A. Other Agents

[0081] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that improve cardiac function.

B. Polypeptide Expression

[0082] In some aspects, there are nucleic acid molecule encoding polypeptides or peptides of the disclosure (e.g TCR genes). These may be generated by methods known in the art, e.g., isolated from B cells of mice that have been immunized and isolated, phage display, expressed in any suitable recombinant expression system and allowed to assemble to form antibody molecules or by recombinant methods.

C. Expression

[0083] The nucleic acid molecules may be used to express large quantities of polypeptides. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for humanization of the TCR genes.

D. Vectors

[0084] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, alpha chain, beta chain, or the antigen-binding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0085] To express the polypeptides or peptides of the disclosure, DNAs encoding the polypeptides or peptides are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences. In some aspects, a vector that encodes a functionally complete human CH or CL immunoglobulin or TCR sequence with appropriate restriction sites engineered so that any variable region sequences can be easily inserted and expressed. In some aspects, a vector that encodes a functionally complete human TCR alpha or TCR beta sequence with appropriate restriction sites engineered so that any variable sequence or CDR1, CDR2, and/or CDR3 can be easily inserted and expressed. Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using the same are well known in the art.

E. Expression Systems

[0086] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.

F. Methods of Gene Transfer

[0087] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patents 5,610,42; 5,322,783, 5,563,55, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,55, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

G. Host Cells

[0088] In another aspect, contemplated are the use of host cells into which a recombinant expression vector has been introduced. Antibodies can be expressed in a variety of cell types, as examples. An expression construct encoding an antibody can be transfected into cells according to a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. In certain aspects, the antibody expression construct can be placed under control of a promoter that is linked to T-cell activation, such as one that is controlled by NFAT-1 or NF- KB, both of which are transcription factors that can be activated upon T-cell activation. Control of antibody expression allows T cells, such as tumor- targeting T cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0089] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts.

H. Isolation

[0090] The nucleic acid molecule encoding either or both of the entire heavy, light, alpha, and beta chains of an antibody or TCR, or the variable regions thereof may be obtained from any source that produces antibodies. Methods of isolating mRNA encoding an antibody are well known in the art. See e.g., Sambrook et al., supra. The sequences of human heavy and light chain constant region genes are also known in the art. See, e.g., Kabat et al., 1991, supra. Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed in a cell into which they have been introduced and the antibody isolated.

III. General Pharmaceutical Compositions

[0091] In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects may involve administering an effective amount of a composition to a subject. In some embodiments, an antibody or antigen binding fragment capable of binding to [protein of interest] may be administered to the subject to protect against or treat a condition (e.g., cancer). Alternatively, an expression vector encoding one or more such antibodies or polypeptides or peptides may be given to a subject as a preventative treatment. Additionally, such compositions can be administered in combination with an additional therapeutic agent (e.g., a chemotherapeutic, an immunotherapeutic, a biotherapeutic, etc.). Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0092] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0093] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0094] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0095] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0096] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [0097] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile inj ectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0098] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0099] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

IV. Combination Therapy

[0100] In certain cases, the therapy of the present disclosure is utilized in conjunction with one or more other therapies for a cardiac medical condition. The one or more other therapies may be directly or indirectly related to the cardiac medical condition (examples of indirectly related therapies include those for pain or infection). In specific embodiments, the additional therapy related to the cardiac medical condition is drug therapy, surgery, coronary bypass, heart valve repair or replacement, an implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) or biventricular pacing, a ventricular assist device (VAD), video assisted thoracotomy (VAT), percutaneous coronary intervention (PCI) and/or heart transplant.

[0101] In specific embodiments, one or more agents that prevent fibrosis and/or enhance or promote angiogenesis may be used as adjuncts to embodiments of the disclosure. They may be provided to an individual in a localized region of the heart, including a region that has tissue damage, loss of cardiomyocyte, scar tissue, and so forth, or they may be provided systemically. The one or more agents may be any composition suitable to facilitate angiogenesis in the desired region. In specific embodiments, the agent may be a protein, peptide, small molecule, nucleic acid, and so forth. Embodiments such as those described in US2003/0103943 or US2001/0041679 may be employed in conjunction with the methods of the disclosure. Specific embodiments include Gata4, fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); angiopoi etins, Angl and Ang2; matrix metalloproteinases (MMP); Delta-like ligand 4 (DIM); Ets variant 2 (ETV2); or peptides thereof; or combinations thereof. ITD-1 is a small molecule that inhibits TGF- beta and thus, fibrosis and cardiac remodeling (Willems E, Cabral-Teixeira J, Schade D, et al. Cell Stem Cell. 2012. pp. 242-252), and it may be utilized. Specific embodiments also include Mef2c, TBX5, Hand2, myocardin, Mesoderm posterior protein 1 (Mespl), miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, 5-azacytidine, Zebularine, or peptides thereof, or combinations thereof.

[0102] The therapy of the present disclosure may precede or follow the other agent treatment by intervals ranging from minutes to hours to days to weeks or months. In embodiments where the other agent and the instant therapy are applied separately to the individual, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapy of the disclosure and the additional therapy would still be able to exert an advantageously combined effect on the individual. In such instances, it is contemplated that one may contact the individual with both modalities simultaneously or within minutes of each other or within about 1-12, 6-12, or 12-24 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0103] In specific embodiments, the therapy of the present disclosure and the additional therapy are provided at the same time or at different times. The separate entities may be within the same compositions or they may be comprised in separate compositions. In cases wherein the therapy of the present disclosure and the second therapy are provided at different times, they may be separated by any suitable range in times, such as minutes, hours, days, or weeks. In embodiments wherein they are provided separately, the order of delivery of two (or more) therapies may be of any suitable order, including delivery of the at least one or more ORL1 -activating agent prior to or subsequent to another therapy.

[0104] Examples of other treatments to be employed with the therapy of the disclosure includes one or more of the following: Angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor plus neprilysin inhibitors (ARNIs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, diuretics, potassium, magnesium, potassium-sparing diuretics or aldosterone antagonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, Digoxin or Lanoxin or digitalis, hydralazine and isosorbide dinitrate (BiDil), Vericiguat or Verquvo or an oral soluble guanylate cyclase (sGC) stimulator, positive inotropes or inotropic therapy, vasodilators, anticoagulant medication, aspirin, nitrates, statins, and/or blood thinners.

