HDAC3 inhibitors to treat arrhythmogenic cardiomyopathy

Abstract

Disclosed herein are methods of treating cardiac disorders, such as heart conditions including cardiac fibrosis, by administering an HDAC3-specific inhibitor to a subject in need thereof. Also disclosed are methods of mitigating cardiac fibrosis and cardiomyocyte apoptosis, such as by treating the cardiac tissue with an HDAC3-specific inhibitor. Further disclosed are pharmaceutical compositions including HDAC3-specific inhibitors and routes of administering the same.

Description

HDAC3 INHIBITORS TO TREAT ARRHYTHMOGENIC CARDIOMYOPATHYSTATEMENT OF GOVERNMENTAL RIGHTS

[0001] This invention was made with government support under HL161741 awarded by the National Institutes of Health. The Government has certain rights in the invention.FIELD OF THE INVENTION

[0002] This disclosure generally relates to methods of treating a heart condition, such as a cardiac disorder involving cardiac fibrosis, abnormal fat formation, and cardiomyocyte death, e.g., arrhythmogenic cardiomyopathy, by administering an HDAC 3 -specific inhibitor to a subject in need thereof. The disclosure further relates to methods of decreasing cardiac fibrosis, abnormal cardiac fat formation, and apoptosis in cardiomyocytes by treating cardiac tissue with an HDAC- 3-specific inhibitor. Additionally, the disclosure relates to pharmaceutical compositions of HDAC- 3-specific inhibitors and routes of administering the same.BACKGROUND

[0003] Cardiovascular diseases remain the major cause of death in the US. Scientific advances in reprogramming somatic cells from human cardiac patients into induced pluripotent stem cells (iPSCs) enable in vitro modelling of human cardiac diseases for pathogenic and therapeutic studies. Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy with an estimated prevalence of 1 in 1000-5000 individuals that predisposes individuals to sudden cardiac death, particularly in young patients and athletes. Most identified mutations (>50%) associated with ACM are in 5 genes encoding cardiac desmosomes, which include plakoglobin (JUP), plakophilin-2 (PKP2), desmoplakin (DSP), desmoglein-2 (DSG2) and desmocollin-2 (DSC2). Pathological hallmarks of ACM are progressive fibro-fatty replacement of cardiomyocytes (CM)with increased CM apoptosis primarily in the right ventricle, leading to sudden cardiac death (SCD) in the young.

[0004] Currently, no curative or disease-slowing therapy is available except inserting cardiac defibrillators into ACM patients to prevent SCD and ablation to decrease ventricular tachycardia burden. Pathogenic processes of human ACM are difficult to study because of the slow progressive nature of this disease, difficulties in obtaining cardiac samples from early stages of human ACM hearts, and risks of cardiac perforation from cardiac biopsy. These limiting factors impose significant constraints in developing therapies for human ACM. Aspects of the invention disclosed herein address these needs.

[0005] How desmosomal mutations lead to ACM pathologies remains poorly understood. Experimental data from animal and cell line models has led to diverse and conflicting results. Using iPSC-derived cardiomyocytes (iPSC-CMs) generated from ACM patients with PKP2 mutations, we (Applicants) established a method to induce adult-like, fatty acid oxidation (FAO) dominant metabolism of primitive iPSC-CMs with three factors [3F= insulin, steroid & 3-isobutyl- 1-methil-xanthine (IBMX)] and established the first metabolic maturation-based in vitro cardiac disease model (Nature, 2013). We showed that ACM iPSC-CMs manifested AC pathologies only after inducing abnormal peroxisome proliferator-activated receptor-gamma (PPARy) activation on normal adult PPARa-mediated metabolic backgrounds by a 5-factor protocol (5F= 3F+ rosiglitazone and indomethacin), resulting in exaggerated lipogenesis / apoptosis, cardiac Na+channel (SCN5A) and Ca2+handling deficits, recapitulating pathological signatures of ACM. PPARy inhibitors rescue all ACM pathologies but carry systemic side effects, e.g., hypertension. Importantly, we also established a mouse model of ACM with cardiac-specific deletion of plakoglobin (JUP) genes, which largely recapitulated the clinical manifestation of human ACM(Hum Mol Genet 2011; 20: 4582-96) and validated the key role of PPARy activation in ACM pathogenesis (data shown in this application). Further understanding how PPARy could be abnormally activated in ACM will likely provide new therapeutic strategies.

[0006] Here, using our in vitro (iPSC-CMs) and in vivo (mouse) ACM models, and mRNA / microRNA (miR) profding of normal and ACM human heart tissues / iPSC-CMs, we unravel a novel key pathogenic network connecting desmosomal mutations to the abnormal PPARy activation and CM death, which allows us to develop novel ACM therapeutics (Fig, 1, please see detailed description section for these novel findings). We show that desmosomal mutations lead to faster degradation of JUP proteins, which is the anchoring protein on the insulin receptor (InsR) for p85 of the PI3 kinase (PI3K) pathway. With less JUP available for p85 binding, insulin is less effective in activating PI3K via InsRs, leading to insulin resistance, less Protein kinase B (Akt) activation, and hyperactive glycogen synthase kinase 3P (GSK3P). Hyperactive GSK3P then phosphorylates and inactivates JUP, [3-catenin (PCat) and YAP (Yes-associated protein- 1), leading to downregulation of a key miR 23b / 27b / 24 cluster that allows abnormal PPARy activation. Abnormally activated PPARy then binds to a novel inhibitory PPAR-responsive element (iPPRE) on the promoter of miR 23b / 27b / 24 cluster and suppresses their expression, setting off a vicious cycle of PPARy over-activation and CM apoptosis. We also show that this key pathogenic pathway occurs in 6 explanted human ACM hearts and in 2 ACM mouse models, validating our novel pathogenic pathway in vivo. Based on our breakthrough findings and because PPARy usually requires histone deacetylase 3 (HD AC3) / nuclear receptor corepressor 2 (NCOR2) complexes to exert its inhibitory promoter regulations, we propose here to use various HDAC3- specific inhibitors as novel therapeutics to treat human ACM (Innovations).INCORPORATION BY REFERENCE

[0007] Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each were incorporated by reference individually, and as if each is fully set forth herein. However, where such reference is made, and whether to patents, publications, non-patent literature, or other sources of information, it is for the general purpose of providing context for discussing features of the invention. Accordingly, unless specifically stated otherwise, the reference is not to be construed as an admission that the document or underlying information, in any jurisdiction, is prior art, or forms part of the common general knowledge in the art.SUMMARY OF THE INVENTION

[0008] In one aspect, the invention includes methods of treating cardiac fibrosis in a subject having a cardiac disease.

[0009] A first embodiment is a method of treating cardiac fibrosis in a subject having a cardiac disease including administering a therapeutically effective amount of a histone deacetylase 3 (HDAC3)-specific inhibitor to the subject.

[0010] A second embodiment is a method of treating cardiac fibrosis in a subject having a cardiac disease, where the cardiac disease is arrhythmogenic cardiomyopathy (ACM).

