Sgl t2 inhibitors and methods of use thereof

Abstract

Disclosed are methods of preventing fibroblast activation in a subject, comprising administering an Sodium-glucose co-transporter-2 (SGLT2) inhibitor to a subject in need thereof. Disclosed are methods of preventing fibrosis in a subject, comprising administering an SGLT2 inhibitor to a subject in need thereof. Disclosed are methods of improving survival of a subject at risk of having a cardiomyopathy comprising administering an SGLT2 inhibitor to the subject. Disclosed are methods of preventing cardiomyopathy in a subject at risk of developing a cardiomyopathy, comprising administering an SGLT2 inhibitor to the subject.

Description

ATTORNEY DOCKET NO. 37759.0634P1SGLT2 INHIBITORS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 733,271, filed on December 12, 2024, which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under BX004861-03S1 and BX004861-04 awarded by Veterans Administration. The government has certain rights in the invention.BACKGROUND

[0003] Local or regional myocardial fibrosis assessed by cardiac MRI (late-gadolinium enhancement) has been shown to be an independent predictor for ventricular arrhythmias (VArr) and SCD. Like tachycardia-induced cardiomyopathy (CM), premature ventricular contractions (PVC)-CM animal models have demonstrated persistent left ventricle (LV) interstitial fibrosis. Persistent interstitial fibrosis in patients with tachycardia-induced CM is thought to be responsible for the increased mortality despite recovery of LV function after treatment. A secondary analysis of the congestive heart failure: survival trial of antiarrhythmic therapy (CHF- STAT) study demonstrated that PVC-CM is not only associated with LV dysfunction but increased mortality if untreated. Thus, persistent interstitial fibrosis in PVC-CM may serve also as a substrate for VArr and sudden cardiac death (SCD) despite recovery of LVEF even after PVC ablation.

[0004] In general, initiation of ventricular arrhythmias requires 2 key elements: a trigger and substrate. PVCs have been well -recognized “triggers’" for ventricular arrhythmias (VArr), especially in the presence of underlying myocardial substrate, increasing the risk of SCD. Preclinical studies have shown that interstitial fibrosis can significantly affect electrophysiological myocardial properties, which can serve as a “substrate” for the development of VArr and SCD. Increased adverse interstitial fibrosis triggered by fibroblast activation in PVC-CM leads to electrophysiological (EP) remodeling (e.g.. conduction velocity, activation and repolarization time, action potential duration).BRIEF SUMMARY

[0005] Disclosed are methods of preventing fibroblast activation in a subject, comprisingATTORNEY DOCKET NO. 37759.0634P1 administering a Sodium-glucose co-transporter-2 (SGLT2) inhibitor to a subject in need thereof.

[0006] Disclosed are methods of preventing fibrosis in a subject, comprising administering a SGLT2 inhibitor to a subject in need thereof.

[0007] Disclosed are methods of improving survival of a subject at risk of having a cardiomyopathy comprising administering a SGLT2 inhibitor to the subject.

[0008] Disclosed are methods of preventing cardiomyopathy in a subject at risk of developing a cardiomyopathy, comprising administering a SGLT2 inhibitor to the subject.

[0009] Disclosed are methods of preventing or minimizing interstitial fibrosis in a subject, comprising administering an SGLT inhibitor to a subject in need thereof.

[0010] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0012] FIGS. 1A-1B show interstital and perivascular collagen in PVC-CM. FIG. 1A) LV samples show increased collagen (optical, Sirius red / fast green staining) (n=6); FIG. IB) Representative cross-sectional TEM images of LVFW with increased interstitial space (yellow), (hierarchical statistics nested t-test *P<0.05, ** P<0.01, *** PO.OOl Sham vs PVC).

[0013] FIG. 2 shows SGLT2 expression in isolated fibroblasts and LV tissue (n=2).

[0014] FIG. 3 shows Empagliflozin (“EMPA” a SGLT2 inhibitor) blunts fibroblast activation and fibrosis in LV samples (n=2 swine) in PVC-CM. noted by WB of Vimentin (VIM) expression (top left); qPCR of Collagen 1 (top right); and fibrosis with Sirus red / fast green staining (bottom).

[0015] FIG. 4 shows a Western Blot form an LV sample showing protein upregulation related to fibrosis and angiogenesis in PVC-CM.

[0016] FIG. 5 shows immunofluorescence of LV samples, aSMA (a marker of pericytes andATTORNEY DOCKET NO. 37759.0634P1 smooth muscle cells); IB4 (endothelial cells); vimentin (fibroblasts and endothelial cells) in the tunica intima of blood vessels using confocal microscopy.

[0017] FIG. 6 shows a number and area of blood vessels are increased in PVC-CM.Artificial intelligence software (AIVIA) identified and quantified different cell types (confocal images). “WGA” quantifies total myocytes. "aS MA" quantifies pericytes and blood vessels from the same image. Many images from several animals (n=5) undergo quantitative analysis and statistical comparisons (hierarchical statistics; nested t-test *P<0.05, **P<0.01 Sham vs PVC). Each dot (graph) represents the mean value of an image.

[0018] FIG. 7 shows an increase in total # of capillaries per myocyte and cross-sectional area of capillaries in PVC-CM. “WGA” quantified myocytes and “IB4” quantified the number of capillaries and cross-sectional area. AIVIA software trained to analyze several images per animal (hierarchical statistics; nested t-test *P<0.05, **P<0.01 Sham vs PVC).

[0019] FIGS. 8A and 8B show interstitial and perivascular collagen in PVC-CM. FIG 8A) LV samples show increased collagen (optical) (n=6); FIG. 8B) Representative cross-sectional TEM images of LVFW with increased interstitial space, (hierarchical statistics; nested t-test *P<0.05, **P<0.01, ***P<0.00I Sham vs PVC).

[0020] FIG. 9 shows LV RNAseq analysis. Extracellular matrix and cytokine signaling transcripts amplified in PVC-CM.

[0021] FIG. 10 shows single nuclei RNAseq (snRNAseq) in Sham and PVC. Clustering using UMAP and analysis of markers show at least 11 different cell types in the LVFW sample. Consisten with the cardiac hypertrophy there is a 10% increase in nuclei other than cardiomyocytes in PVC-CM. Increase in number of cells includes endothelial cells, fibroblasts, lymphocytes, pericytes, macrophages.

[0022] FIG. 11 shows SGLT2 expression in isolated fibroblasts and LV tissue (n=2).

[0023] FIG. 12 shows empagliflozin (SGLT2i) blunts fibroblast activation and fibrosis inLV samples (n=2 swine) in PVC-CM, noted by WB of vimentin (VM) expression (top left); qPCR of Collagen 1 (top right); and fibrosis with sirus red / fast green staining (bottom).

[0024] FIG. 13 shows a group randomization and intervention during 12- wk protocol (5d-t, 5-day telemetry; EPS, electrophysiologic study).

[0025] FIG 14 is a table that shows the methodology of tissue, cellular and molecular remodeling.

[0026] FIGS. 15A-B show antifibrotic and antihypertrophic effects of Empagliflozin (10 mg p.o. / daily) in PVC-CM Swine model. Left ventricular free wall (LVFW) sections were stainedATTORNEY DOCKET NO. 37759.0634P1 with Sirius Red / Fast Green to visualize fibrotic depositions, showed as red collagen fibers (FIG. 15 A). Cardiomyocyte hypertrophy was assessed using WGA- Alexa Fluor 633 red staining. Nuclei were counterstained with DAPI. FIG. 15B shows graphs of interstitial fibrosis and cardiomyoctues from FIG. 15A. One-way ANOVA; & p<0.01 vs Sham, * p< 0.05 vs PVC, # p<0.05 vs Sham.

[0027] FIG. 16 shows Empagliflozin treatment prevents the upregulation of sodium-glucose co-transporter 2 (SGLT2) and vimentin (VIM) in the left free ventricular wall (LFVW) of PVC- CM swine. Data were analyzed using one-way ANOVA. & p < 0.01 vs. Sham; * p < 0.05 vs. PVC; # p < 0.05 vs. Sham.

[0028] FIG. 17 shows the progression of left ventricular ejection fraction in PVC vs. Sham + / - SGLT2 inhibitors (mean + / - SE).DETAILED DESCRIPTION

[0029] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0030] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0031] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a peptide is disclosed and discussed and a number of modifications that can be made to a number of molecules including the amino acids are discussed, each and every combination and permutation of the peptide and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule. A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of theATTORNEY DOCKET NO. 37759.0634P1 combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B. and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.A. Definitions

[0032] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0033] It must be noted that as used herein and in the appended claims, the singular forms "a ", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a SGLT2 inhibitor" includes a plurality of such inhibitors, reference to "the SGLT2 inhibitor” is a reference to one or more inhibitors and equivalents thereof known to those skilled in the art, and so forth.

[0034] The w ord “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

[0035] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. For example, "treating" cardiomyopathy may refer to increasing heart function. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0036] As used herein, “subject” refers to the target of administration, e.g. an animal. ThusATTORNEY DOCKET NO. 37759.0634P1 the subject of the disclosed methods can be a vertebrate, such as a mammal. For example, the subject can be a human. The term does not denote a particular age or sex. Subject can be used interchangeably with “individual” or “patient”.

[0037] As used herein, the term “preventing” refers to partially or completely eliminating a disease, disorder, and / or condition or symptom thereof.

[0038] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0040] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises.” means “including but not limitedATTORNEY DOCKET NO. 37759.0634P1 to,'’ and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.B. Compositions

[0041] Disclosed are compositions comprising a sodium-glucose co-transporter-2 (SGLT2) inhibitor. In some aspects, the SGLT2 inhibitor can be canagliflozin, dapagliflozin, empagliflozin, besaglifloxin, or ertugliflozin.

[0042] In some instances, the composition can be a pharmaceutical composition comprising a SGLT2 inhibitor and a pharmaceutically acceptable earner.

[0043] In some instances, the compositions can further comprise a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0044] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous earners include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0045] The disclosed SGLT2 inhibitors can be formulated and / or administered in or with a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the requiredATTORNEY DOCKET NO. 37759.0634P1 particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug (e.g. peptide) in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0046] Thus, the compositions disclosed herein can comprise lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a peptide and a cationic liposome can be administered to the blood, to a target organ, or inhaled into the respiratory tract to target cells of the respiratory' tract. For example, a composition comprising a peptide or nucleic acid sequence described herein and a cationic liposome can be administered to a subject's lung cells. Regarding liposomes, see, e g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1 :95 100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413 7417 (1987); U.S. Patent No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[0047] In some instances, disclosed are pharmaceutical compositions comprising any of the disclosed SGLT2 inhibitors described herein, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, buffer, or diluent. In various aspects, theATTORNEY DOCKET NO. 37759.0634P1 peptide of the pharmaceutical composition is encapsulated in a delivery vehicle. In a further aspect, the delivery vehicle is a liposome, a microcapsule, or a nanoparticle. In a still further aspect, the delivery vehicle is PEG-ylated.

[0048] In the methods described herein, delivery of the compositions to cells can be via a variety of mechanisms. As defined above, disclosed herein are compositions comprising any one or more of the peptides described herein and can also include a carrier such as a pharmaceutically acceptable carrier. For example, disclosed are pharmaceutical compositions, comprising the peptides disclosed herein, and a pharmaceutically acceptable carrier. In one aspect, disclosed are pharmaceutical compositions comprising the disclosed peptides. That is, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed peptide or at least one product of a disclosed method and a pharmaceutically acceptable carrier.

[0049] In certain aspects, the disclosed pharmaceutical compositions comprise the disclosed SGLT inhibitors (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable earner, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for nasal, oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0050] In practice, the SGLT2 inhibitors described herein, or pharmaceutically acceptable salts thereof, of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil- in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the invention, and / or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and / or delivery devices. TheATTORNEY DOCKET NO. 37759.0634P1 compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

[0051] The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen. Other examples of carriers include dimyristoylphosphatidyl (DMPC), phosphate buffered saline or a multivesicular liposome. For example. PG:PC:Cholesterol:peptide or PC:peptide can be used as carriers in this invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Other examples of the pharmaceutically-acceptable earner include, but are not limited to, saline, Ringer’s solution and dextrose solution. The pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0052] In order to enhance the solubility and / or the stability' of the disclosed peptides in pharmaceutical compositions, it can be advantageous to employ a-, f>- or y-cyclodextrins or their derivatives, in particular hydroxyalkyl substituted cyclodextrins, e.g. 2-hydroxypropyl-P- cyclodextrin or sulfobutyl-[3-cyclodextrin. Also, co-solvents such as alcohols may improve the solubility and / or the stability' of the compounds according to the invention in pharmaceutical compositions.

[0053] Pharmaceutical compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the polypeptide, peptide, nucleicATTORNEY DOCKET NO. 37759.0634P1 acid, vector of the invention is not compromised. Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

[0054] Because of the ease in administration, oral administration can be used, and tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated bystandard aqueous or nonaqueous techniques.