EXAMPLES

[0105] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow present techniques discovered by the inventors to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

EXAMPLE 1

[0106] Heart failure (HF) is a major cardiovascular disease. Although current main therapeutic strategies, targeting HF -related G-protein-coupled receptor (GPCR) such as angiotensin receptor antagonists markedly improved the outcome of HF, its morbidity and mortality remain high. There is a critical need to explore new therapeutic approaches.

[0107] During the past two decades, several researchers including us, reported that Gata4 overexpression (OE) improved cardiac function after myocardial infarction (MI) in murine heart. In 2007, Penn reported that delivery of Gata4 to the post-infarct rat heart induced beneficial ventricular remodeling and improved left ventricular function. In 2010, Ruskoaho reported that the hearts treated by adenovirus expressing Gata4 improved cardiac function after MI in rat heart. It was concluded that Gata4 prevented adverse postinfarction remodeling through myocardial angiogenesis, anti-apoptosis and stem cell recruitment and that Gata4-based gene transfer may represent a novel therapy for HF. In 2017, Rosengart reported that Gata4 OE reduces cardiac fibrosis and improves cardiac function after MI in rat. In 2020, Ruskoaho reported that Gata4 reduced Angiotensin Il-induced remodeling in rat heart by interfering with pro-fibrotic and hypertrophic gene expressions. Despite those positive reports, Gata4 OE has never been pursued as a HF treatment. It was speculated that because Gata4 induces cardiac hypertrophy, and prolonged hypertrophy eventually introduces HF, Gata4 OE is not an option for HF treatment even though it improves cardiac function for a short time. Recent HF research instead focuses on downregulation of Gata4 by post-translational modification.

[0108] After Rosengart reported that Gata4 OE suppressed HF in the post-infarct rat heart, the effects of Gata4 OE in vitro were further investigated. RNAseq analysis revealed that Pnoc gene was significantly upregulated by Gata4 OE in cardiac fibroblasts. Pnoc is a preproprotein and proteolytically processed to generate nociceptin that binds to ORL1. ORL1 is the most recently discovered member of the opioid receptor family. Pnoc and ORL1 have been intensely investigated in neuroscience field. The activated ORL1 is shown to modulate calcium channel in neural cells. Preliminary experiments confirmed that Gata4 OE inhibits calcium influx (FIG. 1) and dephosphorization of NF AT (FIG. 2) in H9C2 cells. Thus, Gata4 OE suppresses NF AT pathway, resulting in downregulation of NF AT downstream genes, NPPA and NPPB in H9C2 cells (FIG. 3 A). The downregulation of NPPA and NPPB by Gata4 OE were diminished by ORL1 antagonist administration (FIG. 3 A). Next it was confirmed that nociceptin agonist, MCOPPB, administration induced the same downregulation of NPPA and NPPB in H9C2 cells (FIG. 3B). MCOPPB also downregulated fibrogenic genes, Collal and Fnl, in cardiac fibroblasts, and ORL1 antagonist diminished the downregulation (FIG. 4).

[0109] ORL1, a GPCR, belongs to an opioid family, has a modulatory role on other opioid GPCRs. Vilardaga et al. (Nat Chem Biol. 2008;4: 126-131) reported that there is a cross-talk between alfa 2-adrenagic and u-opioid receptors and morphine receptor activation caused alfa2- adrenagic receptor’s inactivation.⁵ Preliminary experiments were performed to explore if crosstalk exists between ORL1 and alfa2-adrenagic receptor. ORL1 induced inhibition of ERK1/2 activation which alfa2-adrenagic receptor activated (FIG. 5). Therefore, it seems possible that ORL1 activation suppresses pathological hypertrophy not only by NF AT pathway downregulation but also by hypertrophic hormone receptor inactivation such as alfa 2-adrenagic, ET-1, and Agll receptors.

[0110] In addition, many reports indicate that GPCRs and receptor tyrosine kinases (RTKs) have intense cross communication. IGF1 receptor is an RTK and IGF1 is known as an inducer of physiological hypertrophy. The preliminary experiment showed that ORL1 activates IGF1 receptor (FIG. 6). Thus, it is possible that ORL1 not only inhibits pathological hypertrophy but also induce physiological hypertrophy. RTK, VEGFR experiments were performed to explore if ORL1 activation induces VEGFR activation, considering Ruskoaho’s report in 2010 that Gata4 OE induced angiogenesis and anti-apoptosis.² The data suggest that ORL1 activates VEGF receptor (FIG. 7). [OHl] Based on the preliminary data, an in vivo study was performed to investigate if ORL1 activation inhibits HF progression in a long-term follow-up. Sprague Dawley rats (6 males and 6 females) underwent LAD coronary artery ligation. Three weeks later, normal saline or MCOPPB (ORL1 activator, 2.5mg/kg) intraperitoneal injection was started, and continued 5 days a week, for 3 months. Echocardiographic data showed that ejection fraction significantly improved in MCOPPB group (FIGS. 7A-7B). Echocardiographic data also showed that end-systolic interventricular septum thickness and end-systolic LV posterior wall thickness increased in MCOPPB group (FIGS. 7C-7D). End-systolic LV ventricular volume measured by echocardiography significantly decreased in MCOPPB group (FIG. 7E). Fibrosis area, stained by Masson-Trichrome, significantly decreased in MCOPPB group (FIG. 7F). Cardiomyocyte diameter measured with the Masson-Trichrome stained slides, significantly increased in MCOPPB group (FIGS. 7G-7H). Angiogenesis was assessed by CD31 staining and MCOPPB group had significantly higher vessel counts in border zone (FIGS. 7I-7J).

[0112] Thus, the in vivo experiments demonstrate that activated ORL1 inhibits pathological hypertrophy, induces physiological hypertrophy, suppresses fibrosis, and induces angiogenesis, resulting in attenuating HF. Activation of ORL1 may be a novel therapy to prevent HF progression.