[0011] A third embodiment is a method of treating cardiac fibrosis in a subject, where the HD AC3 -specific inhibitor is RGFP966 ((2E)-N-(2-Amino-4-fluorophenyl)-3-[(2E)-l-(3-phenyl- 2-propen- 1 -y 1)- 1 H-py razol-4-y 1] -2-propenamide, (E)-N -(2-amino-4-fluorophenyl)-3 -( 1 - cinnamyl-lH-pyrazol-4-yl)acrylamide)), compound 11, BRD3308 (4-(acetylamino)-N-(2-amino- 4-fluorophenyl)-benzamide), compound I, compound II, compound III, compound IV, compound V, compound VI, compound VII , an analog thereof, or a pharmaceutically acceptable salt thereof.

[0012] A fourth embodiment is a method of treating cardiac fibrosis in a subject, where the HDAC3-specific inhibitor is RGFP966, compound 11, BRD3308, an analog thereof, or a pharmaceutically acceptable salt thereof.

[0013] A fifth embodiment is a method of treating cardiac fibrosis in a subject including intravenously or locally administering a pharmaceutical composition including the HDAC3- specific inhibitor to the epicardial space of the subject.

[0014] A sixth embodiment is a method of treating cardiac fibrosis in a subject, where treating the cardiac disease includes inhibiting cardiac fibrosis, fibro-fatty replacement of cardiomyocytes, cardiomyocyte apoptosis, or a combination thereof.

[0015] A seventh embodiment is a method of treating cardiac fibrosis in a subject, where the subject is human.BRIEF DESCRIPTION OF THE FIGURES

[0016] The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the description serve to explain the principles of the disclosure.

[0017] FIG. 1A presents a working schematic of arrhythmogenic cardiomyopathy (ACM) pathogenesis, indicating how InsR-JUP-PI3K-Akt-GSK3p and pCat / YAP / JUP axes affect PPARy activation via a miR 23b / 27b / 24 cluster.

[0018] FIG. IB presents administration of an HD AC3 -specific inhibitor as a potential therapeutic strategy to counteract the action of PPARy on the newly identified iPPRE of miR 23b / 27b / 24 cluster, preventing both down-regulation of this miR cluster and subsequent ACM cardiomyocyte (CM) apoptosis.

[0019] FIGS. 2A-2E presents ChlP-qPCR data showing regulation at the miR 23b / 27b / 24 promoter region following exposure to no pathogenic factors (OF, as the baseline control), 3 metabolic maturation-induction factors (3F= insulin, steroid and IBMX), and 5 pathogenic factors (5F = 3F+ rosiglitazone, and indomethacin). These protocols represent a metabolic maturationbased in vitro cardiac disease model. N=3-4 for all data. Asterisks (*) denote p<0.05 by ANOVA.

[0020] FIG. 2A shows a gel and bar graph indicating that 5F leads to reduced binding of YAP to a distal TEA Domain Transcription Factor (TEAD) promoter.

[0021] FIG. 2B shows a gel and bar graph indicating that 5F leads to reduced binding of (3-cat to a distal T-cell factor / lymphoid enhancer factor (TCF / LEF) promoter.

[0022] FIG. 2C shows a gel and bar graph indicating increased PPARy occupancy on the proximal iPPRE following exposure to 5F.

[0023] FIG. 2D shows bar graphs indicating that distal TCF / LEF site is stimulatory but proximal PPRE is inhibitory to the miR 23b / 27b / 24 transcription. Mutation of C to A in the proximal iPPRE converts its inhibitory to stimulatory role in miR 23b / 27b / 24 production using Luciferase (Luc) vector-mediated bioluminescence assays.

[0024] FIG. 2E shows a simple diagram that demonstrates the location and sequences of these promoters.

[0025] FIGS. 3A-3E show the results of drug studies in ACM iPSC-CMs and two ACM mouse models. TSA represents trichostatin, a non-specific class 1 HD AC inhibitor. Vai represents valsartan, an angiotensin II receptor blocker (ARB) without PPARy agonistic activities. Los represents losartan, an ARB with strong PPARy agonistic activities. JUP homozygous knockout(KO) (labelled Mut in Fig 3B) represents an ACM mouse model with cardiac-restricted knockout of JUP. Double KO (DKO) represents an ACM mouse model with tamoxifen (TAM)-inducibleand cardiac-specific (by aMHC-Cre) double knockout of JUP and P-catenin. N=4 for all data unless otherwise indicated, and * denotes p<0.05 (ANOVA or Student t-test).

[0026] FIG. 3A is a bar graph showing the effects of OF, 3F, 5F, TSA and valsartan on apoptosis in ACM iPSC-CMs. Trichostatin or valsartan inhibited CM apoptosis at indicated concentrations.

[0027] FIG. 3B shows bar graphs representing comparative gene expression of PPARy, caspase 3, miR27a, and miR27b in wild-type (WT) and JUP KO ACM (Mut) mouse hearts. JUP KO mice developed cardiac dysfunctions in 1-2 months and usually died from arrhythmia and SCD between 2-6 months after birth. Homozygous JUP KO mouse hearts also showed overactivated PPARy (PPARG mRNA), downregulated mouse miR27a / b (mouse equivalents to the human miR 23b-27b / 24 cluster) and elevated CM apoptotic Caspase 3 expressions.

[0028] FIG. 3C shows a gel and bar graph representing protein levels of PPARy and P-actin (as the loading control) in WT and JUP KO ACM mouse hearts. Both 0.2 mg / ml Valsartan or 2 pg / ml TSA in feeding water for 6 weeks reduced the PPARy protein levels in JUP KO ACM mice.

[0029] FIG. 3D shows bar graphs representing elevated apoptotic BAX / BCL2 ratios in JUP KO mice and DKO mice after rosiglitazone (Rosi) pathogenic induction (DKO+Rosi), which could be suppressed by Vai and TSA administration.

[0030] FIG. 3E shows bar graphs representing relative left ventricle ejection fraction (LV EF), a measure of cardiac function, in DKO and JUP KO mouse models. We fed DKO ACM mice with 0.1 mg / ml rosiglitazone (Rosi, a PPARy activator) for 2 weeks after starting TAM injection (to knockout JUP in cardiomyocytes), which accelerated ACM pathologies within 5 weeks after TAM with decreased LV EF on echocardiograms (+Rosi). The Rosi-induced ACM pathology could be ameliorated by Valsartan (Rosi+Val, Fig. 3E left panel). In JUP KO mice, LV EF levels arepreserved by administration of Vai but worsened by losartan (Los). Data in Fig 3E support strongly that PPARy activation is causal for ACM pathologies.

[0031] FIGS. 4A-4C show that an exemplary HD AC3 -specific inhibitor RGFP966 (RGFP) suppresses pathological fibrogenesis and adipogenesis in ACM iPSC-derived cardiac fibroblasts as well as decreases apoptosis in ACM iPSC-CMs. N=3 biological replicates for all data; * indicates p<0.05 (ANOVA).