[0055] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0056] A tablet containing the compositions of the present invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0057] The pharmaceutical compositions of the present invention comprise a disclosedATTORNEY DOCKET NO. 37759.0634P1SGLT2 inhibitor (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable earner, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity7of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0058] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0059] Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. Typically, the final injectable form should be sterile and should be effectively fluid for easy syringability. The pharmaceutical compositions should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0060] Injectable solutions, for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations.

[0061] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol,ATTORNEY DOCKET NO. 37759.0634P1 polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as. for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0062] Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.

[0063] In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and / or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and / or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e g., as a transdermal patch, as a spot on, as an ointment.

[0064] Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.

[0065] Formulations for optical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be desirable.

[0066] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants canATTORNEY DOCKET NO. 37759.0634P1 be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a disclosed peptide, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0067] The exact dosage and frequency of administration depends on the particular disclosed peptide, a product of a disclosed method of making, a pharmaceutically acceptable salt, solvate, or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof; the particular condition being treated and the severity of the condition being treated; various factors specific to the medical history of the subject to whom the dosage is administered such as the age; weight, sex, extent of disorder and general physical condition of the particular subject, as well as other medication the individual may be taking; as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and / or depending on the evaluation of the physician prescribing the compositions.

[0068] Depending on the mode of administration, the pharmaceutical composition will comprise from 0.05 to 99 % by weight, preferably from 0. 1 to 70 % by weight, more preferably from 0. 1 to 50 % by weight of the active ingredient, and, from 1 to 99.95 % by weight, preferably from 30 to 99.9 % by weight, more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.C. Methods

[0069] The disclosed methods comprise the administration of a sodium-glucose cotransporter-2 (SGLT2) inhibitor. In some aspects, the administration can be of a composition comprising an SGLT2 inhibitor. In some aspects, the composition can be any of the pharmaceutical compositions described herein. For example, the composition can be a pharmaceutical composition comprising a SGLT2 inhibitor and pharmaceutically acceptable carrier.1. Preventing Fibroblast Activation

[0070] Disclosed are methods of preventing fibroblast activation in a subject, comprising administering a sodium-glucose co-transporter-2 (SGLT2) inhibitor to a subject in need thereof.

[0071] In some aspects, fibroblast activation can lead to interstitial fibrosis. In some aspects, fibroblast activation can lead to altering cardiomyocyte function. Thus, the disclosed methods of preventing fibroblast activation can prevent or reduce interstitial fibrosis and / or increase or maintain cardiomyocyte function.ATTORNEY DOCKET NO. 37759.0634P1

[0072] In some aspects, the subject at risk of developing a cardiomyopathy does develop a cardiomyopathy and there is decreased diffuse microscopic scarring after the subject develops the cardiomyopathy. Thus, in some aspects, the SGLT2 inhibitor prevents diffuse microscopic scarring.

[0073] In some aspects, extracellular matrix remodeling is prevented or reduced in the subject after administration of the SGLT inhibitor.

[0074] In some aspects, vimentin expression is reduced in the cardiac tissue of the subject after administration of the SGLT inhibitor.

[0075] In some aspects, the SGLT2 inhibitor can be canagliflozin, dapagliflozin, empagliflozin, besaglifloxin. or ertugliflozin. i. Subject population

[0076] In some aspects, the subject in need thereof is a subject at risk of having a cardiomyopathy. In some aspects, a cardiomyopathy is a disease or condition that affects the heart muscle and results in poor blood flow because it is more difficult for the heart to pump blood. Thus, in some aspects, a subject at risk of having a cardiomyopathy can be determined based on the presence of a risk factor for developing a cardiomyopathy.

[0077] In some aspects, a cardiomyopathy is a condition wherein the heart expels <50% of blood (ejection fraction).

[0078] In some aspects, the subject at risk of having a cardiomyopathy has heart palpitations, premature ventricular contractions, and / or extra heart beats. In some aspects, extra heart beats or premature ventricular contractions refers to abnormal heart beats that result in a feeling of palpitation. Thus, in some aspects, the methods are preventing cardiomyopathy caused by extra heart beats.

[0079] In some aspects, the subject at risk of having a cardiomyopathy is currently or has previously been treated with a chemotherapeutic agent. Thus, in some aspects, the methods are preventing cardiomyopathy caused by a chemotherapeutic agent. In some aspects, a chemotherapeutic agent can be, but is not limited to, anthracy clines. In some aspects, anthracy clines can be, but are not limited to. doxorubicin. XXX

[0080] In some aspects, the subject at risk of having a cardiomyopathy has heart inflammation. Thus, in some aspects, the methods are preventing cardiomyopathy caused by heart inflammation. In some aspects, heart inflammation can be from, but is not limited to, infection.

[0081] In some aspects, the subject at risk of having a cardiomyopathy is taking or hasATTORNEY DOCKET NO. 37759.0634P1 previously taken a drug that weakens the heart. In some aspects, a drug that weakens the heart can be a chemotherapeutic agent.

[0082] In some aspects, the subject at risk of having a cardiomyopathy has tachycardia. Thus, in some aspects, the methods are preventing cardiomyopathy caused by tachycardia.

[0083] In some aspects, the subject at risk of having a cardiomyopathy has sarcoidosis. Thus, in some aspects, the methods are preventing cardiomyopathy caused by sarcoidosis. In some aspects, sarcoidosis can cause cardiomyopathy due to the collection of immune cells that form granulomas in the heart tissue.

[0084] In some aspects, the subject at risk of having a cardiomyopathy has ventricular dysplasia. Thus, in some aspects, the methods are preventing cardiomyopathy caused by ventricular dysplasia (also known as arrhythmogenic cardiomyopathy).

[0085] In some aspects, the subject does not have diabetes.

[0086] In some aspects, the subject has not had a myocardial infarction prior to administering an SGLT inhibitor to the subject.2. Preventing Fibrosis

[0087] Disclosed are methods of preventing fibrosis in a subject, comprising administering a SGLT2 inhibitor to a subject in need thereof.

[0088] In some aspects, fibrosis occurs due to fibroblast activation and / or proliferation. In some aspects of the disclosed methods after administration of the SGLT inhibitor, the subject has is reduced fibroblast activation and / or proliferation compared to a subject not receiving a SGLT2 inhibitor. Thus, in some aspects, the reduction in fibroblast activation and / or proliferation leads to preventing fibrosis.

[0089] In some aspects, the fibrosis is interstitial fibrosis. Interstitial fibrosis is a type of cardiac fibrosis that occurs in cardiomyopathies and can be characterized by the accumulation of collagen between cells. In some aspects, interstitial fibrosis can lead to complications such as, but not limited to, arrhythmias, stiffening of the heart wall, diastolic dysfunction, and heart failure. Thus, also disclosed are methods of preventing interstitial fibrosis in a subject, comprising administering a SGLT2 inhibitor to a subject in need thereof.

[0090] In some aspects, the SGLT2 inhibitor can be canagliflozin, dapagliflozin, empagliflozin, besaglifloxin, or ertugliflozin. i. Subject population

[0091] In some aspects, the subject in need thereof is a subject at risk of having a cardiomyopathy. In some aspects, a cardiomyopathy is a disease or condition that affects theATTORNEY DOCKET NO. 37759.0634P1 heart muscle and results in poor blood flow because it is more difficult for the heart to pump blood. Thus, in some aspects, a subject at risk of having a cardiomyopathy can be determined based on the presence of a risk factor for developing a cardiomyopathy.

[0092] In some aspects, a cardiomyopathy is a condition wherein the heart expels <50% of blood (ejection fraction).

[0093] In some aspects, the subject at risk of having a cardiomyopathy has heart palpitations, premature ventricular contractions, and / or extra heart beats. In some aspects, extra heart beats or premature ventricular contractions refers to abnormal heart beats that result in a feeling of palpitation. Thus, in some aspects, the methods are preventing cardiomyopathy caused by extra heart beats.

[0094] In some aspects, the subject at risk of having a cardiomyopathy is currently or has previously been treated with a chemotherapeutic agent. Thus, in some aspects, the methods are preventing cardiomyopathy caused by a chemotherapeutic agent. In some aspects, a chemotherapeutic agent can be, but is not limited to, anthracy clines. In some aspects, anthracy clines can be, but are not limited to, doxorubicin.

[0095] In some aspects, the subject at risk of having a cardiomyopathy has heart inflammation. Thus, in some aspects, the methods are preventing cardiomyopathy caused by heart inflammation. In some aspects, heart inflammation can be from, but is not limited to, infection.

[0096] In some aspects, the subject at risk of having a cardiomyopathy is taking or has previously taken a drug that weakens the heart. In some aspects, a drug that weakens the heart can be a chemotherapeutic agent.

[0097] In some aspects, the subject at risk of having a cardiomyopathy has tachycardia. Thus, in some aspects, the methods are preventing cardiomyopathy caused by tachycardia.

[0098] In some aspects, the subject at risk of having a cardiomyopathy has sarcoidosis. Thus, in some aspects, the methods are preventing cardiomyopathy caused by sarcoidosis. In some aspects, sarcoidosis can cause cardiomyopathy due to the collection of immune cells that form granulomas in the heart tissue.

[0099] In some aspects, the subject at risk of having a cardiomyopathy has ventricular dysplasia. Thus, in some aspects, the methods are preventing cardiomyopathy caused by ventricular dysplasia (also known as arrhythmogenic cardiomyopathy).

[0100] In some aspects, the subject does not have diabetes.

[0101] In some aspects, the subject has not had a myocardial infarction prior toATTORNEY DOCKET NO. 37759.0634P1 administering an SGLT inhibitor to the subject.3. Improving Survival

[0102] Disclosed are methods of improving survival of a subject at risk of having a cardiomyopathy comprising administering a SGLT2 inhibitor to the subject.

[0103] In some aspects, fibroblast activation and / or proliferation leads to fibrosis, such as interstitial fibrosis, which can have detrimental consequences that lead to death. In some aspects of the disclosed methods fibroblast activation and / or proliferation is decreased in the subject after administration of the SGLT inhibitor. Thus, in some aspects, the reduction in fibroblast activation and / or proliferation leads to improved survival of the subject. In some aspects, fibrosis is decreased in the subject after administration of the SGLT inhibitor. Thus, in some aspects, the decrease in fibrosis leads to improved survival of the subject. In some aspects, the decrease in fibroblast activation / proliferation or decrease in fibrosis is compared to a subject not receiving a SGLT2 inhibitor.

[0104] In some aspects of the disclosed methods diffuse microscopic scarring is decreased in the subject after administration of the SGLT inhibitor. In some aspects, the decrease in diffuse microscopic scarring can be due to the decrease in fibroblast activation and / or proliferation.

[0105] In some aspects, the SGLT2 inhibitor is can be canagliflozin, dapagliflozin, empagliflozin, besaglifloxin, or ertugliflozin. i. Subject population

[0106] In some aspects, the subject in need thereof is a subject at risk of having a cardiomyopathy. In some aspects, a cardiomyopathy is a disease or condition that affects the heart muscle and results in poor blood flow because it is more difficult for the heart to pump blood. Thus, in some aspects, a subject at risk of having a cardiomyopathy can be determined based on the presence of a risk factor for developing a cardiomyopathy.

[0107] In some aspects, a cardiomyopathy is a condition wherein the heart expels <50% of blood (ejection fraction).

[0108] In some aspects, the subject at risk of having a cardiomyopathy has heart palpitations, premature ventricular contractions, and / or extra heart beats. In some aspects, extra heart beats or premature ventricular contractions refers to abnormal heart beats that result in a feeling of palpitation. Thus, in some aspects, the methods are preventing cardiomyopathy caused by extra heart beats.

[0109] In some aspects, the subject at risk of having a cardiomyopathy is currently or has previously been treated with a chemotherapeutic agent. Thus, in some aspects, the methods areATTORNEY DOCKET NO. 37759.0634P1 preventing cardiomyopathy caused by a chemotherapeutic agent. In some aspects, a chemotherapeutic agent can be, but is not limited to, anthracyclines. In some aspects, anthracy clines can be, but are not limited to, doxorubicin.

[0110] In some aspects, the subject at risk of having a cardiomyopathy has heart inflammation. Thus, in some aspects, the methods are preventing cardiomyopathy caused by heart inflammation. In some aspects, heart inflammation can be from, but is not limited to, infection.

[0111] In some aspects, the subject at risk of having a cardiomyopathy is taking or has previously taken a drug that weakens the heart. In some aspects, a drug that weakens the heart can be a chemotherapeutic agent.

[0112] In some aspects, the subject at risk of having a cardiomyopathy has tachycardia. Thus, in some aspects, the methods are preventing cardiomyopathy caused by tachycardia.

[0113] In some aspects, the subject at risk of having a cardiomyopathy has sarcoidosis. Thus, in some aspects, the methods are preventing cardiomyopathy caused by sarcoidosis. In some aspects, sarcoidosis can cause cardiomyopathy due to the collection of immune cells that form granulomas in the heart tissue.