[0113] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosure as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. REFERENCES

[0114] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0115] Bian J, Popovic ZB, Benejam C, Kiedrowski M, Rodriguez LL, Penn MS. Effect of cell-based intercellular delivery of transcription factor Gata4 on ischemic cardiomyopathy. Circ Res. 2007;100: 1622-1633. PMID: 17495229. DOI: 10.1161/01.RES.000026978.75877.68.

[0116] Rysa J, Tenhunen O, Serpi R, Soini Y, Nemer M, Leskinen H, Ruskoaho H. Gata-4 is an angiogenic survival factor of the infarcted heart. Circ Heart Fail. 2010;3:440-450. PMID: 20200331. DOI: 10.1161/CIRCHE ARTFAILURE.109.889642.

[0117] Mathison M, Singh VP, Sanagasetti D, Yang L, Pinnamaneni JP, Yang J, Rosengart TK. Cardiac reprogramming factor Gata4 reduces postinfarct cardiac fibrosis through direct repression of the profibrotic mediator snail. J Thorac Cardiovasc Surg.2017;154: 1601-1610. PMID: 28711329. DOI: 10.1016/j.jtcvs.2017.06.35.

[0118] Acosta AJ, Rysa J, Szabo Z, Moilanen A-M, Serpi R, Ruskoaho H. Phosphorylation of GATA4 at serine 105 is required for left ventricular remodelling process in angiotensin Il-induced hypertension in rats. Basic Clin Pharmacol Toxicol. 2020;127: 178-195. PMID: 32060996. DOI: 10.1111/bcpt.13398.

[0119] Vilardaga J-P, Nikolaev VO, Lorenz K, Ferrandon S, Zhuang Z, Lohse MJ. Conformational cross-talk between alpha2A-adrenergic and mu-opioid receptors controls cell signaling. Nat Chem Biol. 2008;4: 126-131. PMID: 18193048. DOI: 10.1038/nchembio.64.

Claims

CLAIMS What is claimed is:

1. A method of treating a cardiac medical condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more compositions, wherein the one or more compositions comprise one or more 0RL1 -activating agents or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the one or more compositions are a small molecule, a peptide, a protein, a biologic, a carbohydrate, and/or a nucleic acid.

3. The method of any one of claims 1 and 2, wherein the at least one or more 0RL1- activating agents comprise (R)-Ro65-6570, [(pF)Phe4]N/OFQ-(l-13)-NH2, [3H]Leu-N/OFQ, [3H]N/OFQ, [3H]Tyrl4-N/OFQ, [Argl4Lysl5]N/OFQ, [F/G]N/OFQ-(1-13)-NH2, Ac- RYYRIK-NH2, Ac-RYYRWK-NH2, AT-121, AT-403, BU08028, buprenorphine, buprenorphine hydrochloride, cebranopadol, etorphine, JNJ-19385899, MCOPPB, MCOPPB trihydrochloride hydrate, MT-7716, N/OFQ-(1-13)-NH2, N/0FQ-NH2, NNC 63-0532, nociceptin/orphanin FQ (N/OFQ), norbuprenorphine, PWT2-N/OFQ, Ro64-6198, Ro6570, SCH221510, SCH486757, SerlOO (ZP120), SR16835, SR16435, TH-030418, UFP-102, and/or UFP-112.

4. The method of any one of claims 1 to 3, wherein the at least one or more ORL1- activating agents comprise MCOPPB.

5. The method of any one of claims 1 to 4, wherein the cardiac medical condition induces heart failure, and wherein the cardiac medical condition is with aortic stenosis, arrhythmia, cerebrovascular accident, chronic obstructive pulmonary disease, cigarette smoking, congenital heart disease, diabetic cardiomyopathy, dilated cardiomyopathy, a genetic defect, heart failure, hypertension, ischemic coronary disease, kidney disease, low baseline left ventricular ejection fraction, low platelet count, male gender, mitral regurgitation, myocardial infarction, myocarditis, obstructive hypertrophic cardiomyopathy, obesity, old age, peripheral vascular disease, renal disease, rheumatic heart disease, valvular disease, and/or viral myocarditis.

6. The method of any one of claims 1 to 5, wherein the cardiac medical condition is associated with heart failure.

7. The method of any one of claims 1 to 6, wherein the at least one or more 0RL1- activating agents are provided in a pharmaceutical composition.

8. The method of any one of claims 1 to 7, wherein the at least one or more compositions are delivered systemically.

9. The method of any one of claims 1 to 7, wherein the at least one or more compositions are delivered locally.

10. The method of any one of claims 1 to 9, wherein the at least one or more compositions are provided intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, locally, via inhalation, via injection, via infusion, via continuous infusion, via localized perfusion, via a catheter, and/or via a lavage.

11. The method of any one of claims 1 to 10, wherein the at least one or more compositions are provided as a tablet, troche, lozenge, aqueous suspension, oily suspension, emulsion, hard capsule, soft capsule, and/or syrup.

12. The method of any one of claims 1 to 11, further comprising the step of providing one or more additional therapeutic compositions to the subject in need thereof.

13. The method of any one of claims 1 to 12, wherein the one or more compositions further comprise Gata4, fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); angiopoi etins, Angl and Ang2; matrix metalloproteinase (MMP); Delta-like ligand 4 (DIM); ITD-1; Mef2c, TBX5, Hand2, myocardin, Mesoderm posterior protein 1 (Mespl), miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, 5-azacytidine, Zebularine, and/or or peptides thereof.

14. The method of any one of claims 1 to 13, wherein the one or more compositions further comprise Angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor plus neprilysin inhibitors (ARNIs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, diuretics, potassium, magnesium, potassium-sparing diuretics or aldosterone antagonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, Digoxin or Lanoxin or digitalis, hydralazine and isosorbide dinitrate (BiDil), Vericiguat or Verquvo or an oral soluble guanylate cyclase (sGC) stimulator, positive inotropes or inotropic therapy, vasodilators, anticoagulant medication, aspirin, nitrates, statins, and/or blood thinners.

15. The method of any one of claims 1 to 14, wherein administration of the at least one or more compositions occurs before administration of the at least one or more ORL-1 activating agents.

16. The method of any one of claims 1 to 14, wherein administration of the at least one or more compositions occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

17. The method of any one of claims 1 to 14, wherein administration of the at least one or more compositions occurs after administration of the at least one or more ORL-1 activating agents.