[0032] FIG. 4A are bar graphs showing the effects of RGFP on the expression of fibrogenic collagen genes COL1A1 and COL3A1 in ACM DSP R160X (top) and DSP E1159R mutant fibroblasts (bottom).

[0033] FIG. 4B is a bar graph showing the effects of RGFP on the levels (%) of ACM PKP2 DelC mutant fibroblast-derived adipocytes (immune-stained positive for an adipocyte-specific marker, perilipin 1).

[0034] FIG. 4C is a bar graph showing the effects of RGFP on TUNEL-positive apoptotic CMs (%) in 5F-treated pathological PKP2 DelC mutant iPSC-CMs.

[0035] FIG. 5 shows the chemical structures of two exemplary HD AC3 -specific inhibitors RGFP966 and compound 11 (Nl-(2-aminophenyl)-N8-(3-(l-phenyl-lH-l,2,3-triazol-4-yl)- phenyl)-octanediamide).

[0036] FIG. 6 shows bar graphs representing the effects of exemplary HD AC3 -specific inhibitors Compound 11 (Cpdl l) and RGFP966 (RGFP) on fibrogenesis, adipogenesis, and cardiomyocyte (CM) apoptosis in mutant ACM cells. N=3 biological replicates for all data. * indicates p<0.05 (ANOVA).

[0037] FIG. 6A shows bar graphs comparing the effects of Cpdl 1 (blue) to RGFP (red) on the expression of fibrogenic collagen genes COL 1 Al and COL3A1 in ACM DSP R160X (top) and DSP El 159R mutant fibroblasts (bottom).

[0038] FIG. 6B is a bar graph comparing adipogenesis (Perilipin- l+)-suppressing effects of Cpdl 1 to RGFP in PKP2 DelC mutant fibroblasts.

[0039] FIG. 6C is a bar graph comparing apoptosis (TUNEL+)-suppressing effects of Cpdl 1 to RGFP in PKP2 DelC cardiomyocytes (CM).

[0040] FIGS. 7A-7D present data showing the rescuing effects of exemplary HDAC3 -specific inhibitor RGFP966 (RGFP) on apoptosis in human ACM DelC mutant CMs in vitro as well as on cardiac pathologies in JUP KO (Mut) ACM mice in vivo. The numbers in the graphs indicate the number of biological replicates, unless indicated otherwise; * denotes p<0.05 (by ANOVA or Student t-test).

[0041] FIG. 7A is a bar graph showing that RGFP decreases apoptosis levels (% of TUNEL- positive cells) in delC ACM iPSC-CMs induced by the pathogenic 5F protocol (n=4).

[0042] FIG. 7B has bar graphs showing that RGFP (at 10 mg / kg by i. p. injection every 2-3 days for 4 weeks) decreases expression of PPARy (PPARG) and apoptotic caspase 3 but increases miR27b in 4-week-old JUP KO (Mut) ACM mice in vivo when compared to WT mice. JUP KO ACM mouse hearts have elevated PPARG / Caspase 3 and reduced m27b levels when compared to WT mouse hearts at 8 weeks old.

[0043] FIG. 7C is a bar graph showing that RGFP decreases elevated apoptotic BAXBCL2 gene ratios in JUP KO mouse hearts (see Fig. 3D, which showed elevated BAX / BCL2 gene ratios in JUP KO mouse hearts relative to WT mouse hearts.

[0044] FIG. 7D is a bar graph showing that 4 weeks of RGFP treatment prevent the decrease in left ventricle ejection fraction (LV EF), an indicator of cardiac function, in 4-5-week-old JUP KO mice.

[0045] FIG. 8A and 8B show preferred core structures of HD AC3 -specific inhibitors (highlighted by red, blue and cyan circles) in this application. Preferred exemplary compounds have an ICso value that is at least 100-fold more selective to HDAC3 than to other subclasses of class 1 HDACs or HDAC6 (indicated by cyan circles). Compound 11 shown in this application corresponds to compound 10 in this figure (Sarkar et al., Eur. J. Med. Chem. 2020;192: 112-171). Blue- and Cyan-circled compounds might be further chemically modified to create more specific and safer HD AC3 -specific inhibitors.DETAILED DESCRIPTION

[0046] Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy characterized by pathological fibrofatty productions and cardiomyocyte (CM) loss primarily in the right ventricle (RV), leading to lethal arrhythmias and cardiac dysfunction in young adults. Over 50% of ACM patients have mutations in 5 desmosome genes, most commonly in plakophilin-2 (PKP2). How desmosomal mutations lead to ACM pathologies remains poorly understood. Experimental data from animal and cell line models has led to diverse and conflicting results. Scientific advances in reprogramming somatic cells from cardiac patients into induced pluripotent stem cells (iPSCs) enable in vitro modelling of human cardiac diseases for pathogenic and therapeutic studies. Using iPSC-CMs generated from ACM patients with CA7U mutations, applicants developed a method to induce adult-like, fatty acid oxidation (FAO) dominant metabolism of primitive iPSC-CMs with three factors (3F= insulin, steroid & IBMX) and established the first metabolic maturation-based in vitro cardiac disease model (Nature , 2013). We showed that ACM iPSC-CMs manifested ACpathologies only after inducing abnormal PPARy activation on normal adult PPARa-mediated metabolic backgrounds by a 5-factor protocol (5F= 3F+ rosiglitazone and indomethacin), resulting in exaggerated lipogenesis / apoptosis, cardiac Na+channel (SCN5A) and Ca2+handling deficits, recapitulating pathological signatures of ACM. As noted, PPARy inhibitors rescue all ACM pathologies but could carry systemic side effects when used in humans, e g., hypertension. Importantly, applicants also established a mouse model of ACM with cardiac-specific deletion of plakoglobin JUP) genes (JUP KO), which largely recapitulated the clinical manifestation of human ACM (Hum Mol Genet 2011; 20: 4582-96) and validated the key role of PPARy activation in ACM pathogenesis (data shown in this application). Further understanding how PPARy could be abnormally activated in ACM cardiomyocytes will likely provide new therapeutic strategies.

[0047] Using iPSC-CMs generated from ACM patients with PKP2 mutations, we have established the first metabolic maturation-based in vitro cardiac disease model (Nature 2013; 494: 105-110). Applicants have shown that ACM iPSC-CMs manifested ACM pathologies only after inducing abnormal PPARy activation on normal adult PPARa-mediated metabolic backgrounds. Of note, applicants found that PPARy could not be activated in normal hESC- / iPSC- CMs after exposure to two strong PPARy agonists (indomethacin & rosiglitazone) at high concentrations, yet PPARy in ACM iPSC-CMs could be easily activated by these two PPARy agonists, suggesting that inhibitory factors for PPARy activation are likely down-regulated in ACM iPSC-CMs. While PPARy inhibitors can rescue all ACM pathologies, these inhibitors carry systemic side effects if used in humans. Therefore, an alternative method for blocking the effects of PPARy activation is desired.