[0114] In some aspects, the subject at risk of having a cardiomyopathy has ventricular dysplasia. Thus, in some aspects, the methods are preventing cardiomyopathy caused by ventricular dysplasia (also known as arrhythmogenic cardiomyopathy).

[0115] In some aspects, the subject does not have diabetes.

[0116] In some aspects, the subject has not had a myocardial infarction prior to administering an SGLT inhibitor to the subject.4. Preventing Cardiomyopathy

[0117] Disclosed are methods of preventing cardiomyopathy in a subject at risk of developing a cardiomyopathy, comprising administering a SGLT2 inhibitor to the subject. In some aspects, cardiomyopathy is also know n as low heart function.

[0118] In some aspects, the cardiomyopathy can be, but is not limited to, premature ventricular complex (PVC)-induced cardiomyopathy, drug-induced (e.g. chemotherapeutic agent-induced) cardiomyopathy, inflammation-induced cardiomyopathy, tachycardia-induced cardiomyopathy, sarcoidosis-induced cardiomyopathy, or arrhythmogenic cardiomyopathy. In some aspects, any cardiomyopathy that results in fibrosis, or diffuse microscopic scarring, can be prevented by administering a SGLT2 inhibitor to a subject at risk of developing the cardiomyopathy.ATTORNEY DOCKET NO. 37759.0634P1

[0119] In some aspects, the SGLT2 inhibitor can be canagliflozin, dapagliflozin, empagliflozin, besaglifloxin. or ertugliflozin.

[0120] In some aspects, fibrosis is assessed to determine if cardiomyopathy has been prevented. In some aspects, fibrosis can be assessed by histology. In some aspects, extracellular volume (ECV) by cardiac MRI can be correlated to increased fibrosis or inflammation. Thus, in some aspects, ECV by MRI can be a marker of fibrosis to demonstrate that SGLT2 inhibitors prevent or decrease fibrosis deposition. i. Subject population

[0121] In some aspects, the subject in need thereof is a subject at risk of having a cardiomyopathy. In some aspects, a cardiomyopathy is a disease or condition that affects the heart muscle and results in poor blood flow because it is more difficult for the heart to pump blood. Thus, in some aspects, a subject at risk of having a cardiomyopathy can be determined based on the presence of a risk factor for developing a cardiomyopathy.

[0122] In some aspects, a cardiomyopathy is a condition wherein the heart expels <50% of blood (ejection fraction).

[0123] In some aspects, the subject at risk of having a cardiomyopathy has heart palpitations, premature ventricular contractions, and / or extra heart beats. In some aspects, extra heart beats or premature ventricular contractions refers to abnormal heart beats that result in a feeling of palpitation. Thus, in some aspects, the methods are preventing cardiomyopathy caused by extra heart beats.

[0124] In some aspects, the subject at risk of having a cardiomyopathy is currently or has previously been treated with a chemotherapeutic agent. Thus, in some aspects, the methods are preventing cardiomyopathy caused by a chemotherapeutic agent. In some aspects, a chemotherapeutic agent can be, but is not limited to, anthracyclines. In some aspects, anthracy clines can be, but are not limited to, doxorubicin.

[0125] In some aspects, the subject at risk of having a cardiomyopathy has heart inflammation. Thus, in some aspects, the methods are preventing cardiomyopathy caused by heart inflammation. In some aspects, heart inflammation can be from, but is not limited to, infection.

[0126] In some aspects, the subject at risk of having a cardiomyopathy is taking or has previously taken a drug that weakens the heart. In some aspects, a drug that weakens the heart can be a chemotherapeutic agent.

[0127] In some aspects, the subject at risk of having a cardiomyopathy has tachycardia.ATTORNEY DOCKET NO. 37759.0634P1Thus, in some aspects, the methods are preventing cardiomyopathy caused by tachycardia.

[0128] In some aspects, the subject at risk of having a cardiomyopathy has sarcoidosis. Thus, in some aspects, the methods are preventing cardiomyopathy caused by sarcoidosis. In some aspects, sarcoidosis can cause cardiomyopathy due to the collection of immune cells that form granulomas in the heart tissue.

[0129] In some aspects, the subject at risk of having a cardiomyopathy has ventricular dysplasia. Thus, in some aspects, the methods are preventing cardiomyopathy caused by ventricular dysplasia (also known as arrhythmogenic cardiomyopathy).

[0130] In some aspects, the subject does not have diabetesD. Combination Therapy

[0131] In one aspect of the disclosed methods, the compositions can be administered alone or in combination with one or more additional therapeutic agents. The additional therapeutic agents are selected based on the disease or symptom to be treated. A description of the various classes of suitable pharmacological agents and drugs may be found in Goodman and Gilman, The Pharmacological Basis of Therapeutics. (11th Ed., McGraw-Hill Publishing Co.) (2005).

[0132] In some aspects, the disclosed methods can be a combination therapy of a SGLT2 inhibitor and ACE inhibitors, beta blockers, Angiotensin receptor neprilysin inhibitor, angiotensin receptor blocker (ARB), diuretics, or aldosterone inhibitors.Examples

[0133] It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, 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 invention.A. Example 1

[0134] The prevalence of premature ventricular contractions (PVCs) is estimated from 40 to 75% in ambulatory ECG monitors and increases with age and comorbidities. PVCs are commonly associated with HF, CM, VArr and SCD. PVCs have been reported as an independent predictor of heart failure (HF), morbidity7, and cardiovascular mortality7.

[0135] Frequent PVCs have been reported in 48% of patients with HF regardless of etiology. Until 2016. AHA recognized frequent PVCs as a cause of a reversible LV systolic dysfunctionATTORNEY DOCKET NO. 37759.0634P1(LVEF <50%) referred to as “PVC-induced cardiomyopathy’' (PVC-CM), where PVC suppression can improve and even normalize left ventricular (LV) function. The prevalence of PVC-CM in patients with frequent PVCs and LV systolic dysfunction is estimated at 30%. Unfortunately, diagnosis of PVC-CM is only suspected and treated in patients with LV ejection fraction (LVEF) <50% and PVC burden >15%. A secondary' analysis of the CHF-STAT study supports that PVC-CM is not only associated with LV dysfunction but if untreated leads to increased mortality7.

[0136] Local or regional myocardial fibrosis assessed by cardiac MRI (late-gadolinium enhancement) has been shown to be an independent predictor for ventricular arrhythmias (VArr) and sudden cardiac death (SCD). Like tachycardia-induced CM, PVC-CM animal models have demonstrated persistent LV interstitial fibrosis (FIG. 1). Persistent interstitial fibrosis in tachycardia-induced CM is thought to be responsible for the increased mortality despite recovery of LV function after treatment. Thus, persistent interstitial fibrosis in PVC-CM may sen e also as a substrate for VArr and SCD despite recovery of LVEF even after PVC ablation. Preclinical studies have shown that interstitial fibrosis can significantly affect electrophysiological myocardial properties. Finding therapies to prevent or minimize extracellular matrix remodeling can minimize LV systolic dysfunction and improve long-term outcomes in patients with frequent PVCs and PVC-CM.

[0137] Interstitial fibrosis is the product of fibroblast proliferation and activation (FIG. 1) and conveys detrimental consequences such as tissue stiffness and altered EP properties (e.g., conduction velocity). Fibroblasts can also secrete cytokines affecting cardiomyocyte function.

[0138] A translational animal model of frequent PVC has been recognized as the proof of concept that established PVC-CM as a unique clinical entity'. Over the past few years, this model has led to the characterization and understanding of the triggers and potential mechanism(s) associated with PVC-CM. Like in the clinical setting, the canine PVC-CM model has demonstrated 1) a gradual and linear decrease in LV systolic function that is directly proportional to the PVC burden, and 2) not all animals develop LV systolic dysfunction despite similar PVC burden. The PVC-CM model has been reproduced in swine, moving away from canine research due to the lack of acceptance by the American society.

[0139] Recently, SGLT2i clinical trials have significantly improved survival in patients with LV systolic dysfunction and HF. How ever, the molecular mechanism(s) of this effect has not been elucidated. Recently, animal models (including swine) have demonstrated that SGLT2i significantly decreased fibrosis in myocardial infarction when compared to Sham. Data showsATTORNEY DOCKET NO. 37759.0634P1 the expression of SGLT2 in cardiac fibroblasts and SGLT2 inhibition decrease their activation (FIG. 2). The next step is to test SGLT2i as a therapy to prevent or minimize interstitial fibrosis in the animal model of PV C-CM.

[0140] SGLT2i (Empagliflozin) as an antifibrotic therapy. Fibroblasts and fibrosis are in the center of PVC-CM. The expression of SGLT2 protein was detected in the isolated cardiac fibroblast and LV tissue (FIG. 2). VIM, a marker of fibroblasts, was elevated in PVC-CM, and empagliflozin treatment inhibited its increase in two swine exposed to PVCs (FIG. 3).Moreover, interstitial fibrosis and collagen mRNA (qPCR) elevation observed in PVC-CM was reduced by empagliflozin (FIG. 3). Finally, these data show that empagliflozin (SGLT2i) inhibits fibroblasts in vivo. SGLT2 inhibitors e.g., empagliflozin) can be used, due to the newly discovered inhibitory effects of fibroblasts, to interfere with the structural remodeling (i.e.. interstitial fibrosis) and used to study their role in preventing PVC-CM and VArr.B. Example 2

[0141] Premature ventricular contractions (PVCs) are the most prevalent ventricular arrhythmia and frequent PVCs are associated with heart failure (HF) and sudden cardiac death (SCD). Modified pacemakers can deliver PVCs. Clinical and preclinical studies position left ventricular dyssynchrony (LVD) as a likely trigger of PVC-CM. Experimentally, 12 weeks of frequent PVCs in otherwise healthy animals resulted in adverse cardiac remodeling including: 1) a decrease in LV ejection fraction (LVEF) in association with a reduced expression of Ca2+ handling proteins, 2) eccentric hypertrophy (with increased angiogenesis), and 3) interstitial fibrosis.

[0142] The current study was aimed to (1) study the role of LVD in this adverse LV structural remodeling observed in PVC-CM. An unexpected cellular player in PVC-CM is the cardiac fibroblast. The mechanistic role can be studied by using SGLT2 inhibitors (SGLT2i), an inhibitory effect of fibroblast activation with antifibrotic effect has been identified that can impact subsequent cascade of events that likely lead to LV dysfunction and adverse remodeling observed in PVC-CM. In addition, this study can (2) test the role of fibroblast and the interstitial adverse remodeling on alterations in electrophysiological (EP) properties of the heart, prompting to life-threatening ventricular arrhythmias (VArr).

[0143] Dyssynchronous LV contraction (LVD) in PVCs elicits mechanical stress resulting in fibroblast activation. Activated fibroblasts, through paracrine signaling, decreases myocyte contractility responsible for LV systolic dysfunction. Activated fibroblasts directly promote interstitial matrix remodeling, leading to EP anomalies that are ultimately responsible for VArr.ATTORNEY DOCKET NO. 37759.0634P1The two main independent variables are: 1) PVCs in two locations promoting different degrees of LVD and 2) use of SGLT2i (empagliflozin. EMPA) as a therapy to prevent fibroblast activation and adverse cardiac remodeling.

[0144] The degree of LV dyssynchrony as a main trigger of interstitial fibrosis and development of PVC-CM can also be explored. By varying PVC location, different degrees of LVD will be studied in a swine PVC-CM model. Right ventricle (RV) apical PVCs (RV-PVCs, most LVD) can result in a higher degree of adverse cardiac organ, tissue, and molecular remodeling including LV function QLVEF), fibroblast activation, interstitial fibrosis and angiogenesis when compared to conduction system PVCs (CS-PVCs, least LVD), while CS- PVCs can also demonstrate adverse cardiac remodeling when compared to those without PVCs (Sham group, no LVD).

[0145] Inhibition of fibroblast activation to prevent cardiac remodeling in PVC-CM can also be assesssed. Using SGLT2i (EMPA), found to inhibit fibroblast activation, the role of fibroblast in the development of adverse cardiac remodeling in PVC-CM. Inhibition of fibroblast activation via SGLT2 inhibitors can minimize or prevent organ, tissue, cellular, and molecular remodeling caused by RV-PVCs (most LVD) when compared to those without PVCs (sham group, no LVD).

[0146] The effects of diffuse interstitial fibrosis in cardiac electrophysiological properties and arrhythmia induction in PVC-CM can also be evaluated. Using optical mapping, changes in EP tissue properties and interstitial fibrosis can be correlated and the effects of SGTL2i in this EP remodeling. Areas of interstitial fibrosis in animals exposed to RV-PVCs (most LVD) can correlate with areas of slow conduction velocity (CV), higher dispersion of CV, activation- and repolarization- time (AT, RT) with higher inducibility of VArr compared to sham (without interstitial fibrosis). In silico studies can be developed to model the optical mapping results and to assess the contribution of variable degrees of interstitial fibrosis to the inducibility of VArr.