18. The method of any one of claims 1 to 17, wherein the at least one or more compositions are delivered via a same route of administration as the at least one or more ORL-1 activating agents.

19. The method of any one of claims 1 to 17, wherein the at least one or more compositions are delivered via a different route of administration as the at least one or more ORL-1 activating agents.

20. The method of any one of claims 1 to 19, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in one formulation.

21. The method of any one of claims 1 to 19, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in separate formulations.

22. The method of any one of claims 1 to 21, further comprising treatment with at least one or more co-therapies.

23. The method of claim 22, wherein the at least one or more co-therapies further comprise drug therapy, surgery, coronary bypass, heart valve repair or replacement, an implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) or biventricular pacing, a ventricular assist device (VAD), VAT, percutaneous coronary intervention (PCI) and/or heart transplant.

24. The method of any one of claims 22 and 23, wherein administration of the at least one or more co-therapies occurs before administration of the at least one or more ORL-1 activating agents.

25. The method of any one of claims 22 and 23, wherein administration of the at least one or more co-therapies occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

26. The method of any one of claims 22 and 23, wherein administration of the at least one or more co-therapies occurs after administration of the at least one or more ORL-1 activating agents.

27. A method of inhibiting pathological hypertrophy in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of one or more compositions or a pharmaceutically acceptable salt thereof, wherein the one or more compositions comprises at least one or more 0RL1 -activating agents.

28. The method of claim 27, wherein the one or more compositions are a small molecule, a peptide, a protein, a biologic, a carbohydrate, and/or a nucleic acid.

29. The method of 27 or 28, wherein the at least one or more 0RL1 -activating agents comprise (R)-Ro65-6570, [(pF)Phe4]N/OFQ-(l-13)-NH2, [3H]Leu-N/0FQ, [3H]N/0FQ, [3H]Tyrl4-N/OFQ, [Argl4Lysl5]N/OFQ, [F/G]N/OFQ-(1-13)-NH2, Ac-RYYRIK-NH2, Ac- RYYRWK-NH2, AT-121, AT -403, BU08028, buprenorphine, buprenorphine hydrochloride, cebranopadol, etorphine, JNJ-19385899, MCOPPB, MCOPPB trihydrochloride hydrate, MT- 7716, N/OFQ-(1-13)-NH2, N/OFQ-NH2, NNC 63-0532, nociceptin/orphanin FQ (N/OFQ), norbuprenorphine, PWT2-N/OFQ, Ro64-6198, Ro6570, SCH221510, SCH486757, Seri 00 (ZP120), SR16835, SR16435, TH-030418, UFP-102, and/or UFP-112.

30. The method of any one of claims 27 to 29, wherein the at least one or more ORL1- activating agents comprise MCOPPB.

31. The method of any one of claims 27 to 30, wherein the at least one or more ORL1- activating agents are provided in a pharmaceutical composition.

32. The method of any one of claims 27 to 31, wherein the at least one or more compositions are delivered systemically.

33. The method of any one of claims 27 to 31, wherein the at least one or more compositions are delivered locally.

34. The method of any one of claims 27 to 33, wherein the at least one or more compositions are provided intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, locally, via inhalation, via injection, via infusion, via continuous infusion, via localized perfusion, via a catheter, and/or via a lavage.

35. The method of any one of claims 27 to 34, wherein the at least one or more compositions are provided as a tablet, troche, lozenge, aqueous suspension, oily suspension, emulsion, hard capsule, soft capsule, and/or syrup.

36. The method of any one of claims 27 to 35, further comprising the step of providing one or more additional therapeutic compositions to the subject in need thereof.

37. The method of any one of claims 27 to 36, wherein the one or more compositions further comprise Gata4, fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); angiopoi etins, Angl and Ang2; matrix metalloproteinase (MMP); Delta-like ligand 4 (DIM); ITD-1; Mef2c, TBX5, Hand2, myocardin, Mesoderm posterior protein 1 (Mespl), miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, 5-azacytidine, Zebularine, and/or or peptides thereof.

38. The method of any one of claims 27 to 37, wherein the one or more compositions further comprise Angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor plus neprilysin inhibitors (ARNIs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, diuretics, potassium, magnesium, potassium-sparing diuretics or aldosterone antagonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, Digoxin or Lanoxin or digitalis, hydralazine and isosorbide dinitrate (BiDil), Vericiguat or Verquvo or an oral soluble guanylate cyclase (sGC) stimulator, positive inotropes or inotropic therapy, vasodilators, anticoagulant medication, aspirin, nitrates, statins, and/or blood thinners.

39. The method of any one of claims 27 to 38, wherein administration of the at least one or more compositions occurs before administration of the at least one or more ORL-1 activating agents.

40. The method of any one of claims 27 to 38, wherein administration of the at least one or more compositions occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

41. The method of any one of claims 27 to 38, wherein administration of the at least one or more compositions occurs after administration of the at least one or more ORL-1 activating agents.

42. The method of any one of claims 27 to 41, wherein the at least one or more compositions are delivered via a same route of administration as the at least one or more ORL-1 activating agents.

43. The method of any one of claims 27 to 41, wherein the at least one or more compositions are delivered via a different route of administration as the at least one or more ORL-1 activating agents.

44. The method of any one of claims 27 to 43, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in one formulation.

45. The method of any one of claims 27 to 43, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in separate formulations.

46. The method of any one of claims 27 to 45, further comprising treatment with at least one or more co-therapies.

47. The method of claim 46, wherein the at least one or more co-therapies further comprise drug therapy, surgery, coronary bypass, heart valve repair or replacement, an implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) or biventricular pacing, a ventricular assist device (VAD), VAT, percutaneous coronary intervention (PCI) and/or heart transplant.

48. The method of any one of claims 46 and 47, wherein administration of the at least one or more co-therapies occurs before administration of the at least one or more ORL-1 activating agents.

49. The method of any one of claims 46 and 47, wherein administration of the at least one or more co-therapies occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

50. The method of any one of claims 46 and 47, wherein administration of the at least one or more co-therapies occurs after administration of the at least one or more ORL-1 activating agents.