[0048] One of the common suppressors of gene expression is via microRNAs (miRs).Therefore, using NanoString miR arrays, applicants compared miR expression patterns betweenpathogenic 5F-treated, 3F-treated (m etab oli cal ly mature without ACM pathologies), and baseline (OF) ACM iPSC-CMs. Using the metabolic maturation-based ACM model (Nature 2013; 494: 105-110) and mRNA / microRNA (miR) profding of normal and ACM human heart tissues / iPSC- CMs, Applicants show here that desmosomal mutations lead to faster degradation of plakoglobin proteins (JUP), which is the anchoring protein on the insulin receptor (InsR) for p85 of the PI3 kinase (PI3K) pathway. With less JUP available for p85 binding, insulin is less effective in activating PI3K via InsRs, leading to insulin resistance, less Akt activation, and hyperactive GSK3[3. Hyperactive GSK3[3 then phosphorylates and inactivates JUP, P-catenin (PCat) and YAP, leading to downregulation of a key miR 23b / 27b / 24 cluster that allows abnormal PP A Ry activation and subsequent ACM pathologies (FIG. 2A-D; See overall pathway disclosed in FIG. 1A).

[0049] Abnormally activated PPARy then binds to a novel inhibitory PPAR-responsive element (PPRE) on the promoter of miR 23b / 27b / 24 cluster and suppresses their expression, allowing a self-perpetuating vicious cycle of PPARy over-activation and CM apoptosis. Applicants also show that this key pathogenic pathway occurs in 6 explanted human ACM hearts, validating this novel pathogenic pathway in vivo.

[0050] In Figure 3, we show that a prototype non-specific Class 1 HD AC inhibitor, trichostatin A (TSA) decreases apoptosis in PKP2 mutant ACM iPSC-CMs treated by pathogenic 5F as well as decreases PPARy mRNA and proteins levels and prevents CM apoptosis in two AC mouse models. In addition to our new data in Figure 3, Class 1 HDACs (including HDAC1, 2, 3 & 8) had been shown to affect adipogenesis (especially HDAC1 & 3, Biochem Biophys Res Commun. 2012; 428:271-7) and TSA can reduce ATIR-mediated cardiac fibrosis (Handb Exp Pharmacol. 2011; 206:57-78). Because 1) abnormally activated PPARybinds to a novel inhibitory PPAR-responsive element (iPPRE) on the promoter of miR 23b / 27b / 24 cluster and further suppresses theirexpression (FIG. 2) and 2) PPARy usually requires histone deacetylase 3 (HDAC3) / nuclear receptor corepressor 2 (NC0R2) complexes to exert its inhibitory promoter regulations, we propose here to use various HD AC3 -specific inhibitors as novel therapeutics to treat human ACM.

[0051] First, we hypothesized that a HD AC3 -specific inhibitor might be effective in inhibiting fibrogenesis / adipogenesis of ACM mutant fibroblasts and in preventing ACM CM apoptosis. Therefore, we tested a prototype HDAC3-specific inhibitor, RGFP966 (RGFP, IC50= 60-80 nM), in terms of its efficacies in decreasing fibrogenesis and adipogenesis of cardiac fibroblasts and in reducing CM apoptosis in our in vitro ACM iPSC-CM models. We used cardiac fibroblasts with a heterozygous nonsense mutation [desmoplakin (DSP) c.478 C>T (p. R160X)] or with a heterozygous insertion of an adenine nucleotide that results in frame-shifted C-terminal with premature stop codons [DSP c.3474_3475insA (p. Glul 159ArgfsX3), termed E1159R here for brevity] to test their fibrogenic responses to a known fibrosis inducer, 1 ng / ml TGFJ31 because DSP mutant fibroblasts had strong fibrogenic potentials in our preliminary research. We used PKP2 DelC mutant fibroblasts to test adipogenic responses of ACM fibroblasts to our 3F adipogenic protocol because PKP2 mutant fibroblasts have high adipogenic potentials. We also used PKP2 DelC mutant iPSC-CMs to test drug effects on CM apoptosis induced by our 5F protocol. In FIG. 4, RGFP at 0.5 pM effectively decreased fibrogenic collagen genes [collagen 1A1 (COL1AP) and 3A1 (COL3AP), FIG. 4A] expressions, reduced adipogenesis (adipocyte formation by immunostaining for adipocyte-specific perilipin 1 -positive cells, FIG. 4B), and prevented CM apoptosis (by TUNEL staining, FIG. 4C). These results support strongly our key pathogenic pathway and therapeutic inventions. All experiments were from 3 biological replicates.

[0052] Second, another HD AC3 -specific inhibitor, termed compound 11 (chemical structures of RGFP966 and compound 11 are shown in FIG. 5), effectively reduced pathological cardiacfibrosis (FIG. 6A), pathological adipogenesis (FIG. 6B), and CM apoptosis (FIG. 6C) at 1 pM in our ACM in vitro models. Compound 11 appeared to have less detrimental effects at >1 pM when compared to RGFP 966 (Red histograms in all subfigures of Fig. 6).

[0053] Third, we used RGFP to study its therapeutic effects in JUP KO ACM mice in vivo because RGFP decreased ACM iPSC-CM apoptosis (FIG. 7A) in vitro (also see Fig. 4). As expected, RGFP also decreased PPARy and apoptotic gene levels (Caspase 3 and BAX / BCL2 ratios, FIGS. 7B-C) as well as preserved cardiac ejection fraction (EF) in JUP KO mice (FIG. 7D) after 4 weeks of 10 mg / kg RGFP treatment (by i.p. injection 3 times per week for 4 weeks).

[0054] In accordance with these novel pathogenic findings, Applicants previously identified three therapeutic strategies to prevent disease progression in human ACM, which involved 1) use of Class I HDAC inhibitors to prevent ACM disease progression; 2) use of CRISPR / CAS9 or CRISPR / deaminase technology to convert the inhibitory PPRE to stimulatory PPRE by a single nucleotide change, which prevents the key PPARy activation and subsequent pathogenic disease processes; and 3) over-expression of the miR 23b / 27b / 24 cluster with various technologies to block PPARy activation-mediated ACM pathologies. Applicants now advance previous findings that Class 1 HDAC could prevent ACM disease progression by specifically identifying HDAC3 as the target for developing novel and improved therapeutic strategies for human ACM. As set forth herein, the improved strategy involves administering an HD AC3 -specific inhibitor to a subject in need thereof, such as a subject suffering from cardiomyocyte death and / or cardiac fibrosis, e.g., an ACM patient.

[0055] Using an established metabolic maturation-based ACM in vitro model, 2 ACM mouse models, and mRNA / miR genomic profiling of normal and ACM human heart tissues / iPSC-CMs,Applicants show here that the metabolic maturation-based iPSC-CM model provides a unifyingpathogenic scheme (see FIG. 1A). Specifically, desmosomal mutations are proposed to lead toJUP deficiencies, which downregulate insulin signalling pathways and a miR cluster, leading to abnormal PPARy activation and CM apoptosis.