[0147] These studies can be randomized and compare 5 different PVC swine groups including 1) SHAM without PVCs (no LVD), 2) RV apical PVCs (RV-PVCs = most LVD), 3) conduction system PVCs (CS-PVC = least LVD). 4) SHAM plus EMPA. and 5) RV-PVC plus EMPA.

[0148] Three conceptual innovations can be explored, including: 1) understanding the pivotal role of LVD and fibroblast activation promoting PVC-CM, 2) providing preclinical evidence of SGLT2i therapy to prevent adverse cardiac remodeling in PVC-CM, and 3) demonstrating the relationship between structural, tissue and EP remodeling and the impact ofATTORNEY DOCKET NO. 37759.0634P1SGLT2i to decrease the risk of VArr and SCD in PVC-CM. SGLT2i could have substantial benefits through a novel “antifibrotic” effect that improve outcomes regardless of LV function. This study proposes a paradigm shift by recognizing the role of the myocyte microenvironment related to fibroblasts activation and extracellular matrix, serving as potential mechanism for PVC-CM and substrate for VArr. This provides mechanistic insight to the clinical and survival benefits shown in recent SGLT2i clinical trials of systolic HF.1. Background and Scientific Purpose

[0149] Premature ventricular contractions (PVCs) are the most frequent ventricular arrhythmia and prevalence increases in adults and elderly. Moreover, frequent PVCs are associated with cardiomyopathy (CM), known as PVC-induced CM (PVC-CM), increased heart failure (HF), and sudden cardiac death (SCD).

[0150] The physiological electrical signal originating from sinus rhythm relays in the atrioventricular node and propagates though the Purkinje conduction system resulting in a coordinated ventricular contraction. In contrast, PVCs are initiated prematurely and ectopically (outside Purkinje) producing an abnormal ventricular propagation and uncoordinated contraction associated with mechanical stress, a phenomenon termed '‘LV dyssynchrony” (LVD). Acutely, PVCs increase ventricular contractility by a mechanism known as post-extrasystolic potentiation (PESP), but overtime persistent frequent PVCs result in a reduction in LV ejection fraction (LVEF), a hallmark of PVC-CM. Preclinical and clinical studies indicate that LVD plays a major role in the development of PVC-CM, since QRS duration and epicardial origin with greater LVD, are key clinical predictors of the development of PVC-CM. Like PVCs, pacing the right ventricle (RV) results in a wide QRS associated with LVD. In contrast, pacing from the conduction system (CS, including the left bundle branch) creates a wide yet narrower QRS with less LVD. Regardless of location, ventricular pacing results in less LVD than PVCs due to the lack of prematurity. Comparing effects of frequent PVC originating from conduction system (CS-PVC) vs. RV-PVC in an animal model identifies the role of LVD as a trigger for cardiac remodeling at organ, tissue, and molecular levels in PVC-CM.

[0151] A large canine model of frequent PVCs and PVC-CM has led the characterization and understanding of the triggers and potential mechanism of PVC-CM. Similar to the canine, the swine PVC-CM model has shown diffuse interstitial fibrosis that persists despite elimination of PVCs. Recently, the PVC-CM model has shown an increase in IL-10 and other mediators of inflammation and fibrosis, known to impact the L-lype Ca2+ current (LTCC) and myocyte contractility. Yet, one local source of IL-1 (3 in the heart are activated fibroblasts, likely activatedATTORNEY DOCKET NO. 37759.0634P1 by mechanical stress during PVCs. Expression of the sodium glucose co-transporter-2 (SGLT2) has been identified in the cardiac fibroblast and shown that an SGLT2 inhibitor (SGLT2i) can significantly decrease activation of fibroblasts and subsequent extracellular remodeling. LVD due to frequent PVCs are key to trigger fibroblast activation and fibrosis which can be significantly lowered by SGLT2i.

[0152] In general, initiation of arrhythmias requires 2 key elements: a trigger and substrate. PVCs have been well-recognized ‘'triggers” for ventricular arrhythmias (VArr), especially in the presence of underlying myocardial substrate, increasing the risk of SCD42. Recent studies have shown that fibroblast activation and increased extracellular matrix (interstitial fibrosis) can significantly affect electrophysiological (EP) myocardial properties that can serve as a "substrate” for the development of VArr and SCD. Increased adverse interstitial fibrosis triggered by fibroblast activation in PVC-CM leads to EP remodeling (e g., conduction velocity, activation and repolarization time, action potential duration) and inhibition of fibroblast activation with SGLT2i can prevent such electrical remodeling.

[0153] The consequences of chronic PVCs and pathophysiology of PVC-CM have come to the forefront as potential causes of HF and SCD. This study aims to understand (1) the role of LV dyssynchrony in PVC-CM as well as (2) the potential mechanistic role of fibroblast not only on the development of CM, but also in LV structural and EP remodeling. Furthermore, this study tests (3) whether inhibiting fibroblast activation can lead to prevention of adverse LV remodeling and prevent or minimize the development of PVC-CM.2. Arms of the Study

[0154] The following three elements can be addressed in this study.

[0155] The degree of LV dyssynchrony as a main trigger of interstitial fibrosis and development of PVC-CM. RV-PVCs (most LVD) can demonstrate a higher degree of adverse cardiac organ, tissue, and molecular remodeling (i.e., LVEF, eccentric and cellular hypertrophy, fibroblast activation, interstitial fibrosis and angiogenesis) when compared to CS- PVCs (least LVD), while CS-PVCs can also demonstrate adverse cardiac remodeling when compared to those without PVCs (sham group, no LVD).

[0156] Fibroblast inhibition can be assessed to prevent cardiac remodeling in PVC-CM. Inhibition of fibroblast activation via SGLT2i can minimize or prevent organ, tissue, cellular and molecular remodeling caused by RV-PVCs (most LVD) when compared to Sham (no PVCs, no LVD).

[0157] The effects of diffuse interstitial fibrosis can be evaluated in cardiacATTORNEY DOCKET NO. 37759.0634P1 electrophysiological properties and arrhythmia induction in PVC-CM. Using optical mapping, areas of interstitial fibrosis in animals exposed to RV-PVCs (most LVD) can correlate with areas of slow conduction velocity (CV), higher dispersion of CV, activation, and repolarization time (AT, RT) with higher inducibility of VArr compared to sham (without interstitial fibrosis). In silico studies can be developed to model optical mapping results and to assess the contribution of variable degrees of interstitial fibrosis to inducibility of VArr.3. Significance ! in pact

[0158] The prevalence of PVCs is estimated from 40 to 75% in ambulatory ECG monitors and increases with age and comorbidities. PVCs are commonly associated with HF, CM, VArr and SCD. PVCs have been reported as an independent predictor of HF, morbidity’, and cardiovascular mortality.

[0159] Frequent PVCs have been reported in 48% of patients with HF regardless of etiology. Until 2016, AHA recognized frequent PVCs as a cause of a reversible LV systolic dysfunction (LVEF <50%) referred to as “PVC-induced cardiomyopathy’' (PVC-CM), where PVC suppression can improve and even normalize LV function. The prevalence of PVC-CM in patients with frequent PVCs and LV systolic dysfunction is estimated at 30%. Unfortunately, diagnosis of PVC-CM is only suspected and treated in patients with LVEF <50% and PVC burden >15%. A secondary’ analysis of the CHF-STAT study supports that PVC-CM is not only associated with LV dysfunction but if untreated leads to increased mortality’.

[0160] Local or regional myocardial fibrosis assessed by cardiac MRI (late-gadolimum enhancement) has been shown to be an independent predictor for VArr and SCD. Like tachycardia-induced CM, PVC-CM animal models have demonstrated persistent LV interstitial fibrosis. Persistent interstitial fibrosis in tachycardia-induced CM is thought to be responsible for the increased mortality despite recovery of LV function after treatment. Thus, persistent interstitial fibrosis in PVC-CM may serve also as a substrate for VArr and SCD despite recovery’ of LVEF even after PVC ablation. Preclinical studies have shown that interstitial fibrosis can significantly affect electrophysiological myocardial properties. Finding therapies to prevent or minimize extracellular matrix remodeling could minimize LV systolic dysfunction and improve long-term outcomes in patients with frequent PVCs and PVC-CM.

[0161] A translational animal model of frequent PVC has been recognized as the proof of concept that established PVC-CM as a unique clinical entity’ (2016 AHA Scientific Statement). Over the past few years, this model has led to the characterization and understanding of the triggers and potential mechanism(s) associated with PVC-CM. Like in the clinical setting, aATTORNEY DOCKET NO. 37759.0634P1 canine PVC-CM model has demonstrated 1) a gradual and linear decrease in LV systolic function that is directly proportional to the PVC burden, and 2) not all animals develop LV systolic dysfunction despite similar PVC burden. The PVC-CM model has been reproduced in swine, moving away from canine research due to the lack of acceptance by American society.

[0162] Recently, SGLT2i clinical trials have significantly improved survival in patients with LV systolic dysfunction and HF. However, the molecular mechanism(s) of this effect has not been elucidated. Recently, animal models (including swine) have demonstrated that SGLT2i significantly decreased fibrosis in myocardial infarction when compared to Sham. Data shows the expression of SGLT2 in cardiac fibroblasts and SGLT2 inhibition decrease their activation. Next. SGLT2i can be tested as a therapy to prevent or minimize interstitial fibrosis in the animal model of PVC-CM.

[0163] The main significance of the proposed study is to identify the role of (1) fibroblast activation in the development of PVC-CM and (2) interstitial fibrosis as the substrate of EP remodeling responsible for increased risk of ventricular arrhythmias and SCD. In contrast to other studies, this study addresses the mechanism and role of fibrosis as fibroblast activation is inhibited using SGLT2i to prevent interstitial fibrosis as a substrate for VArr associated with PVC-CM. The clinical implications are substantial since SGLT2i could improve outcomes regardless of LV function in PVC-CM by preventing or minimizing adverse cardiac remodeling due to frequent PVCs through a newly identified '’anti fibrotic’' effect. This could impact longterm mortality risk in PVC-CM and other non-ischemic cardiomyopathies. This study proposes a paradigm shift by recognizing the role of the myocyte microenvironment related to fibroblasts activation and extracellular matrix. This can provide mechanistic insight into the clinical and survival benefits shown in recent SGLT2i clinical trials of systolic HF (DAPA-HF, EMPEROR- reduced).4. Results

[0164] A canine model of frequent PVC (50% RV-PVCs) characterized PVC-CM by LV systolic dysfunction, increased LV end-systolic and end-diastolic dimensions (LVESD, LVEDD). The original studies showed changes in Ca2+ handling dynamics and electrical remodeling (e.g., LTCC downregulation, increased ventricular effective refractory period (VERP) and action potential duration (APD).

[0165] Echocardiographic analysis show ed that PVC-CM is characterized by an increase in the LV mass index with a decrease in the relative wall thickness, consistent with eccentric hypertrophy. To understand the effects of LVD and PESP associated with PVCs. early PVCsATTORNEY DOCKET NO. 37759.0634P1(200ms, prominent PESP and less LVD) were compared to late PVCs (330ms, reverse pattern with a prominent LVD and lower PESP). However, no significant difference in phenotype (i.e., LVEF, LVESD, LVEDD, and LV mass) was demonstrated in early vs. late PVCs supporting that both LVD and PESP play a significant role as a trigger of PVC-CM.

[0166] In situ analysis of LV cardiomyocyte dimensions showed that width and length are increased in PVC-CM. consistent with an eccentric hypertrophy phenotype. Molecular markers of pathological hypertrophy such as Store Operated Ca2+ Entry (SOCE), calcineurin, ACTA1 and 0-MHC were unaltered, but pro-hypertrophic and anti-apoptotic signaling pathways such as ERK and mTOR / AKT were overexpressed in PVC-CM hearts. Growth factors (VEGF-A and VEGF-B) and their receptors were elevated in PVC-CM (FIG. 4). Therefore, frequent PVCs activate a molecular program responsible for LV remodeling. These studies also showed alterations in the Ca2+ handling machinery and the sympathetic autonomic response in PVC- CM.

[0167] The LTCC I Na+-Ca2+ exchanger (NCX) balance is altered revealing a -30% reduction in the Ca2+ channel expression, whereas the NCX expression is unaltered. The RyR2 / SERCA2 balance appears also to be altered, in which RyR2 expression is unchanged but SERCA2 expression is -20% reduced. SERCA activity showed a -13% reduction in PVC-CM. These changes point to a decrease in Ca2+ cycling mainly driven by a reduction in LTCC expression and function, and to a lesser extent SERCA alterations. Importantly, sympathetic activity is chronically hyperactivated by PVCs but it is accompanied by a decrease in phospholamban and cardiac troponin I phosphorylation, indicating that downregulation and / or desensitization of the 0-adrenergic signaling may contribute to the decrease in LVEF. Moreover, the recent finding of prolongation of electromechanical latency (EML) in PVC-CM can also be explained by this Ca2+ handing remodeling.