51. A method of inducing physiological hypertrophy in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of one or more compositions or a pharmaceutically acceptable salt thereof, wherein the one or more compositions comprises at least one or more ORL1 -activating agents.

52. The method of claim 51, wherein the one or more compositions are a small molecule, a peptide, a protein, a biologic, a carbohydrate, and/or a nucleic acid.

53. The method of any one of claims 51 and 52, wherein the at least one or more ORL1- activating agents comprise (R)-Ro65-6570, [(pF)Phe4]N/OFQ-(l-13)-NH2, [3H]Leu-N/OFQ, [3H]N/OFQ, [3H]Tyrl4-N/OFQ, [Argl4Lysl5]N/OFQ, [F/G]N/OFQ-(1-13)-NH2, Ac- RYYRIK-NH2, Ac-RYYRWK-NH2, AT-121, AT-403, BU08028, buprenorphine, buprenorphine hydrochloride, cebranopadol, etorphine, JNJ-19385899, MCOPPB, MCOPPB trihydrochloride hydrate, MT-7716, N/OFQ-(1-13)-NH2, N/OFQ-NH2, NNC 63-0532, nociceptin/orphanin FQ (N/OFQ), norbuprenorphine, PWT2-N/OFQ, Ro64-6198, Ro6570, SCH221510, SCH486757, SerlOO (ZP120), SR16835, SR16435, TH-030418, UFP-102, and/or UFP-112.

54. The method of any one of claims 51 to 53, wherein the at least one or more ORL1- activating agents comprise MCOPPB.

55. The method of any one of claims 51 to 54, wherein the at least one or more ORL1- activating agents are provided in a pharmaceutical composition.

56. The method of any one of claims 51 to 55, wherein the at least one or more compositions are delivered systemically.

57. The method of any one of claims 51 to 55, wherein the at least one or more compositions are delivered locally.

58. The method of any one of claims 51 to 57, wherein the at least one or more compositions are provided intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, locally, via inhalation, via injection, via infusion, via continuous infusion, via localized perfusion, via a catheter, and/or via a lavage.

59. The method of any one of claims 51 to 58, wherein the at least one or more compositions are provided as a tablet, troche, lozenge, aqueous suspension, oily suspension, emulsion, hard capsule, soft capsule, and/or syrup.

60. The method of any one of claims 51 to 59, further comprising the step of providing one or more additional therapeutic compositions to the subject in need thereof.

61. The method of any one of claims 51 to 60, wherein the one or more compositions further comprise Gata4, fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); angiopoi etins, Angl and Ang2; matrix metalloproteinase (MMP); Delta-like ligand 4 (DIM); ITD-1; Mef2c, TBX5, Hand2, myocardin, Mesoderm posterior protein 1 (Mespl), miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, 5-azacytidine, Zebularine, and/or or peptides thereof.

62. The method of any one of claims 51 to 61, wherein the one or more compositions further comprise Angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor plus neprilysin inhibitors (ARNIs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, diuretics, potassium, magnesium, potassium-sparing diuretics or aldosterone antagonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, Digoxin or Lanoxin or digitalis, hydralazine and isosorbide dinitrate (BiDil), Vericiguat or Verquvo or an oral soluble guanylate cyclase (sGC) stimulator, positive inotropes or inotropic therapy, vasodilators, anticoagulant medication, aspirin, nitrates, statins, and/or blood thinners.

63. The method of any one of claims 51 to 62, wherein administration of the at least one or more compositions occurs before administration of the at least one or more ORL-1 activating agents.

64. The method of any one of claims 51 to 62, wherein administration of the at least one or more compositions occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

65. The method of any one of claims 51 to 62, wherein administration of the at least one or more compositions occurs after administration of the at least one or more ORL-1 activating agents.

66. The method of any one of claims 51 to 65, wherein the at least one or more compositions are delivered via a same route of administration as the at least one or more ORL-1 activating agents.

67. The method of any one of claims 51 to 65, wherein the at least one or more compositions are delivered via a different route of administration as the at least one or more ORL-1 activating agents.

68. The method of any one of claims 51 to 67, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in one formulation.

69. The method of any one of claims 51 to 67, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in separate formulations.

70. The method of any one of claims 51 to 69, further comprising treatment with at least one or more co-therapies.

71. The method of claim 70, wherein the at least one or more co-therapies further comprise drug therapy, surgery, coronary bypass, heart valve repair or replacement, an implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) or biventricular pacing, a ventricular assist device (VAD), VAT, percutaneous coronary intervention (PCI) and/or heart transplant.

72. The method of any one of claims 70 and 71, wherein administration of the at least one or more co-therapies occurs before administration of the at least one or more ORL-1 activating agents.

73. The method of any one of claims 70 and 71, wherein administration of the at least one or more co-therapies occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

74. The method of any one of claims 70 and 71, wherein administration of the at least one or more co-therapies occurs after administration of the at least one or more ORL-1 activating agents.

75. A method of suppressing fibrosis in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of one or more compositions or a pharmaceutically acceptable salt thereof, wherein the one or more compositions comprises at least one or more ORL1 -activating agents.

76. The method of claim 75, wherein the one or more compositions are a small molecule, a peptide, a protein, a biologic, a carbohydrate, and/or a nucleic acid.

77. The method of any one of claims 75 and 76, wherein the at least one or more ORL1- activating agents comprise (R)-Ro65-6570, [(pF)Phe4]N/OFQ-(l-13)-NH2, [3H]Leu-N/OFQ, [3H]N/OFQ, [3H]Tyrl4-N/OFQ, [Argl4Lysl5]N/OFQ, [F/G]N/OFQ-(1-13)-NH2, Ac- RYYRIK-NH2, Ac-RYYRWK-NH2, AT-121, AT-403, BU08028, buprenorphine, buprenorphine hydrochloride, cebranopadol, etorphine, JNJ-19385899, MCOPPB, MCOPPB trihydrochloride hydrate, MT-7716, N/OFQ-(1-13)-NH2, N/OFQ-NH2, NNC 63-0532, nociceptin/orphanin FQ (N/OFQ), norbuprenorphine, PWT2-N/OFQ, Ro64-6198, Ro6570, SCH221510, SCH486757, SerlOO (ZP120), SR16835, SR16435, TH-030418, UFP-102, and/or UFP-112.