[0056] For human in vivo evidence, Applicants’ findings in metabolic deregulation from desmosome mutations in ACM iPSC-CMs would predict that early ACM patients should have higher FAO, which conferred ACM patients with advantages for endurance exercise but could lead to energy spills into ketone productions. Abnormally high FAO flux in ACM lead to elevated ketones in blood, which can be used for diagnosing adverse progression of ACM even in relatively asymptomatic patients and in ACM mice (Sci Transl Med, 2020; 12, eaay8329). Applicants’ lab had also found that testosterone worsened clinical outcomes in ACM patients and estradiol protected female patients from ACM pathologies likely by reducing the FAO flux (Eur. Heart J, 2017; 38:1498-1508). Recognition of the pathways leading to abnormal PPARy over-activation has led to Applicants’ novel therapeutic approaches in this application for treating human ACM patients.

[0057] As disclosed herein, desmosomal mutations lead to downregulation of JUP and subsequent insulin resistance and hyperactive GSK3|3, which decreases YAP and Peat activities. Reduced nuclear YAP and Peat activities decrease the expression of an inhibitory miR 23b / 27b / 24 cluster allowing abnormal PPARy over-activation and ACM CM apoptosis. Furthermore, Applicants identified a novel inhibitory PPAR responsive element (iPPRE; having the sequence of TGGTCACAAGTCT in the sense direction, as represented in SEQ ID NO:1) associated with ACM patients, which is located on the promoter of the miR 23b / 27b / 24 cluster. (See FIG. 2E) Abnormally activated PPARy binds to this novel inhibitory PPRE on the promoter of miR 23b / 27b / 24 cluster and further suppresses their expression, setting up a self-perpetuating anddetrimental cycle that enables PPARy over-activation and CM apoptosis. Most importantly,Applicants show that this key pathogenic pathway occurs in 6 explanted human ACM hearts with various desmosome gene mutations and in 2 mouse models of ACM, validating our novel pathogenic pathway in vivo. As disclosed herein, the atypical PPRE present in the miR cluster of ACM patients represents an inhibitory PPRE. In the atypical PPRE, the nucleotide located at position 7 of the response element is a C (cytosine) (SEQ ID NO:1), whereas that position is usually an A (adenine) (SEQ ID NO:2), G (guanine) (SEQ ID NO:3), or T (thymidine) (SEQ ID NO:4) in the typical PPRE. The classic theory is that association of PPARy with co-repressors (SMART) or co-activators (PGC1) determines the functional roles of PPRE. It is surprising that the nucleotide sequences in PPRE could determine whether the PPRE is stimulatory or inhibitory.

[0058] In addition, Applicants also elucidated the pathogenic pathways for exaggerated fibrosis and abnormal adipogenesis in human ACM hearts using patient-specific iPSC-derived cardiac fibroblasts (manuscripts are currently submitted for potential publication). Using the in vitro ACM and in vivo mouse ACM models disclosed herein, preventing activation of the iPPRE by PPARy was shown to break this vicious cycle and inhibit disease progression. Because PPARy usually requires HDAC3 / NCOR2 complexes to exert its inhibitory promoter regulations, we show here that HDAC3-specific inhibitors could slow down the ACM disease progression as well as inhibit exaggerated fibrosis and pathological adipogenesis in our in vitro ACM models. We also showed that a prototype of HD AC3 -specific inhibitors, RGFP966, could prevent ACM disease progression in an ACM mouse model. See FIG. 7A-D.

[0059] “HDAC3” refers to an enzymatic histone deacetylase 3 protein, an important zincdependent metalloenzyme that is implicated in various mammalian diseases through epigenetic modulations. HDAC3 is a member of the class I HD AC and is distinctive within the Class 1 HD ACfamily. HDAC3 is also a component of the nuclear receptor co-repressor complex and thus has distinct molecular and physiological functions. The role of HDAC3 is correlated in several lifethreatening diseases like cancers, inflammatory diseases, cardiovascular diseases, neurodegenerative disorders, learning and memory dysfunctions, Huntington’s disease (HD), diabetes, etc.

[0060] HDAC3 inhibitors such as selective HDAC3 inhibitors are described, e.g., by US 2017 / 0204094 Al, US 2019 / 0216754 Al, WO 2023 / 049798 Al, WO 2019 / 032652 Al, WO 2018 / 223122 Al, WO 2014 / 018979 Al, WO 2015 / 069693 Al, WO 2018 / 071740 Al, US 2023 / 0096380 Al, WO 2022 / 058405 A2, and WO 2014 / 143666 Al. Cao et al., Molecules. 2018 Mar; 23(3): 551 and Sarkar et al., Eur J Med Chem. 2020; 192: 112171, also describe HDAC3- specific inhibitors, exemplary structures of which are shown in FIGS. 8A and 8B.

[0061] As referenced herein, selective HDAC3 inhibitors may be referred to interchangeably as HD AC3 -selective or HD AC3 -specific inhibitors. In some examples, a selective HDAC3 inhibitor of the disclosure is a compound that lacks binding affinity for or antagonistic effects on other classes or subclasses of HD AC families, e.g., HDAC1 and HDAC2, but inhibits the activity of HDAC3 with very low IC50 values (see FIGS. 8A-8B). In other examples, an HD AC3 -specific inhibitor of the disclosure has an ICso value that is at least about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80- fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 600-fold lower than its corresponding ICso values for other classes or subclasses of HDACs, such as HDAC1 , HDAC2, and HDAC8 in Class I HDACs, or HDAC6 in Class IIB HDACs. In preferred examples, an HDAC3-specific inhibitor of the disclosure has an ICso value that is about 50-fold, about 75- fold, about 100-fold, or > 100-fold lower than the ICso values to other subclasses of class I HDACsor HDAC6. HDAC3 selectivity can be determined according to methods available to one of skill in the art, such as with use of HDAC enzymatic assays or HDAC inhibition assays. Histone deacetylase activity assays are described, e.g., by Arts et al., Br J Cancer . 2007 Nov 19; 97(10): 1344-1353 and Schafer et al., Bioorg Med Chem. 2008 Feb 15;16(4):2011-33.

[0062] For illustrative purposes, a preferred HD AC3 -selective inhibitor of the disclosure is RGFP966, which is a highly selective HDAC3 inhibitor but shows negligible inhibition of other HDACs at concentrations up to 15 pM. In another preferred example, the HDAC3-selective compound 11 has an IC50 of 120 nM at HDAC3 but an IC50 in excess of 30,000 nM at HDAC1, HDAC2, HDAC6, and HDAC8, as shown in Sarkar et al. and FIGS. 8A-8B.

[0063] Examples of HDAC3 inhibitors useful in the present disclosure include: RGFP966, BRD3308 (4-(acetylamino)-N-(2-amino-4-fluorophenyl)-benzamide), and SR-4370. Other HDAC3 inhibitors, for example, selective HDAC3 inhibitors useful in the present disclosure, are known in the art, and may also include those described in WO2023049798A1, US20170204094A1, US20150299130A1, US20140249148A1, and US20140243345A1, US20190216754A1, US20170204094A1, US20150299130A1, US20140249148A1,US20140243345A1, the disclosures of all of the preceding patent references are incorporated by reference herein in their entireties.