[0168] Fibroblasts are key players in extracellular matrix remodeling leading to fibrosis. Fibroblasts (vimentin (VIM) positive cells) have been identified and VIM increased in several PVC-CM samples (FIGs. 4 and 5). In addition, the concerted interplay between fibroblasts, pericytes, and endothelial cells are all in play during the angiogenic response. New capillaries are key to provide adequate blood supply during an adaptive hypertrophic response in PVC- CM66. While diffuse interstitial fibrosis has been demonstrated by prior groups on a swine PVC-CM model, the data demonstrates for the first-time increased angiogenesis (FIGs. 6 and 7).

[0169] Interstitial fibrosis is the product of fibroblast proliferation and activation (FIG. 8) and conveys detrimental consequences such as tissue stiffness and altered EP properties (e.g.,ATTORNEY DOCKET NO. 37759.0634P1 conduction velocity). Fibroblasts can also secrete cytokines affecting cardiomyocyte function. For instance. IL-1 (FIG. 4) (implicated in reducing LTCC expression in cardiomyocytes) could contribute to the pathological phenotype of PVC — CM described above.

[0170] VEGFs, strong pro-hypertrophic and pro-angiogenic factors, were elevated in PVC- CM together with an increase in blood vessels and capillaries (FIG. 4). The increase in endothelial cells forming capillaries (isolectin B4 (IB4) staining, FIG. 7) is balanced with a trend of increase in cells morphologically identified as pericytes (positive a-SMA staining, FIG. 6).

[0171] Global RNAseq results and preliminary single nuclei RNAseq studies. RNAseq was performed to identify potential pathway s / mechanisms altered in PVC-CM. This showed an upregulation of genes involved in extracellular matrix remodeling (FIG. 9), correlating strongly with fibrosis in the PVC-CM model (FIG. 8). In addition, genes related to cytokine activity were upregulated (FIG. 9). Although the global RNAseq data has been invaluable, transcriptional data at the single cell level is lacking. Single-nuclei RNAseq technology' is revolutionizing the study of heart remodeling. A single nuclei RNAseq analysis was performed in PVC-CM (FIG. 10, one sample each). Data reduction using Uniform Manifold Approximation and Projection (UMAP) identified 14 clusters, assigning cell identity to them based on gene expression (FIG. 10). Non- cardiomyocyte nuclei corresponded to 18% in Sham and 31% in PVC. This increase in cells other than cardiomyocytes is consistent with the cardiac hypertrophic remodeling observed in PVC-CM. Note that in the swine, cardiomyocytes may have up to 4 nuclei thus number of nuclei is not directly proportional to the number of cardiomyocytes. Interestingly, the increase in non- cardiomyocyte cells was driven by macrophages / monocytes, lymphocytes, pericytes, fibroblasts, and endothelial cells (FIG. 10), which is consistent with changes in the cytokine landscape, diffuse fibrosis, and angiogenic adaptations observed in PVC-CM. In addition, myocytes were identified forming cluster 3 in the Sham were transformed into cluster 2 in the PVC-CM (FIG. 10). Gene expression between these two clusters shows a reduction in CACNA1C (CaV1.2), JPH2 (junctophilin 2), and KCND3 (Kv4.3, Itof) mRNA in PVC-CM, consistent with reductions previously reported, strongly validating the snRNAseq technology.

[0172] SGLT2i (Empagliflozin) is an antifibrotic therapy. These findings place fibroblasts and fibrosis in the center of PVC-CM. The expression of SGLT2 protein was discovered in the isolated cardiac fibroblast and LV tissue (FIG. 11). VIM, a marker of fibroblasts, was elevated in PVC-CM, and empagliflozin treatment inhibited its increase in two swine exposed to PVCs (FIG. 12). Moreover, interstitial fibrosis and collagen mRNA (qPCR) elevation observed in PVC-CM is reduced by empagliflozin (FIG. 12). Finally, these data show that empagliflozinATTORNEY DOCKET NO. 37759.0634P1(SGLT2i) inhibits fibroblasts in vivo. SGLT2 inhibitors (i.e., empagliflozin FDA approved), due to inhibitory effects of fibroblasts, can be used to interfere with the structural remodeling (i.e., interstitial fibrosis) and role in preventing PVC-CM and VArr.5. Research Strategy and Methods

[0173] The mechanism(s) responsible for interstitial fibrosis in PVC-CM has not been elucidated, yet it can serve as a substrate for VArr with significant implications for SCD in patients with frequent PVCs and PVC-CM. The main purpose of this study is to compare the adverse cardiac, tissue, cellular and molecular remodeling due to LV dyssynchrony (LVD) with different PVCs originating from RV apex (RV-PVCs; most LVD), CS (CS-PVC; least LVD) and sham (no PVCs, no LVD). While premature atrial contractions (PACs) lack dyssynchrony, they are not a great comparison since ventricular coupling interval (CI) cannot be controlled due to AV nodal decremental conduction, frequently resulting in a variable prematurity. Moreover, inhibition of fibroblasts activation with SGLT2i (empagliflozin) can be assessed to prevent or minimize adverse cardiac remodeling and its impact on minimizing EP remodeling and VF vulnerability associated with interstitial fibrosis. To achieve this goal, swine (male and female) can be exposed to chronic states of bigeminal PVCs (50% burden) with different degrees of LV dyssynchrony (RV-PVCs and CS-PVCs) and sham (no dyssynchrony) for 12-weeks. i. Animal preparation and Pacemaker implantation

[0174] Under general anesthesia, 60 animals (power analysis below) will undergo a transvenous pacemaker implant, inserting a bipolar endocardial lead (Medtronic lead 3830, and sheath CS31536; via right external jugular vein) lead to reproduce either RV-PVCs or CS-PVCs connected to an experimental device (Abbott, Inc., St. Paul, MN, USA) with the premature pacing algorithm. CS pacing lead implant (conduction system pacing) in swine is feasible. A biopotential DSI transmitter (Li l, DSI, St. Paul, MN. U.S.A) can be implanted subcutaneously to obtain ambulatory cardiac telemetry'. ii. Group randomization

[0175] After a 2-4 week post-operative period, all swine can be randomly assigned to 5 different groups: 1) RV-PVCs (50% burden; CI=350ms, n=12). 2) CS-PVC (50% burden; CI=350ms, n=12), 3) Sham (no PVCs, n=12) and 4) RV-PVCs+SGLT2i (50% burden; CI=350ms, n=12), and 5) Sham+SGLT2i (no PVCs, n=12). Regardless of group assignment, all animals will undergo echo, telemetry' and electrophysiologic study (EPS) at baseline and during the 12-week protocol (FIG. 13).ATTORNEY DOCKET NO. 37759.0634P1 iii. Simulation of bigeminal PVCs (50% burden)

[0176] After all baseline procedures are completed, the device can be enabled to initiate a 12-week exposure based on group assignment. Experimental devices with the premature pacing algorithm can allow us to reproduce 50% burden with PVC origin depending on randomization group. A PVC CI of 350ms was chosen since swine has a longer effective refractory period up to 280ms and will guarantee the aim of 50% burden with significant LVD. Sham group can have all pacing features disabled. iv. Invasive and non-invasive procedures

[0177] Echocardiographic evaluation. Serial echocardiograms (Vivid-7, 5MHz cardiac probe, General Electric, U.S.A.) will be performed in each animal at selected time points (FIG. 13). All echocardiographic parameters (FIG. 14) will be obtained as per guidelines without PVCs at least 15 minutes after disabling pacing algorithm. Different parameters of LV mechanics will be obtained (Table 1). LV dyssynchrony will be assessed by the dispersion of QRS-to-Peak LV strain using speckle-tracking analysis (EchoP AC, GE, U.S.A) in short axis view as recent publications. Electromechanical latency (EML) will be assessed as previously- reported (QRS onset to first radial strain peak regardless of segment).

[0178] Electrophysiologic study (EPS). Programmed ventricular stimulation with singles, doubles and triples (S1-S2, S3 and S4) will be performed from RV apex and LV free w all to obtain VERP and assess arrhythmia inducibility during survival and final surgeries.

[0179] Cardiac telemetry (DSI transmitter). Ambulatory single-lead ECG throughout the 12-week period (FIG. 13) will assess true PVC burden and changes in spontaneous atrial and ventricular arrhythmias. v. Euthanasia and sample collection:

[0180] All animals will be euthanized after completing the 12-week protocol. The heart will be harvested to undergo either a) whole-heart Langendorf perfusion for optical mapping60 to obtain EP properties including mean and dispersion (max - min) of conduction velocity (CV), activation time (AT), repolarization time (RT) and APD; and b) tissue collection for molecular and snRNA seq (snap frozen in liquid nitrogen, stored at -8042), tissue staining and immunochemistry analysis (formalin fixation).6. Explore the degree of LV dyssynchrony as a main trigger of interstitial fibrosis and development of PVC-CM.

[0181] Many patients do not develop PVC-CM despite high burden PVCs. LVD has been shown to be a more consistent predictor for the development of PVC-CM in the clinical setting.ATTORNEY DOCKET NO. 37759.0634P1LVD also correlates with the degree of LV dysfunction and interstitial fibrosis in the swine PVC-CM mode, but the mechanism(s) behind-and implications of- interstitial fibrosis have not been elucidated.

[0182] LVD causes mechanical myocardial stress that triggers adverse cardiac remodeling. It is known that fibroblasts are key hubs constantly sensing and assessing the microenvironment via mechanoreceptors sensitive to mechanical stress. Fibroblasts are directly secreting metalloproteinases needed to remodel the extracellular matrix, producing collagens, and working concertedly with pericytes and endothelial cells in angiogenic responses to support hypertrophy. Data has show n an increase in IL- 10 that can be released by activated fibroblasts. Thus, fibroblasts play a key role in the development of PVC-CM and its phenotype. More specifically, RV-PVCs (most LVD) can demonstrate a higher degree of adverse cardiac remodeling including LV dysfunction QLVEF), eccentric hypertrophy, fibroblast activation, interstitial fibrosis, cellular hypertrophy and angiogenesis when compared to CS-PVCs (least LVD), whereas CS- PVCs can demonstrate a lower degree of adverse cardiac remodeling when compared to those without PVCs (sham group, no LVD). i. Evaluate severity of cardiac structural remodeling (in-vivo) and spontaneous arrhythmias in PVC-CM between different degrees of LV dyssynchrony. a. Method

[0183] PVCs that have identical CI and subsequently identical PESP with significantly different LVD, which can be achieved by comparing 50% PVC burden originating from RV apex (RV-PVCs, most LVD) vs. from CS (CS-PVCs, least LVD). Thus, a comparison of swine randomized to the following groups: 1) sham (lacking PVCs; no LVD), 2) CS-PVCs (least LVD), 3) RV-PVCs (most LVD).

[0184] After a 2-week recovery from pacemaker implant (Section A), device will be enabled to delivered PVCs based on group assignment. A series of invasive and non-invasive procedures will be performed (FIG. 13) to assess the cardiac remodeling of PVC-CM after 12 weeks in all 3 groups. Echocardiograms, ECG, and telemetry will be reviewed offline by a trained cardiologist blinded to group assignment. ECG and telemetry will be analyzed for the presence of spontaneous VArr, QRS duration. QT duration with and without PVCs. Echo parameters (Table 1) and number of spontaneous non-sustained and sustained VArr will be quantified in all 3 groups.Table 1. Echocardiographic parametersATTORNEY DOCKET NO. 37759.0634P1S, septal; P, posterior; LVEDD, LV end-diastolic dimension; LVESD, LV end-systolyc dimension b. Data Analysis / interpretation and Expected Outcomes.

[0185] Final echo parameters, QRS duration, QT duration and VArr for each group (RV- PVCs, CS-PVC, sham) will be compared to their baseline, while these same variables will be compared between the 3 groups at 3 months. The RV-PVC group candevelop a more severe PVC-CM phenotype (decrease in LVEF, increase in LVEDD LVESD and LV mass consistent with eccentric hypertrophy and delay in EML) compared to baseline and the other groups, while CS-PVCs will develop a minimal or mild phenotype compared to baseline and sham group. Spontaneous VArr can be higher in the RV-PVC, while CS-PVCs can be similar to sham with minimal or no VArr. ii. Assess severity of adverse tissue, cellular and molecular remodeling (in-situ) between different degrees of LV dyssynchrony. a. Approach / Methodology

[0186] 3 groups (RV-PVC, CS-PVC, Sham) can be studied assessing tissue, cellular and molecular remodeling (FIG. 14). After euthanasia, tissue can be collected and analyzed for:

[0187] Cardiomyocyte hypertrophy in situ can be assessed by measuring cardiomyocyte dimensions in cross-sections as previously described66. LV fixed tissue (10% neutral buffered formalin. NBF) longitudinal sections will be stained with WGA conjugated to AlexaFluor633 (WGA-AF633) and DAPI. WGA stains the contour of cells and DAPI is used as counterstain.