78. The method of any one of claims 75 to 77, wherein the at least one or more ORL1- activating agents comprise MCOPPB.

79. The method of any one of claims 75 to 78, wherein the at least one or more 0RL1- activating agents are provided in a pharmaceutical composition.

80. The method of any one of claims 75 to 79, wherein the at least one or more compositions are delivered systemically.

81. The method of any one of claims 75 to 79, wherein the at least one or more compositions are delivered locally.

82. The method of any one of claims 75 to 81, wherein the at least one or more compositions are provided intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, locally, via inhalation, via injection, via infusion, via continuous infusion, via localized perfusion, via a catheter, and/or via a lavage.

83. The method of any one of claims 75 to 82, wherein the at least one or more compositions are provided as a tablet, troche, lozenge, aqueous suspension, oily suspension, emulsion, hard capsule, soft capsule, and/or syrup.

84. The method of any one of claims 75 to 83, further comprising the step of providing one or more additional therapeutic compositions to the subject in need thereof.

85. The method of any one of claims 75 to 84, wherein the one or more compositions further comprise Gata4, fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); angiopoi etins, Angl and Ang2; matrix metalloproteinase (MMP); Delta-like ligand 4 (DIM); ITD-1; Mef2c, TBX5, Hand2, myocardin, Mesoderm posterior protein 1 (Mespl), miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, 5-azacytidine, Zebularine, and/or or peptides thereof.

86. The method of any one of claims 75 to 85, wherein the one or more compositions further comprise Angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor plus neprilysin inhibitors (ARNIs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, diuretics, potassium, magnesium, potassium-sparing diuretics or aldosterone antagonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, Digoxin or Lanoxin or digitalis, hydralazine and isosorbide dinitrate (BiDil), Vericiguat or Verquvo or an oral soluble guanylate cyclase (sGC) stimulator, positive inotropes or inotropic therapy, vasodilators, anticoagulant medication, aspirin, nitrates, statins, and/or blood thinners.

87. The method of any one of claims 75 to 86, wherein administration of the at least one or more compositions occurs before administration of the at least one or more ORL-1 activating agents.

88. The method of any one of claims 75 to 86, wherein administration of the at least one or more compositions occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

89. The method of any one of claims 75 to 86, wherein administration of the at least one or more compositions occurs after administration of the at least one or more ORL-1 activating agents.

90. The method of any one of claims 75 to 89, wherein the at least one or more compositions are delivered via a same route of administration as the at least one or more ORL-1 activating agents.

91. The method of any one of claims 75 to 89, wherein the at least one or more compositions are delivered via a different route of administration as the at least one or more ORL-1 activating agents.

92. The method of any one of claims 75 to 91, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in one formulation.

93. The method of any one of claims 75 to 91, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in separate formulations.

94. The method of any one of claims 75 to 93, further comprising treatment with at least one or more co-therapies.

95. The method of claim 94, wherein the at least one or more co-therapies further comprise drug therapy, surgery, coronary bypass, heart valve repair or replacement, an implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) or biventricular pacing, a ventricular assist device (VAD), VAT, percutaneous coronary intervention (PCI) and/or heart transplant.

96. The method of any one of claims 94 and 95, wherein administration of the at least one or more co-therapies occurs before administration of the at least one or more ORL-1 activating agents.

97. The method of any one of claims 94 and 95, wherein administration of the at least one or more co-therapies occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

98. The method of any one of claims 94 and 95, wherein administration of the at least one or more co-therapies occurs after administration of the at least one or more ORL-1 activating agents.

99. A method of inducing angiogenesis in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of one or more compositions or a pharmaceutically acceptable salt thereof, wherein the one or more compositions comprises at least one or more 0RL1 -activating agents.

100. The method of claim 99, wherein the one or more compositions are a small molecule, a peptide, a protein, a biologic, a carbohydrate, and/or a nucleic acid.

101. The method of any one of claims 99 and 100, wherein the at least one or more ORL1- activating agents comprise (R)-Ro65-6570, [(pF)Phe4]N/OFQ-(l-13)-NH2, [3H]Leu-N/OFQ, [3H]N/OFQ, [3H]Tyrl4-N/OFQ, [Argl4Lysl5]N/OFQ, [F/G]N/OFQ-(1-13)-NH2, Ac- RYYRIK-NH2, Ac-RYYRWK-NH2, AT-121, AT-403, BU08028, buprenorphine, buprenorphine hydrochloride, cebranopadol, etorphine, JNJ-19385899, MCOPPB, MCOPPB trihydrochloride hydrate, MT-7716, N/OFQ-(1-13)-NH2, N/OFQ-NH2, NNC 63-0532, nociceptin/orphanin FQ (N/OFQ), norbuprenorphine, PWT2-N/OFQ, Ro64-6198, Ro6570, SCH221510, SCH486757, SerlOO (ZP120), SR16835, SR16435, TH-030418, UFP-102, and/or UFP-112.

102. The method of any one of claims 99 to 101, wherein the at least one or more ORL1- activating agents comprise MCOPPB.

103. The method of any one of claims 99 to 102, wherein the at least one or more ORL1- activating agents are provided in a pharmaceutical composition.

104. The method of any one of claims 99 to 103, wherein the at least one or more compositions are delivered systemically.

105. The method of any one of claims 99 to 103, wherein the at least one or more compositions are delivered locally.

106. The method of any one of claims 99 to 105, wherein the at least one or more compositions are provided intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, locally, via inhalation, via injection, via infusion, via continuous infusion, via localized perfusion, via a catheter, and/or via a lavage.

107. The method of any one of claims 99 to 106, wherein the at least one or more compositions are provided as a tablet, troche, lozenge, aqueous suspension, oily suspension, emulsion, hard capsule, soft capsule, and/or syrup.

108. The method of any one of claims 99 to 107, further comprising the step of providing one or more additional therapeutic compositions to the subject in need thereof.

109. The method of any one of claims 99 to 108, wherein the one or more compositions further comprise Gata4, fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); angiopoi etins, Angl and Ang2; matrix metalloproteinase (MMP); Delta-like ligand 4 (DIM); ITD-1; Mef2c, TBX5, Hand2, myocardin, Mesoderm posterior protein 1 (Mespl), miR- 133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, 5-azacytidine, Zebularine, and/or or peptides thereof.