[0064] In some embodiments, the HD AC3 -selective inhibitor is selected from the group consisting of RGFP966, compound 11, BRD3308, compound I, compound II, compound III, compound IV, compound V, compound VI, and compound VII, the structures of which are shown below. In other embodiments, the HD AC3 -selective inhibitor is an analog of a structure shown below, such as a chemical analog having enhanced HDAC3 specificity with better safety profiles relative to the base chemical structure.BRD3308;Compound III;Compound VII.

[0065] In preferred embodiments, the HD AC3 -selective inhibitor is RGFP966 ((2E)-N-(2- Amino-4-fluorophenyl)-3-[(2E)-l-(3-phenyl-2-propen-l-yl)-lH-pyrazol-4-yl]-2-propenamide, (E)-N-(2-amino-4-fluorophenyl)-3-(l-cinnamyl-lH-pyrazol-4-yl)acrylamide), having the structure pictured below, or an analog thereof.

[0066] In further preferred embodiments, the HD AC3 -selective inhibitor is compound 11, having the structure pictured below, or an analog thereof.

[0067] Method of Treatment

[0068] In some aspects, provided herein are methods of treating a subject having a cardiac disease with pathological cardiac fibrosis, fibro-fatty replacement of cardiomyocytes, cardiomyocyte apoptosis, or a combination thereof, by administering an HD AC3 -specific inhibitor to the subject. In further aspects, provided herein are methods of preventing cardiac fibrosis, fibro-fatty replacement of cardiomyocytes, cardiomyocyte apoptosis, or a combination thereof in a subject, by administering an HD AC3 -specific inhibitor to the subject. In some embodiments, the subject has cardiomyopathy. In some embodiments, the subject has cardiac fibrosis. In preferred embodiments, the subject has arrhythmogenic cardiomyopathy (ACM). Herein, a “subject,” such as a subject having ACM or a “subject in need thereof,” may be referred to interchangeably as a “patient.” In some embodiments, the subject is a mammal. In preferred embodiments, the subject is human.

[0069] In some embodiments, disclosed methods comprise decreasing fibro-fatty replacement of cardiomyocytes (CM), decreasing CM apoptosis, or a combination thereof, in a subject. In some embodiments, the subject is experiencing various degrees of clinical signs and / or symptoms of ACM (e.g., heart failure, frequent PVCs, syncope, sudden death, and / or sustained arrhythmia). In some embodiments, disclosed methods comprise administering an HD AC3 -specific inhibitor to the subject, such as a patient having pathological cardiac fibrosis or a patient having ACM.

[0070] In some embodiments, administering one or more HDAC3-specific inhibitor(s) to a patient, such as a patient having cardiac fibrosis or ACM, decreases fibro-fatty replacement of cardiomyocytes (CM) and / or decreases CM apoptosis in patients identified with arrhythmogenic cardiomyopathy.

[0071] In some embodiments, the HDAC3-specific inhibitor comprises RGFP966, compound 11 , a pharmaceutically acceptable salt thereof, an analog thereof, or a combination thereof. In some embodiments, the HDAC3-specific inhibitor is RGFP966 or an analog thereof. In some embodiments, the HD AC3 -specific inhibitor is compound 11 or an analog thereof. In one embodiment, the HD AC3 -specific inhibitor is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0072] In some embodiments, disclosed methods comprise administering two or moreHDAC3-specific inhibitors to a subject in need thereof, such as RGFP966, compound 11, an analog thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0073] Disclosed HD AC3 -specific inhibitors may be administered as compounds per se or may be formulated as pharmaceutical / pharmacological compositions or medicaments. The pharmaceutical compositions / medicaments may comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, and / or antioxidants.

[0074] Pharmaceutical compositions, such as compositions comprising HD AC3 -specific inhibitors, can be formulated by techniques known to the person skilled in the art, such as the techniques published in “Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22ndedition. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, mucosal, rectal, nasal, topical, aerosol or vaginal administration.

[0075] In some embodiment HDAC3-specific inhibitors are used in the manufacture of a pharmaceutical / pharmacological medicament for a treatment of cardiac disease. In some embodiments, the HD AC3 -specific inhibitor for use in the medicament is RGFP966 ((2E)-N-(2- Amino-4-fluorophenyl)-3 - [(2E)- 1 -(3 -phenyl-2-propen- 1 -y 1)- lH-pyrazol-4-yl]-2-propenamide, (E)-N-(2-amino-4-fluorophenyl)-3-(l-cinnamyl-lH-pyrazol-4-yl)acrylamide)), compound 11, BRD3308 (4-(acetylamino)-N-(2-amino-4-fluorophenyl)-benzamide), compound I, compound II, compound III, compound IV, compound V, compound VI, compound VII , an analog thereof, or a pharmaceutically acceptable salt thereof. Preferably, the HDAC3-specific inhibitor for use in the medicament is RGFP966, compound 11, BRD3308, an analog thereof, or a pharmaceuticallyacceptable salt thereof. In some embodiments, the HDAC 3 -specific inhibitor is used in the manufacture of a pharmaceutical / pharmacological medicament for treating arrhythmogenic cardiomyopathy (ACM). In some embodiments the medicament containing HDAC3-speicific inhibitors will inhibiting cardiac fibrosis, fibro-fatty replacement of cardiomyocytes, cardiomyocyte apoptosis, or a combination thereof of the cardiac disease.

[0076] In some embodiments, disclosed methods comprise administering a pharmaceutical composition comprising a therapeutically effective amount of an HD AC3 -specific inhibitor to a subject in need thereof by any standard route of administration, such as oral administration, parenteral administration, intravenous administration, mucosal administration, and transdermal administration. In some embodiments, disclosed methods comprise administering an HDAC3- specific inhibitor to a subject in need thereof by local injection to the myocardial tissues of said patient, such as the subject’s myocardium. In some embodiments, disclosed methods comprise administering an HDAC3-specific inhibitor to a subject in need thereof via intramyocardial, intracoronary arterial injection, epicardial injection via the epicardial space, or a combination thereof.

[0077] The term “about” as used herein means greater or lesser than the value or range of values stated by 10 percent but is not intended to limit any value or range of values to only this broader definition. Each value or range of values preceded by the term “about” is also intended to encompass the embodiment of the stated absolute value or range of values.

[0078] “Subject” refers to any mammal for whom diagnosis, treatment, or therapy is desired including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits and guinea pigs), livestock (e.g., cows, sheep, goats, and pigs), household pets (e.g., dogs, cats, and rodents), and horses. In preferred embodiments, the subject is human.