[0188] Confocal images (Zeiss LSM 700) will be analyzed using artificial intelligence (AI)- powered AIVIA software (Leica microsystems, Bellevue, WA, USA). Width and length of each myocyte will be measured automatically for each image.

[0189] Interstitial and perivascular fibrosis will require the following: (a). Sirius Red / Fast Green staining. 10% NBF fixed tissue will be paraffin embedded, sectioned (10 pm-thick), rehydrated, and stained with sirius red / fast green to visualize collagen fiber86. Images will be acquired using a color camera (Axio Imager Z2 microscope). Area covered (%) by collagen fibers will be quantified using ImageJ software in at least 20 images per animal, (b.) Transmission electron microscopy (TEM by Dr. Samso). LV samples fixed with 4%ATTORNEY DOCKET NO. 37759.0634P1 glutaraldehyde, 0.1M cacodylate, pH 7.4. will be stained using 2% osmium tetroxide and uranyl acetate. Images can be acquired on a Tecnai F20 or a Jeol JEM-1230 TEM with a Gatan UltraScan 4K x 4K. CCD camera. The size of the interstitial space will be visualized (FIG. 8) from at least 20 sections per animal, ii.c. Western blot (WB) and qPCR. WB will measure vimentin (VIM), MMP9, expanding to other markers including metalloproteinases MMP8, MMP3, ADAMTS9, and ADAMTS4 upregulated in PVC-CM (FIG. 9). qPCR will measure mRNA levels of collagen I and collagen III as an orthogonal method to determine fibroblast activation and fibrosis. WB will be performed following protocols previously published using horseradish peroxidase conjugated-secondary antibodies and enhanced chemiluminescent (ECL) reagent for detection. qPCR, total RNA will be extracted from LV samples (RNeasy mini kit, Qiagen). Genomic DNA elimination and reverse transcription will be performed with iScript gDNA Clear cDNA Synthesis kit (BioRad). qPCR will be performed on a CFX Opus 96 Realtime PCR System (BioRad). Data will be analyzed by the 2-AACt method using GAPDH as a housekeeping gene.

[0190] ii.d. Immunofluorescence for quantitative analysis (AIVIA software). Fibroblasts will be quantified in cross-sections of NBF-fixed tissue slices (40 pm thick) of LV tissue samples stained with an anti-vimentin antibody and using Alexa Fluor-488 conjugated secondary antibody (FIG. 5). In addition, the total number of cardiomyocytes will be quantified in the same image using WGA-AF633 staining (FIGS. 6 and 7) to assess the ratio of fibroblasts per myocyte. Confocal images (Zeiss LSM 700) will be analyzed using AIVIA software trained to identify shapes compatible with fibroblasts while filtering out blood vessels. Hundreds of cells per image in at least 20 images per animal will be analyzed allowing a robust statistical analysis.

[0191] Angiogenesis will require the following:

[0192] Immunofluorescence. Number of pericytes, number and dimensions of capillaries, and small blood vessels will be quantified providing further evidence of increased angiogenesis. NBF-fixed tissue slices will be stained using IB4 (selective for endothelial cells, Fig 5). a- Smooth muscle actin (aSMA) stains both smooth muscle in blood vessels and cells morphologically compatible to pericytes (intimately attached to capillaries. FIG. 5). By using the AIVIA software these two structures can be easily separated for quantitative analysis (FIG. 6). WGA staining will be used to quantify' the number of myocytes and pericyte per cardiomyocyte, number of blood vessels per field, and the total surface covered by blood vessels per image. Pericytes can be a heterogeneous cell population and will be carefully identified with markers such as neuron-glial antigen 2 (NG2), EGF-Like, Fibronectin Type-Ill and Laminin G-LikeATTORNEY DOCKET NO. 37759.0634P1Domain-Containing Protein (EGFLAM), and Guanylate Cyclase 1 Soluble Subunit Alpha 2 (GUCY1A2).

[0193] WB and qPCR (Section ii.c). Angiogenesis markers will include aSMA (smooth muscle and pericyte markers); NG2, EGFLAM, GUCY1A2 (pericyte marker); Platelet Endothelial Cell Adhesion Molecule (CD31) and endothelial nitric oxide synthase (eNOS) (endothelial cell makers).

[0194] Cell-type populations and transcriptomics (Dr. Dozmorov). snRNAseq experimental and computational analysis will be performed following best practices guidelines87,88. The 10X Genomics Chromium system enables 3’ mRNA digital counting of thousands of single cells / nuclei89. Resulting cDNAs containing unique molecular identifiers (UMIs) and GEM- specific barcodes will be pooled for amplification and library construction. Chromium Single Cell 3’ Reagent Kit (v3 Chemistry) will allow library preparation.

[0195] Raw FASTQ files will be processed with CellRanger software (v7.2) to perform alignment, filtering, barcode counting and UMI counting and utilize the Nextflow90 “scmaseq” pipeline for reproducibility. After several quality control steps, the data will be normalized using global scaling or a single-cell-specific robust normalization approach. The data will then be log- transformed before further downstream analysis with Seurat. Subpopulations of cells will be detected using Principal Components Analysis (PCA) coupled with t-distributed Stochastic Neighbor Embedding (t-SNE) and UMAP. Cell types will be annotated with SingleR and SCimilarity. Loupe Cell browser v.6.57.0 (10X Genomics) will be used for data visualization and subpopulation selection. Genes DE between subpopulations will be performed using edgeR58 R package, as well as Wilcoxon test and MAST, implemented in Seurat. P-values for DE genes will be corrected using a False Discovery Rate (FDR) multiple-testing correction method. Functional interpretation will be performed using IPA and the R packages enrichR and clusterProfiler using the MSigDb gene signatures. The pipeline will be implemented using the Department of Biostatistics High-Performance Computing cluster (see Resources at VCU). b. Data Analysis / interpretation and Expected Outcomes.

[0196] Using all methods mentioned above, cardiomyocyte hypertrophy, interstitial and perivascular fibrosis, angiogenesis and cell type / transcriptomics can be compared between the 3 different groups at 12-weeks. The RV-PVC group can develop a more severe adverse remodeling phenotype (cellular hypertrophy, increased interstitial and perivascular fibrosis and angiogenesis) compared to other groups (CS-PVCs and Sham), while CS-PVCs will develop a minimal or mild phenotype compared to sham, supporting the hypothesis that LV dyssynchronyATTORNEY DOCKET NO. 37759.0634P1 is a primary' trigger for adverse structural remodeling in PVC-CM.7. Assess fibroblast inhibition to prevent cardiac remodeling in PVC-CM.

[0197] Fibroblast activation is responsible for the adverse cardiac remodeling including fibrosis, angiogenesis and hypertrophy observed in PVC-CM. Therefore, it is possible that this adverse remodeling could be prevented by inhibition of fibroblast activation with SGLT2i (empagliflozin, FIG. 12). Empagliflozin has been shown to reduce cardiovascular mortality and hospitalizations in patients with HF without a clear mechanism. SGLT2i can prevent or minimize fibroblast activation, interstitial fibrosis, LV systolic dysfunction, eccentric (echo) and cellular hypertrophy and angiogenesis caused by RV-PVCs (most LVD) with a similar phenot pe to those without PVCs (sham, no LVD). A swine study showing that adverse cardiac remodeling (LV dilatation, fibrosis) was prevented with empagliflozin after myocardial infarction. i. Approach / Methodology

[0198] All 2 PVC groups (RV-PVC. RV-PVC+SGLT21) with identical CI (350ms, identical PESP) are compared with 2 Sham groups with and without SGLT2i (Sham, and Sham+SGLT2i). SGT2i (empagliflozin). A dose of 0.1 -0.5 mg / kg of empagliflozin will be administered (p.o.) daily throughout the 12-week PVC protocol61 to inhibit both fibroblast activation and subsequent fibrosis. Specially designed veterinary' grade empagliflozin treats can be used to ensure the effective delivery of the drug and “placebo” pills will be administered to the Sham animals. Echocardiography, ECG, telemetry', tissue, cellular and molecular analysis will be identical to that described above. This aim will provide a thorough description of the effect of SGLT2i on fibroblasts and LV cardiac remodeling induced by 12-weeks of frequent PVCs. ii. Data Analysis / interpretation and Expected Outcomes.

[0199] LV remodeling data from RV-PVC and Sham groups in Aim la and lb (Table 1 and FIG. 14) will be compared to those randomized to interv ention with SGLT2i (RV-PVC+SGLT2i and Sham+SGLT2i) at 12 weeks. RV-PVC+SGLT2i group will allow' us to assess inhibition of fibroblast activation when compared to RV-PVC, while Sham+SGLT2i will allow us to assess the effects of SGLT2i alone when compared to Sham (without SGLT2i). Based on the data, markers of adverse cardiac remodeling in RV-PVC including interstitial fibrosis and fibroblast activation, adaptive eccentric and cellular hypertrophy, and angiogenesis can be markedly attenuated with SGLT2i despite similar PVC exposure (RV-PVC+SGLT2i). SGLT2i (empagliflozin) can attenuate or prevent LV dysfunction (LVEF below 50%). Any adverseATTORNEY DOCKET NO. 37759.0634P1 effect of empagliflozin (Sham+ SGLT2i) can be monitored compared to Sham. As reported in preclinical studies, interstitial fibrosis in PVC-CM remains after elimination of PVCs despite recovery of LVEF. Therefore, even if LVEF declines in RV-PVC+SGLT2i group, reduction in cellular and molecular remodeling (namely interstitial fibrosis and hypertrophy) may provide strong preclinical information for its potential clinical application to improve outcomes in PVC- CM22-24. The snRNAseq data can provide a high throughput orthogonal method to support the physiological, structural, and molecular outcomes described above. snRNAseq can show changes in cell population that would support conclusions whether adverse remodeling is increasing by 12-weeks of frequent PVCs. In addition, the effect of Empagliflozin on snRNAseq results will be fundamental to better understanding the mechanisms by which this drug interferes with cell type population changes in this cardiac remodeling. Also, the study of up- and down- regulated genes in each cluster can provide an infinite source of information to design new hypothesis of how PVCs induce the pathological changes in the heart and how SGLT2i can modify or prevent their pathological effect for future studies.8. Evaluate the effects of diffuse interstitial fibrosis in cardiac electrophysiological properties and arrhythmia induction in PVC-CM.

[0200] The initial studies demonstrated cellular EP remodeling characterized by APD prolongation and dispersion with changes in ion currents (Ito, IKr, ICa) and increased VERP. In vivo and in silico studies show that interstitial fibrosis impacts propagation of depolarizing electrical waves, resulting in late activation zones and dispersion of CV, repolarization gradients, and generation of spatially discordant voltage altemans or Ca2+ altemans43. These together with impaired Ca2+ mishandling and APD prolongation, provides a composite substrate for VArr and increased risk of SCD in PVC-CM and many other non-ischemic CM42. Thus, it is paramount to understand the EP remodeling associated with interstitial fibrosis as well as the effects of preventing or minimizing interstitial fibrosis by inhibition of fibroblast activation using SGLT2i. Changes in CV across RV and LV are different leading to increased dispersion of CV, AT, and RT in PVC-CM, which in turn increases VT / VF vulnerability. Finally, inhibition of fibroblasts with SGLT2i can minimize such electrical remodeling in PVC- CM and VT / VF vulnerability. i. Approach / Methodology

[0201] Optical mapping with dual dyes (near-infra red transmembrane voltage CryoVolt-2 and green excitation intra_,cellular Ca2+ concentration dye Rhod-2) recordings will be performed from the RV-PVC (n=5). RV-PVC+SGLT2i (n=5), and 2 sham groups (Sham andATTORNEY DOCKET NO. 37759.0634P1Sham+SGLT2i, n=5 each) with a whole-heart Langendorf preparation as previously performed60. EP parameters including APD, CV. AT, RT, VERP will be obtained (high-speed EMCCD Evolve 128 camera / multiple IMX273 sensor-based CMOS cameras) from different ventricular epicardial regions (RV and LV outflow, RV and LV free wall and apical and inferior LV). In contrast, endocardial recordings of RV and LV will be limited to free wall with light guides inserted into the ventricles. As previously performedl06, different excitation wavelength (520 nm and 650 nm) and dyes (Di-4-ANNEPS and CryoVolt-2) will be recorded from different depths across the tissue to quantify the difference across the transmural wall. Dispersion of these EP parameters between different regions (max - min) will be calculated. EP properties (CV, AT, RT, APD) will be obtained during atrial pacing in 3 different conditions: 1) intrinsic conduction (normal activation), 2) bigeminal PVCs at different CI and 3) EPS (Section D above) to assess VERP and VF vulnerability. Once EPS protocol is completed, the heart will be fixed by perfusing with formaldehyde for assessment of interstitial fibrosis with specific interest in areas with documented slow conduction, re-entry and prolongation of APD. Finally, in silico models will be developed using openCARP cardiac electrophysiology simulation software!07. These models will simulate the same three pacing protocols described above (intrinsic, bigeminal PVCs and EPS) in 3D slabs of myocardial tissue with varying degrees of fibrosis and cellular EP remodeling. EP parameters described above will be evaluated as a function of the severity of phenofypical changes of the myocardium. In silico experiments will enhance the interpretation of optical mapping data and facilitate a mechanistic assessment that reduction in fibrosis can reduce inducibility of VArr. ii. Data Analysis / interpretation and Expected Outcomes.