110. The method of any one of claims 99 to 109, wherein the one or more compositions further comprise Angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor plus neprilysin inhibitors (ARNIs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, diuretics, potassium, magnesium, potassium-sparing diuretics or aldosterone antagonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, Digoxin or Lanoxin or digitalis, hydralazine and isosorbide dinitrate (BiDil), Vericiguat or Verquvo or an oral soluble guanylate cyclase (sGC) stimulator, positive inotropes or inotropic therapy, vasodilators, anticoagulant medication, aspirin, nitrates, statins, and/or blood thinners.

111. The method of any one of claims 99 to 110, wherein administration of the at least one or more compositions occurs before administration of the at least one or more ORL-1 activating agents.

112. The method of any one of claims 99 to 110, wherein administration of the at least one or more compositions occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

113. The method of any one of claims 99 to 110, wherein administration of the at least one or more compositions occurs after administration of the at least one or more ORL-1 activating agents.

114. The method of any one of claims 99 to 113, wherein the at least one or more compositions are delivered via a same route of administration as the at least one or more ORL-1 activating agents.

115. The method of any one of claims 99 to 113, wherein the at least one or more compositions are delivered via a different route of administration as the at least one or more ORL-1 activating agents.

116. The method of any one of claims 99 to 115, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in one formulation.

117. The method of any one of claims 99 to 115, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in separate formulations.

118. The method of any one of claims 99 to 117, further comprising treatment with at least one or more co-therapies.

119. The method of claim 118, wherein the at least one or more co-therapies further comprise drug therapy, surgery, coronary bypass, heart valve repair or replacement, an implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) or biventricular pacing, a ventricular assist device (VAD), VAT, percutaneous coronary intervention (PCI) and/or heart transplant.

120. The method of any one of claims 118 and 119, wherein administration of the at least one or more co-therapies occurs before administration of the at least one or more ORL-1 activating agents.

121. The method of any one of claims 118 and 119, wherein administration of the at least one or more co-therapies occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

122. The method of any one of claims 118 and 119, wherein administration of the at least one or more co-therapies occurs after administration of the at least one or more ORL-1 activating agents.

123. A method of preventing HF progression in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of one or more compositions or a pharmaceutically acceptable salt thereof, wherein the one or more compositions comprises at least one or more ORL1 -activating agents.

124. The method of claim 123, wherein the one or more compositions are a small molecule, a peptide, a protein, a biologic, a carbohydrate, and/or a nucleic acid.

125. The method of any one of claims 123 and 124, wherein the at least one or more ORL1- activating agents comprise (R)-Ro65-6570, [(pF)Phe4]N/OFQ-(l-13)-NH2, [3H]Leu-N/OFQ, [3H]N/OFQ, [3H]Tyrl4-N/OFQ, [Argl4Lysl5]N/OFQ, [F/G]N/OFQ-(1-13)-NH2, Ac- RYYRIK-NH2, Ac-RYYRWK-NH2, AT-121, AT-403, BU08028, buprenorphine, buprenorphine hydrochloride, cebranopadol, etorphine, JNJ-19385899, MCOPPB, MCOPPB trihydrochloride hydrate, MT-7716, N/OFQ-(1-13)-NH2, N/OFQ-NH2, NNC 63-0532, nociceptin/orphanin FQ (N/OFQ), norbuprenorphine, PWT2-N/OFQ, Ro64-6198, Ro6570, SCH221510, SCH486757, SerlOO (ZP120), SR16835, SR16435, TH-030418, UFP-102, and/or UFP-112.

126. The method of any one of claims 123 to 125, wherein the at least one or more ORL1- activating agents comprise MCOPPB.

127. The method of any one of claims 123 to 126, wherein the at least one or more 0RL1- activating agents are provided in a pharmaceutical composition.

128. The method of any one of claims 123 to 127, wherein the at least one or more compositions are delivered systemically.

129. The method of any one of claims 123 to 127, wherein the at least one or more compositions are delivered locally.

130. The method of any one of claims 123 to 129, wherein the at least one or more compositions are provided intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, locally, via inhalation, via injection, via infusion, via continuous infusion, via localized perfusion, via a catheter, and/or via a lavage.

131. The method of any one of claims 123 to 130, wherein the at least one or more compositions are provided as a tablet, troche, lozenge, aqueous suspension, oily suspension, emulsion, hard capsule, soft capsule, and/or syrup.

132. The method of any one of claims 123 to 131, further comprising the step of providing one or more additional therapeutic compositions to the subject in need thereof.

133. The method of any one of claims 123 to 132, wherein the one or more compositions further comprise Gata4, fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); angiopoi etins, Angl and Ang2; matrix metalloproteinase (MMP); Delta-like ligand 4 (DIM); ITD-1; Mef2c, TBX5, Hand2, myocardin, Mesoderm posterior protein 1 (Mespl), miR-

133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, 5-azacytidine, Zebularine, and/or or peptides thereof.

134. The method of any one of claims 123 to 133, wherein the one or more compositions further comprise Angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor plus neprilysin inhibitors (ARNIs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, diuretics, potassium, magnesium, potassium-sparing diuretics or aldosterone antagonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, Digoxin or Lanoxin or digitalis, hydralazine and isosorbide dinitrate (BiDil), Vericiguat or Verquvo or an oral soluble guanylate cyclase (sGC) stimulator, positive inotropes or inotropic therapy, vasodilators, anticoagulant medication, aspirin, nitrates, statins, and/or blood thinners.

135. The method of any one of claims 123 to 134, wherein administration of the at least one or more compositions occurs before administration of the at least one or more ORL-1 activating agents.

136. The method of any one of claims 123 to 134, wherein administration of the at least one or more compositions occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

137. The method of any one of claims 123 to 134, wherein administration of the at least one or more compositions occurs after administration of the at least one or more ORL-1 activating agents.

138. The method of any one of claims 123 to 137, wherein the at least one or more compositions are delivered via a same route of administration as the at least one or more ORL-1 activating agents.