[0079] As used herein, “treat,” “treating” or “treatment” refer to an action to obtain a beneficial or desired clinical result including, but not limited to, alleviation or amelioration of one or more signs or symptoms of a disease (e.g., regression, partial or complete), diminishing the extent of disease, stability (i .e., not worsening, achieving stable disease) of the state of disease, amelioration or palliation of the disease state, diminishing rate of or time to progression, and remission (whether partial or total).

[0080] As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate-buffered saline solution, water, emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.

[0081] As used herein, the term “inhibitor” refers to a molecule that impedes or decreases a biological action. For the purpose of the present disclosure, an inhibitor generally has a specific target in the cell that it inhibits, e g., targeting HDAC3 with an HDAC3-specific inhibitor as disclosed herein. Inhibition of that specific target will have indirect effects on other biological molecules or processes that are regulated by the specific target. This inhibition may happen directly or indirectly.

[0082] As used herein, the term “RGFP966” and “compound 11” are benzamide ligands as outlined in Kozikowski, ChemMedChem 2009; 4:842-852. “RGFP966” and “compound 11” are pictured in FIG. 5 and FIG. 8A-B.

[0083] As used herein, the term “HDAC” refers to histone deacetylases, which are enzymes that remove the acetyl groups from the lysine residues in core histones, thus leading to the formation of a condensed and transcriptionally silenced chromatin. There are currently 18 knownhistone deacetylases, which are classified into four groups. Class I HDACs, which include HDAC1, HDAC2, HDAC3, and HDAC8, are related to the yeast RPD3 gene. Class II HDACs, which include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, are related to the yeast Hdal gene. Class III HDACs, which are also known as the sirtuins, are related to the Sir2 gene and include SIRT1-7. Class IV HDACs, which contain only HDAC11, have features of both Class I and II HDACs. The term “HD AC” refers to any one or more of the 18 known histone deacetylases, unless otherwise specified, such as with reference to HDAC3 in Class I HDACs.

[0084] As used herein, the term “subject” as used herein refers to a mammal. A subject therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like. Preferably the subject is a human. When the subject is a human, the subject may be referred to herein as a patient.

[0085] As used herein, the terms “therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g., a composition comprising an HD AC3 -specific inhibitor) refers to an amount that is effective to achieve a desired therapeutic result (e.g., treating or preventing cardiac fibrosis in an ACM patient). Therapeutically effective amounts will typically depend upon the IC50 and safety profile of the specific agent being administered. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent described herein may vary depending on such factors as the condition being treated, the mode of administration, and the age, body composition, and health of the subject. Suitable dosage ranges are readily determinable by one skilled in the art.EXAMPLES

[0086] Example 1: Identification of the key roles of plakoglobin in insulin-mediated metabolic signaling

[0087] FIG. 1A illustrates a working scheme for ACM pathogenesis involving the effects of InsR-JUP-PI3K-Akt-GSK3p and pCat / YAP / JUP axes on PPARy activation via promoter regulation of a miR 23b / 27b / 24 cluster. We show for the first time that JUP is the docking protein of p85 on InsR for activating PI3 kinases of the Insulin metabolic pathway. The p85 subunit regulates metabolic actions of InsR by either activating the catalytic pl 10 subunit of the PI3K or binds PTEN to inhibit the PI3K pathway. What determines the activating vs. inhibiting role of p85 in InsR-mediated signaling remains unclear. We show here that JUP is the key protein deciding the activating role of p85 on InsRs, which is a major breakthrough in understanding p85-mediated InsR signaling.

[0088] Example 2: Micro-RNA modulation of PPARy is implicated in apoptosis of ACM iPSC-CMs

[0089] Applicants’ research showed that PPARy bound to the inhibitory peroxisome proliferator-activated receptor response element (iPPRE), as shown in FIG. 2, likely via the HDAC3 / NCOR2 complex to suppress the expression of miR 23b / 27b / 24 cluster. Gene expression of ACM iPSC-CMs was evaluated to determine the relationship between PPARy activation / inhibition and apoptosis. Applicants found that PPARy could not be activated in normal hESC - / iPSC-CMs after exposure to two strong PPARy agonists (indomethacin & rosiglitazone) at high concentrations, yet PPARy in ACM iPSC-CMs could be easily activated by these two agonists, suggesting that inhibitory factors for PPARy activation are likely down-regulated in ACM iPSC-CMs (manuscript submitted for publication).

[0090] One of the common suppressors of gene expression is miR. Therefore, usingNanoString miR arrays, we compared miR expression patterns between pathogenic 5F-treated, 3F- treated and baseline (OF) ACM iPSC-CMs. Because 4 weeks of 5F treatment led to significant CMapoptosis (30-40%), we also profiled miR expressions of ACM iPSC-CMs after 2.5 weeks of 5F treatment (representing the mid-stage of pathological processes). Among the top 10 down- regulated miRs, miR24 and 23b of the miR 23b / 27b / 24 cluster are linked to several lipogenic gene regulations. Also, we used qPCR to confirm the miR expression patterns found in miR arrays. FIG. 2 shows regulation at the miR 23b / 27b / 24 promoter region, as determined by ChlP-qPCR. Downregulation of miR 23b / 27b / 24 expressions in 5F-treated ACM CMs likely enabled the abnormal activation of PPARG in pathological ACM iPSC-CMs.

[0091] FIGS. 2A and 2B show decreased expression of YAP and Peat by 5F, which led to lower binding of YAP and Peat to the distal TEAD or distal TCF / LFE promoter, respectively. FIG. 2C shows that the 5F protocol increased PPARy expression and PPARy occupancy on the proximal iPPRE Additionally, an atypical PPRE sequence in the promoter region of the miR23b / 27b / 24 cluster was found. The atypical PPRE of the miR cluster is an inhibitory PPRE as shown in FIG. 2D. In the atypical PPRE, the middle nucleotide is a C (cytosine at position 7 for the sequence TGGTCACAAGTCT) and it is usually an A (adenine), G (guanine) or T (thymidine) in typical PPRE. (FIG. 2E). The classic theory is that association of PPARy with co-repressors (HDAC3 / NCOR2) or co-activators (PGC1) determines the functional roles of PPRE.

[0092] Example 3: Drug studies in ACM iPSC-CMs and two ACM mouse models

[0093] Compounds, such as PPARy activators and HD AC inhibitors, were evaluated in vitro and in vivo for their effects on ACM pathogenesis in in vitro and in vivo models. In a proof of concept study, Applicants show herein that a non-selective class 1 HD AC inhibitor, trichostatin A (TSA) effectively prevented ACM pathologies in an in vitro ACM iPSC-CM-based model and in two in vivo mouse models of ACM.

[0094] Trichostatin A (TSA), valsartan (Vai), losartan (Los), and rosiglitazone (Rosi), were tested in vitro or in vivo to determine their effects on cardiomyocyte (CM) apoptosis, gene expression, and cardiac function (as indicated by LV EF). TSA is a reversible inhibitor of Class 1 HDAC, whereas Rosi is a PPARy activator that could elicit ACM pathologies. Vai and Los are angiotensin II receptor blockers (ARBs) that agonize angiotensin II actions.