[0202] EP parameters from different ventricular regions (RV and LV outflow, RV and LV free wall and apical and inferior LV), dispersion of these parameters and VF inducibility will be compared between the RV-PVC, RV-PVC+SGLT2i and Sham (no PVCs, no fibrosis).Moreover, EP parameters during all 3 different conditions will be correlated with interstitial fibrosis. Areas of interstitial fibrosis in animals exposed to RV-PVCs (most LVD) can correlate with areas of slow CV. prolongation of APD, AT and RT, higher dispersion of CV, AT and RT. In contrast, RV-PVC+SGLT2i group can have similar EP parameters compared to Sham due to minimal or lack of interstitial fibrosis despite exposure to identical PVC burden than RV-PVC group. Interstitial fibrosis in RV-PVC group can correlate with inducibility' of VArr when compared to Sham (without interstitial fibrosis), while RV-PVC+SGLT2i group can have similar EP properties to Sham. Using in silico experiments. EP variables can be manipulated toATTORNEY DOCKET NO. 37759.0634P1 determine the interaction between fibrosis and expression of ion current channels and Ca2+ handling dynamics. Moreover, a wide range of scenarios will be tested including a parametric sweep for the level of EP adaptation (minimal to extreme changes in related ionic current / channels) to map out correlations between cellular-level EP adaptations and the tissuelevel phenomena measured experimentally (APD, CV, AT, RT, VERP and VArr). Minimal EP adaptation would be needed to match experimental measurements from the RV-PVC+SGLT2i group, while more substantial adaptations would be required to match the RV-PVC group. iii. Statistical analysis, sample size I power calculations

[0203] Statistical analysis is supported by VCU School of Medicine, Dept, of Biostatistics using SAS / STAT® Software (SAS Institute, Inc. Cary, NC). Paired t-tests will compare variables at baseline vs. 12-weeks (end of protocol) for each PVC group. Multi comparison test such one-way and two-way ANOVA will compare variables between different experimental groups. For immunofluorescence studies and TEM analyzed using the AIVIA software, several images will be analyzed per animal (at least 20 fields from 5 slides) and a robust statistical analysis using hierarchical statistics such as nested one-way ANOVA (GraphPad Prism 10.1.0) will be used to determine differences between experimental groups.

[0204] Detailed sample size estimation and power analysis is described in the Animal subject section. Based on prior echocardiographic data from prior swine PVC-model, 10 animals in each group can provide a power 0.85 (Type I error probability of 0.05). Based on SD from previous WB and fibrosis determinations, a sample size of at least 7 animals in each group with an alpha-error of 5%, can provide a power above 85% to identify differences in cardiac remodeling. For snRNAseq studies, 5,000 nuclei per experiment will give over 50 cells in a rarest cell population (~2%, assuming 10 cell types), providing >80% power to detect >1.5 foldchange (Wilcoxon test, at a<0.05 level) in cell-type-specific gene expression. Based on CV studies in fibrosis, 5 ventricular samples per animal (n=5) for each group (RV-PVC vs. Sham) are needed to demonstrate effects of fibrosis in CV (a-error 5%; 85% power). iv. Animal Procedures

[0205] Animals will undergo survival and non-survival surgeries and non-invasive procedures outlined in Fig 10. Further details of procedures are provided below:

[0206] Survival surgery. Animals will be anesthetized with sodium thiopental (30-35mg / kg) followed by inhaled isoflurane to achieve a surgical plane of anesthesia. Durapen 3ml SC will be administered 12 hours prior to surgery. All animals will be intubated and mechanically ventilated. ECG will be monitored continuously during this procedure. Animals will be placedATTORNEY DOCKET NO. 37759.0634P1 on a heating pad during the protocols and body temperatures will be monitored. All animals will have venous access through which fluids can be given to prevent dehydration. The level of anesthesia will be continuously monitored by observation of heart rate during surgical manipulations. Any increase in heart rate in response to surgical stimuli will be interpreted as indicative of inadequate anesthesia and supplemental doses will be given. In addition, corneal, palpebral, and toe-pinch responses will be monitored every 15 minutes and supplemental anesthesia given as needed. While under general anesthesia, left heart catheterization, ECG and pacemaker and DSI transmitter implant will be performed as outlined in research plan.

[0207] At the end of all planned procedures, all animals will be monitored for return of spontaneous respirations and movement (i.e. lifting head). When animals are able to breathe without the assistance of a ventilator for at least 10 minutes and when they show the ability to swallow, the endotracheal tube will be removed. The animal will be monitored for an additional 10-15 minutes to ensure that secretions are being adequately handled. Postoperatively, all animals will be examined at least twice a day for the first 5 days. Standard wound care with furizolidone spray will be provided. Temperature and intake / output will be assessed. If oral intake is limited, supplemental feedings or intravenous fluids will be given. The animal’s behavior will be used to assess for post-op pain. Durapen 3cc will be administered SQ daily for a total of two doses as prophylaxis. Buprenorphine 0. l-0.2mg / kg IM every 8-12 hours will be administered as needed for post-surgical pain. Any animal in pain or distress that cannot be adequately treated with analgesics will be euthanized. Otherw ise, the animals are expected to tolerate the procedure well, begin eating again usually on the first post-op day and recover rapidly. Signs of distress and pain can be suspected in the presence of weight loss, lethargy, limping, vocalizing, excessive licking and even aggression.

[0208] Pacemaker implant. The jugular vein will be accessed to implant the endocardial leads. The lead(s) will be guided and positioned with fluoroscopy and screwed the interior surface of the RV apex. The risk of ventricular arrhythmias including VF is present during implantation of this ventricular lead. An incision will be made, and a pocket will be created between the muscular fascia and subcutaneous tissue in the side of the neck. The device can be implanted in this pocket and the lead(s) can be tunneled then secured to the device. The pacemaker will be positioned and sutured if necessary, with a 0-silk to muscular fascia of the pocket. Appropriate device and lead function will be confirmed again prior to w ound closure. Optionally, prolene mesh can be used subcutaneously to help secure devices and wound closures. Prolene mesh is a stenle. non-absorbable material that high tensile strength for strongATTORNEY DOCKET NO. 37759.0634P1 wound repair. It can be sutured to the muscle and deep skin layers to help support subcutaneous devices and to help keep the sides of the incision correctly opposed.

[0209] Radiotelemetry device implant. A Data Science International (DSI) telemetry device will be implanted after pacemaker implant. One or 2-biopotential channel transmitter will be implanted to obtain ambulatory cardiac telemetry. A pocket on the side of the neck could be created for the device and the leads tunneled to the carotid incision. Optionally, prolene mesh can be used subcutaneously to help secure devices and wound closures. Prolene mesh is a sterile, non-absorbable material that has high tensile strength for strong wound repair. It can be sutured to the muscle and deep skin layers to help support subcutaneous devices and to help keep the sides of the incision correctly opposed.

[0210] Electrophysiologic study (EPS)

[0211] A short EP study may be performed under general anesthesia during survival surgery and final surgery7to observe hemodynamic changes and / or arrhythmia susceptibility before and after development of PVC-cardiomyopathy. VPS can be performed on all animals through the implanted cardiac device and / or lead(s) to determine ventricular effective refractory period (VERP) and test susceptibility of ventricular arrhythmias. VPS consists on a series of painless pacing stimuli using to understand the refractoriness of the ventricular myocardium and vulnerability of ventricular arrhythmias. VPS has a generic protocol, where a total of 8-10 pacing stimuli are delivered at 400 or 600ms, followed by a single or double extra stimulus with a sequential 10ms decrement from 400 to 150ms. This procedure is painless in humans and has been performed in canines and swine as well without any discomfort or pain. However, in the rare event, sustained ventricular arrhythmias are induced, an external defibrillator will be used to restore normal rhythm.

[0212] Non-Survival surgery7. Swine will be anesthetized with sodium thiopental (30- 35mg / kg) followed by inhaled isoflurane to achieve a surgical plane of anesthesia. All animals will be intubated and mechanically ventilated. ECG will be monitored continuously during this procedure. Perioperative care will be similar to survival surgery, as they will be placed on a heating pad and have venous access. Similar to survival surgery, the level of anesthesia will be continuously monitored by observation of heart rate during surgical manipulations. Procedures (electrophysiologic study, ECG) as indicated by7protocol will be performed during non-survival surgery7. Animals will be euthanized as described below.

[0213] Pacemaker interrogation. After pacemaker implantation, device will be interrogated every 2-4 weeks to assess for appropriate function, evaluating R wave amplitude (amplitude ofATTORNEY DOCKET NO. 37759.0634P1 ventricular signal), pacing thresholds, histograms and percentage of pacing. This will be performed via a St. Jude Medical programmer. The programmer is an external device similar to laptop that has a '‘wand”. The wand is positioned close to the device and allows pacemaker evaluation and programming. This process is not painful and will not represent any distress to the animal. No restraint is expected to be required to perform pacemaker interrogation. Pacemaker interrogation will last from 5 to 20 minutes depending on the findings.

[0214] Echocardiogram. This is a painless non-invasive procedure and should not cause any distress to the animal. However, it requires that the animal lays still on the right and left side for at least 10 minutes to obtain accurate cardiac images. Therefore, 2 different approaches can be required to obtain echocardiogram: 1) animal training with or without restraint, and / or 2) general anesthesia. Animal training to lay still on the right and left side for at least 10 minutes is one approach. However, if the animal does not cooperate, echocardiograms can be performed under general anesthesia. Sodium thiopental (15-20 mg / kg) can be administered for short-term anesthesia, which appears to have minimal cardiovascular effects without change in LV ejection fraction. Animals will be continuously monitored by ECG. Baseline echocardiogram will be performed at least 2 weeks after pacemaker implantation and appropriate recovery of the animal. Subsequent echocardiograms will be performed at 4-, 8- and 12-weeks until completion of the protocol. Echocardiogram can take from 15-20 minutes to obtain all required data. Each animal can undergo anywhere from 4 echocardiograms as described in the protocol.

[0215] Continuous cardiac telemetry. This is a painless noninvasive cardiac rhythm monitoring performed commonly in patients and is expected to cause any distress to the animal. Through the subcutaneously implanted DSI transmitter, continuous telemetry recordings can be obtained at week baseline, week 4, 8 and 12. This is performed by activating (through a magnet) the transmitter to initiate cardiac telemetry recording when animal is placed in a DSI telemetry capable dog run (3 x 6 ft. cage). After 5 day -cardiac telemetry is completed, Dataquest ART analysis system will allow analysis of heart rate and PVC burden or percentage.C. Example 3 - SGLT2 inhibitors as a Novel Therapy to Prevent Interstitial Fibrosis and Ventricular Arrhythmias

[0216] Premature ventricular contractions (PVCs) are the most prevalent ventricular arrhythmia and frequent PVCs are associated with heart failure (HF), ventricular arrhythmias and sudden cardiac death (SCD).

[0217] Modified pacemakers can be used to deliver PVCs. The current group has developed the first large animal model of PVC-induced cardiomyopathy (PVC-CM). The large canineATTORNEY DOCKET NO. 37759.0634P1 model of frequent PVCs and PVCCM has led the characterization and understanding of the triggers and potential mechanism of PVC-CM. Similar to the canine, the swine PVC-CM model has shown diffuse interstitial fibrosis that persists despite elimination of PVCs. Recently, the PVC-CM model has show n an increase in IL- ip and other mediators of inflammation and fibrosis, known to impact the L-type Ca2+ current (LTCC) and myocyte contractility . Yet, one local source of IL- i in the heart are activated fibroblasts, likely activated by mechanical stress during PVCs. Thus, an unexpected cellular player in PVC-CM is the cardiac fibroblast. Our group has identified expression of the sodium glucose co-transporter-2 (SGLT2) in the cardiac fibroblast and show n that an SGLT2 inhibitor (SGLT2i) can significantly decrease activation of fibroblasts and subsequent interstitial fibrosis. Thus, one aspect of this study is that frequent PVCs, through an uncoordinated contraction so-called LV dyssynchrony, are key to trigger fibroblast activation and fibrosis w hich can be significantly lowered by SGLT2i.

[0218] Local or regional myocardial fibrosis assessed by cardiac MRI (late-gadolinium enhancement) has been shown to be an independent predictor for ventricular arrhythmias (VArr) and SCD. Like tachycardia-induced CM, PVC-CM animal models have demonstrated persistent LV interstitial fibrosis. Persistent interstitial fibrosis in patients with tachycardia-induced CM is thought to be responsible for the increased mortality despite recovery of LV function after treatment. A secondary analysis of the CHF-STAT study demonstrated that PVC-CM is not only associated with LV dysfunction but increased mortality if untreated. Thus, persistent interstitial fibrosis in PVC-CM may serve also as a substrate for VArr and SCD despite recovery of LVEF even after PVC ablation.