139. The method of any one of claims 123 to 137, wherein the at least one or more compositions are delivered via a different route of administration as the at least one or more ORL-1 activating agents.

140. The method of any one of claims 123 to 139, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in one formulation.

141. The method of any one of claims 123 to 140, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in separate formulations.

142. The method of any one of claims 123 to 141, further comprising treatment with at least one or more co-therapies.

143. The method of claim 142, wherein the at least one or more co-therapies further comprise drug therapy, surgery, coronary bypass, heart valve repair or replacement, an implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) or biventricular pacing, a ventricular assist device (VAD), VAT, percutaneous coronary intervention (PCI) and/or heart transplant.

144. The method of any one of claims 142 and 143, wherein administration of the at least one or more co-therapies occurs before administration of the at least one or more ORL-1 activating agents.

145. The method of any one of claims 142 and 143, wherein administration of the at least one or more co-therapies occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

146. The method of any one of claims 142 and 143, wherein administration of the at least one or more co-therapies occurs after administration of the at least one or more ORL-1 activating agents.

147. A method of attenuating or preventing hypertension progression in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of one or more compositions or a pharmaceutically acceptable salt thereof, wherein the one or more compositions comprises at least one or more 0RL1 -activating agents.

148. The method of claim 147, wherein the one or more compositions are a small molecule, a peptide, a protein, a biologic, a carbohydrate, and/or a nucleic acid.

149. The method of any one of claims 147 and 148, wherein the at least one or more 0RL1- activating agents comprise (R)-Ro65-6570, [(pF)Phe4]N/OFQ-(l-13)-NH2, [3H]Leu-N/0FQ, [3H]N/0FQ, [3H]Tyrl4-N/OFQ, [Argl4Lysl5]N/OFQ, [F/G]N/OFQ-(1-13)-NH2, Ac- RYYRIK-NH2, Ac-RYYRWK-NH2, AT-121, AT-403, BU08028, buprenorphine, buprenorphine hydrochloride, cebranopadol, etorphine, JNJ-19385899, MCOPPB, MCOPPB trihydrochloride hydrate, MT-7716, N/OFQ-(1-13)-NH2, N/0FQ-NH2, NNC 63-0532, nociceptin/orphanin FQ (N/OFQ), norbuprenorphine, PWT2-N/OFQ, Ro64-6198, Ro6570, SCH221510, SCH486757, SerlOO (ZP120), SR16835, SR16435, TH-030418, UFP-102, and/or UFP-112.

150. The method of any one of claims 147 to 149, wherein the at least one or more ORL1- activating agents comprise MCOPPB.

151. The method of any one of claims 147 to 150, wherein the at least one or more ORL1- activating agents are provided in a pharmaceutical composition.

152. The method of any one of claims 147 to 151, wherein the at least one or more compositions are delivered systemically.

153. The method of any one of claims 147 to 151, wherein the at least one or more compositions are delivered locally.

154. The method of any one of claims 147 to 153, wherein the at least one or more compositions are provided intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, locally, via inhalation, via injection, via infusion, via continuous infusion, via localized perfusion, via a catheter, and/or via a lavage.

155. The method of any one of claims 147 to 154, wherein the at least one or more compositions are provided as a tablet, troche, lozenge, aqueous suspension, oily suspension, emulsion, hard capsule, soft capsule, and/or syrup.

156. The method of any one of claims 147 to 155, further comprising the step of providing one or more additional therapeutic compositions to the subject in need thereof.

157. The method of any one of claims 147 to 156, wherein the one or more compositions further comprise Gata4, fibroblast growth factor (FGF); vascular endothelial growth factor (VEGF); angiopoi etins, Angl and Ang2; matrix metalloproteinase (MMP); Delta-like ligand 4 (DIM); ITD-1; Mef2c, TBX5, Hand2, myocardin, Mesoderm posterior protein 1 (Mespl), miR- 133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, 5-azacytidine, Zebularine, and/or or peptides thereof.

158. The method of any one of claims 147 to 157, wherein the one or more compositions further comprise Angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor plus neprilysin inhibitors (ARNIs), beta blockers, calcium channel blockers, cholesterol-lowering drugs, diuretics, potassium, magnesium, potassium-sparing diuretics or aldosterone antagonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, Digoxin or Lanoxin or digitalis, hydralazine and isosorbide dinitrate (BiDil), Vericiguat or Verquvo or an oral soluble guanylate cyclase (sGC) stimulator, positive inotropes or inotropic therapy, vasodilators, anticoagulant medication, aspirin, nitrates, statins, and/or blood thinners.

159. The method of any one of claims 147 to 158, wherein administration of the at least one or more compositions occurs before administration of the at least one or more ORL-1 activating agents.

160. The method of any one of claims 147 to 158, wherein administration of the at least one or more compositions occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

161. The method of any one of claims 147 to 158, wherein administration of the at least one or more compositions occurs after administration of the at least one or more ORL-1 activating agents.

162. The method of any one of claims 147 to 161, wherein the at least one or more compositions are delivered via a same route of administration as the at least one or more ORL-1 activating agents.

163. The method of any one of claims 147 to 161, wherein the at least one or more compositions are delivered via a different route of administration as the at least one or more ORL-1 activating agents.

164. The method of any one of claims 147 to 163, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in one formulation.

165. The method of any one of claims 147 to 163, wherein the at least one or more compositions are delivered together with the at least one or more ORL-1 activating agents in separate formulations.

166. The method of any one of claims 147 to 165, further comprising treatment with at least one or more co-therapies.

167. The method of claim 166, wherein the at least one or more co-therapies further comprise drug therapy, surgery, coronary bypass, heart valve repair or replacement, an implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) or biventricular pacing, a ventricular assist device (VAD), VAT, percutaneous coronary intervention (PCI) and/or heart transplant.

168. The method of any one of claims 166 and 167, wherein administration of the at least one or more co-therapies occurs before administration of the at least one or more ORL-1 activating agents.

169. The method of any one of claims 166 and 167, wherein administration of the at least one or more co-therapies occurs simultaneously to administration of the at least one or more ORL-1 activating agents.

170. The method of any one of claims 166 and 167, wherein administration of the at least one or more co-therapies occurs after administration of the at least one or more ORL-1 activating agents.