[0219] In general, initiation of ventricular arrhythmias requires 2 key elements: a trigger and a substrate. PVCs have been well-recognized ‘‘triggers’' for ventricular arrhythmias (VArr), especially in the presence of underlying myocardial substrate, increasing the risk of SCD. Preclinical studies have shown that interstitial fibrosis can significantly affect electrophysiological myocardial properties, which can serve as a “substrate” for the development of VArr and SCD. Another aspect of the current study is that increased adverse interstitial fibrosis triggered by fibroblast activation in PVC-CM leads to EP remodeling (e.g., conduction velocity, activation & repolarization time, action potential duration) and inhibition of fibroblast activation with SGLT2i can prevent such electrical remodeling.

[0220] SGLT2i (empagliflozin, dapagliflozin), initially developed as an antiglicemic agent to treat patients with diabetes. Recently, SGLT2i clinical trials (DAPA-HF, EMPEROR- reduced) have shown a significant survival benefit in patients with LV systolic dysfunction andATTORNEY DOCKET NO. 37759.0634P1HF regardless of the etiology. Currently, SGLT2i are indicated in patients with systolic HF and cardiomyopathy even without diabetes. However, the molecular mechanism(s) of this effect has not been elucidated. Recently, animal models (including swine) have demonstrated that SGLT2i significantly decreased fibrosis in myocardial infarction when compared to Sham. This data shows the expression of SGLT2 in cardiac fibroblasts and SGLT2 inhibition decrease their activation.

[0221] The obvious next step is to test SGLT2i as a therapy to prevent or minimize interstitial fibrosis in PVC-CM and other CM animal models, as well as test in patients at risk to develop CM associated with fibrosis (e.g., post-chemotherapy, tachycardia, left bundle branch block, high ventricular pacing, PVCs, etc.).

[0222] Finding therapies to prevent or minimize extracellular matrix remodeling (i.e., interstitial fibrosis) could not only minimize LV systolic dysfunction but also improve long-term outcomes in patients with frequent PVCs, PVC-CM and other cardiomyopathies associated with fibrosis.

[0223] A crude analogy of the potential use and impact of SGLT2i in clinical care is that of statins. Statins were introduced as a treatment for high cholesterol. Initially, these medications were only indicated in patients after a myocardial infarction (MI) and severe coronary artery disease (CAD). While statins are known to decrease risk of recurrent MI and halt progression of CAD, there is no evidence that these can reverse CAD. Over the last decades, a body of evidence has shown that these medications provide significant morbidity and mortality benefit to patients at risk of CAD and MI. Nowadays, statins are prescribed as a preventive measure in patients with abnormal cholesterol profiles without CAD or MI considered at high-risk based on comorbidities and / or family history. Today, SGLT2i are used only to treat patients with HF and CM. While it improves mortality, it is not clear that SGLT2i reverses fibrosis. Moreover, no treatment is available to prevent or minimize the development of interstitial fibrosis responsible for increasing mortality risk. Based on these studies SGLT2i can have new clinical indications to prevent or minimize the adverse cardiac remodeling including interstitial fibrosis and mortality risk in patients known to be at risk to develop CM and HF such as post-chemotherapy, ostpartum, tachycardia, left bundle branch block, high ventricular pacing and pacemakerdependent, and PVCs.

[0224] Premature ventricular contractions (PVCs) are ectopic ventricular beats that originate in ventricular tissue and result in dyssynchronous contraction due to a delayed activation in some cardiac segments. High burden PVCs progressively lead to cardiac remodelingATTORNEY DOCKET NO. 37759.0634P1 characterized by eccentric hypertrophy and reactive fibrosis, resulting in systolic dysfunction, known as PVC-induced cardiomyopathy (PVC-CM). Recently, it as demonstrated that empagliflozin — a sodium-glucose co-transporter 2 (SGLT2) inhibitor — effectively prevented cardiac remodeling in a swine translational model of PVC-induced cardiomyopathy (PVC-CM) (Fig. 15). This effect was associated with a reduced expression of SGLT2 and vimentin (VIM) in the left ventricular free wall (LVFW) (Fig. 16).

[0225] The potential impact of SGLT2i in the pathophysiology of cardiomyopathy / heart failure and sudden cardiac death is by (1) inhibiting fibroblast activation to halt adverse cardiac remodeling primarily by preventing or minimizing interstitial fibrosis, and (2) decreasing interstitial fibrosis as the substrate of EP remodeling responsible for increased risk of ventricular arrhythmias and SCD.

[0226] The clinical implications are substantial since SGLT2i improves outcomes regardless of LV function in PVC-CM and other ty pes of cardiomyopathies by preventing or minimizing adverse cardiac remodeling through our newly identified "anti 11 brotic" effect. This impacts longterm mortality risk of many patients with a variety’ of cardiomyopathies with interstitial fibrosis as the common feature.

[0227] All of the methods disclosed and claimed herein can be made and executed w ithout undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be w ithin the spirit, scope and concept of the invention as defined by the appended claims.

Claims

ATTORNEY DOCKET NO. 37759.0634P1CLAIMSWe claim:

1. A method of preventing fibroblast activation in a subject comprising administering a sodium-glucose co-transporter-2 (SGLT2) inhibitor to a subject in need thereof.

2. The method of claim 1, wherein the subject in need thereof is a subject at risk of having a cardiomyopathy.

3. The method of any one of claims 1-2, wherein the subject develops a cardiomyopathy and there is decreased diffuse microscopic scarring after the subject develops the cardiomyopathy.

4. The method of claim 2, wherein the subject at risk of having a cardiomyopathy has heart palpitations, premature ventricular contractions, and / or extra heart beats.

5. The method of any one of claims 2-4, wherein the subject at risk of having a cardiomyopathy is currently or has previously been treated with a chemotherapeutic agent.

6. The method of any one of claims 2-5, wherein the subject at risk of having a cardiomyopathy has a heart inflammation.

7. The method of any one of claims 2-6, wherein the subject at risk of having a cardiomyopathy is taking a drug that weakens the heart.

8. The method of any one of claims 2-7, wherein the subject at risk of having a cardiomyopathy has tachycardia9. The method of any one of claims 2-8, wherein the subject at risk of having a cardiomyopathy has sarcoidosis.

10. The method of any one of claims 2-9, wherein the subject at risk of having a cardiomyopathy has ventricular dysplasia.

11. The method of any one of claims 1-10, wherein the SGLT inhibitor is one or more of canagliflozin, dapagliflozin, empagliflozin, besaglifloxin, or ertugliflozin.ATTORNEY DOCKET NO. 37759.0634P112. The method of any one of claims 1-11. wherein vimentin expression is reduced in the cardiac tissue of the subject after administration of the SGLT inhibitor.

13. The method of any one of claims 1-12, wherein the subject does not have diabetes.

14. The method of any one of claims 1-12, wherein extracellular matrix remodeling is prevented or reduced in the subject after administration of the SGLT inhibitor.

15. A method of preventing fibrosis in a subject, comprising administering an SGLT inhibitor to a subject in need thereof.

16. The method of claim 15, wherein the subject in need thereof is a subject at risk of having a cardiomyopathy.

17. The method of any one of claims 15-16, wherein after administration of the SGLT inhibitor, the subject has reduced fibroblast activation compared to a subject not receiving a SGLT inhibitor.

18. The method of any one of claims 15-17, wherein after administration of the SGLT inhibitor, the subject has reduced fibroblast proliferation compared to a subject not receiving a SGLT inhibitor.

19. The method of any one of claims 16-18, wherein the subject at risk of having a cardiomyopathy has heart palpitations, premature ventricular contractions, and / or extra heart beats.

20. The method of any one of claims 16-19, wherein the subject at risk of having a cardiomyopathy is currently or has previously been treated with a chemotherapeutic agent.

21. The method of any one of claims 16-20, wherein the subject at risk of having a cardiomyopathy has a heart inflammation.

22. The method of any one of claims 16-21, wherein the subject at risk of having a cardiomyopathy is taking a drug that weakens the heart.

23. The method of any one of claims 16-22, wherein the subject at risk of having a cardiomyopathy has tachycardiaATTORNEY DOCKET NO. 37759.0634P124. The method of any one of claims 16-23, wherein the subject at risk of having a cardiomyopathy has sarcoidosis.

25. The method of any one of claims 1 -24, wherein the subject at risk of having a cardiomyopathy has ventricular dysplasia.

26. A method of improving survival of a subject at risk of having a cardiomyopathy comprising administering an SGLT inhibitor to the subject.

27. The method of claim 26, wherein fibroblast activation is decreased in the subject after administration of the SGLT inhibitor.

28. The method of any one of claims 26-27, wherein fibrosis is decreased in the subject after administration of the SGLT inhibitor.

29. The method of any one of claims 26-28, wherein diffuse microscopic scarring is decreased in the subject after administration of the SGLT inhibitor.

30. The method of any one of claims 26-29, wherein the subject at risk of having a cardiomyopathy has heart palpitations, premature ventricular contractions, and / or extra heart beats.

31. The method of any one of claims 26-30, wherein the subject at risk of having a cardiomyopathy is currently or has previously been treated with a chemotherapeutic agent.

32. The method of any one of claims 26-31, wherein the subject at risk of having a cardiomyopathy has a heart inflammation.

33. The method of any one of claims 26-32, wherein the subject at risk of having a cardiomyopathy is taking a drug that weakens the heart.

34. The method of any one of claims 26-33, wherein the subject at risk of having a cardiomyopathy has tachycardia35. The method of any one of claims 26-34, wherein the subject at risk of having a cardiomyopathy has sarcoidosis.ATTORNEY DOCKET NO. 37759.0634P136. The method of any one of claims 26-35, wherein the subject at risk of having a cardiomyopathy has ventricular dysplasia.

37. The method of any one of claims 1-36, wherein the subject has not had a myocardial infarction prior to administering an SGLT inhibitor to the subject.

38. A method of preventing cardiomyopathy in a subject at risk of developing a cardiomyopathy, comprising administering an SGLT inhibitor to the subject.

39. The method of claim 38, wherein the cardiomyopathy is premature ventricular complex (PVC)-induced cardiomyopathy.

40. The method of any one of claims 38-40, wherein the subject at risk of having a cardiomyopathy has heart palpitations / premature ventricular contractions / extra heart beats.

41. The method of any one of claims 38-40, wherein the subject at risk of having a cardiomyopathy is currently or has previously been treated with a chemotherapeutic agent.

42. The method of any one of claims 38-41, wherein the subject at risk of having a cardiomyopathy has a heart inflammation.

43. The method of any one of claims 38-42, wherein the subject at risk of having a cardiomyopathy is taking a drug that weakens the heart.

44. The method of any one of claims 38-43, wherein the subject at risk of having a cardiomyopathy has tachycardia45. The method of any one of claims 38-44, wherein the subject at risk of having a cardiomyopathy has sarcoidosis.

46. The method of any one of claims 38-45, wherein the subject at risk of having a cardiomyopathy has ventricular dysplasia.

47. The method of any one of claims 38-46, wherein the SGLT inhibitor is one or more of canagliflozin, dapagliflozin, empagliflozin, besaglifloxin, or ertugliflozin.ATTORNEY DOCKET NO. 37759.0634P148. A method of preventing or minimizing interstitial fibrosis in a subject, comprising administering an SGLT inhibitor to a subject in need thereof.

49. The method of claim 48, wherein the subject in need thereof is a subject at risk of having a cardiomyopathy.

50. The method of any one of claims 48-49, wherein after administration of the SGLT inhibitor, the subject has reduced fibroblast activation compared to a subject not receiving a SGLT inhibitor.

51. The method of any one of claims 48-50, wherein after administration of the SGLT inhibitor, the subject has reduced fibroblast proliferation compared to a subject not receiving a SGLT inhibitor.

52. The method of any one of claims 48-51, wherein the subject at risk of having a cardiomyopathy has heart palpitations, premature ventricular contractions, and / or extra heart beats.

53. The method of any one of claims 48-52, wherein the subject at risk of having a cardiomyopathy is currently or has previously been treated with a chemotherapeutic agent.

54. The method of any one of claims 48-53, wherein the subject at risk of having a cardiomyopathy has a heart inflammation.

55. The method of any one of claims 48-54, wherein the subject at risk of having a cardiomyopathy is taking a drug that weakens the heart.

56. The method of any one of claims 48-55, wherein the subject at risk of having a cardiomyopathy has tachycardia57. The method of any one of claims 48-56, wherein the subject at risk of having a cardiomyopathy has sarcoidosis.

58. The method of any one of claims 48-57, wherein the subject at risk of having a cardiomyopathy has ventricular dysplasia.ATTORNEY DOCKET NO. 37759.0634P159. The method of any one of claims 1-58. wherein the SGLT inhibitor is one or more of canagliflozin, dapagliflozin, empagliflozin, besaglifloxin, or ertugliflozin.

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