[0095] FIG. 3A shows the results of in vitro data indicating that TSA and Vai inhibit CM apoptosis induced by a 5-factor protocol (5F) involving exposure of JK#11 AC iPSC-CMs to insulin, steroids, IB MX, Rosi, and indomethacin.

[0096] In the JUP KO ACM mouse model, desmosomal mutations downregulate JUP, leading to hyperactive GSK3[3 and suppressed miRs 23b / 27b / 24, which permit abnormal PPARy activation and ACM pathologies. The JUP KO model (Hum Mol Genet 2011 ; 20: 4582-96) is an efficient ACM mouse model, as JUP KO mice develop cardiac dysfunctions in 1-2 months and usually die from arrhythmia and sudden cardiac death (SCD) between 2-6 months after birth. As indicated by FIG. 3B, homozygous JUP KO mouse hearts also showed overactivated PPARy (mRNA and proteins), downregulated mouse miR27a / b (mouse equivalents to the human miR 23b-27b / 24 cluster) and elevated CM apoptotic genes, such as caspase 3, strongly supporting key pathogenic pathways in ACM.

[0097] 0.2 mg / ml Valsartan or 2 pg / ml TSA in feeding water for 6 weeks reduced PPARy proteins and apoptotic genes, as represented by BAX / BCL2 ratios in FIGS. 3C and 3D, respectively. Vai also preserved cardiac function marker LV EF in JUP KO ACM model, yet losartan (LOS), an ARB with PPARy agonist activity, led to one mouse death and one mouse with reduced LV EF (FIG. 3E, right panel).

[0098] A tamoxifen (TAM)-inducible and cardiac-specific (by aMHC-Cre) double KO (DKO) of JUP and 0-catenin ACM mouse model was also tested (Mol Cell Biol. 2012; 32:1056- 67). The DKO model mice developed ACM pathologies at 2-3 months after 5 days of tamoxifen injection. DKO mice were fed with 0.1 mg / ml PPARy activator Rosi for 2 weeks after initiation of TAM injection, which accelerated ACM pathologies within 5 weeks after TAM with decreased LV EF on cardiac echo (+Rosi), which could be ameliorated by Vai in water (FIG. 3E, left panel), supporting that PPARy activation is causal for ACM pathologies.

[0099] Together, these data indicate that the prototype non-specific Class 1 HD AC inhibitor TSA decreased apoptosis in plakophilin 2 (PKP2) mutant JK#11 ACM iPSC-CMs treated by pathogenic 5F as well as decreased PPARy mRNA / protein levels and prevented CM apoptosis in two AC mouse models.

[0100] Example 4: Exemplary HDAC3-selective inhibitor RGFP966 (RGFP) diminished pathogenesis of ACM in vitro

[0101] Class 1 HDACs (including HDAC1, 2, 3 & 8) have been shown to mediate adipogenesis, especially inhibition of HD AC 1 and HD AC, Biochem Biophys Res Common. 2012; 428:271-7). Additionally, TSA can reduce Type 1 angiotensin receptors (ATI Remediated cardiac pathology (Handb Exp Pharmacol. 2011; 206:57-78). We hypothesized that an HDAC3-specific inhibitor might be effective in inhibiting fibrogenesis / adipogenesis of ACM mutant fibroblasts and in preventing ACM CM apoptosis because 1) HDAC3 (a subclass of Class 1 HDACs) is usually associated with inhibitory gene promoter regulations and 2) based on our novel findings, such as presented in FIG. 2, abnormally activated PPARy binds to a novel inhibitory PPAR-responsive element (iPPRE) on the promoter of miR 23b / 27b / 24 cluster and further suppresses theirexpression, setting up a self-perpetuating vicious cycle that enables PPARy over-activation and CM apoptosis.

[0102] I. Exemplary HDAC-3 selective inhibitor RGFP reduced pathological fibrosis in DSP mutant MSCs: A prototype HDAC3-specific inhibitor, RGFP966 (RGFP, IC5o= 60-80 nM), was tested in terms of its efficacies in decreasing fibrogenesis and adipogenesis of cardiac fibroblasts and in reducing CM apoptosis in our in vitro ACM iPSC-CM models. We used cardiac fibroblasts with a heterozygous nonsense mutation [desmoplakin (DSP) c.478 C>T (p. R160X)] or with a heterozygous insertion of an adenine nucleotide that results in frame-shifted C-terminal with premature stop codons [DSP c.3474_3475insA (p. Glut 159 ArgfsX3), termed E1159R here for brevity] to test their fibrogenic responses to a known fibrosis inducer, 1 ng / ml TGF|31. DSP mutant fibroblasts had strong fibrogenic potentials in our preliminary research. In FIG. 4A, RGFP at 0.5 pM effectively decreased fibrogenic collagen gene expressions [collagen 1A1 COL1AP) and 3 Al (COL3A1 FIG. 4A],

[0103] II. Exemplary HDAC-3 selective inhibitor RGFP reduced pathological adipogenesis and apoptosis in PKP2 DelC mutant cells: We used PKP2 DelC mutant fibroblasts to test adipogenic responses of ACM fibroblasts to our 3F adipogenic protocol because PKP2 mutant fibroblasts have high adipogenic potentials. We also used PKP2 DelC mutant iPSC-CMs to test drug effects on CM apoptosis induced by our 5F protocol. In FIG. 4, RGFP at 0.5 pM effectively reduced adipogenesis (adipocyte formation by immunostaining for adipocyte-specific perilipin 1 -positive cells, FIG. 4B), and prevented CM apoptosis (by TUNEL staining, FIG. 4C). These results support strongly our key pathogenic pathway and therapeutic inventions. All experiments were from 3 biological replicates.

[0104] Example 5: Exemplary HDAC3-selective inhibitors Compound 11 (Cpd 11) and RGFP966 (RGFP) diminished pathogenesis of ACM in vitro

[0105] Cardiac fibrosis is a common finding in all cardiovascular diseases and is usually characterized by excessive production and accumulation of cytosolic vimentin (VIM) and extracellular matrices (ECM, particularly fibrillar collagens) in pathological hearts (Circ Res. 2016, Br J Pharmacol 2022, Inflamm. Regen 2017, and Biochemistry 2016) and may lead to lethal ventricular arrhythmia and heart failure. Resident cardiac fibroblasts are generally considered the principal source of cardiac fibrosis. However, cardiac fibroblasts display heterogeneity and lack commonly accepted markers for identification. In contrast, mesenchymal stromal / stem cells (MSCs), morphologically indistinguishable from fibroblasts, have internationally defined characteristics, can be isolated reproducibly from various organs, and are known adult resident progenitor cells for myofibroblasts (MBs, activated fibroblasts, Adv Drug Deliv Rev. 2017) that produce fibrosis in most organs (Cell Stem Cell, 2017), including the heart. However, obtaining cardiac MSCs or fibroblasts from cardiac patients is rarely performed.

[0106] In ACM, pathogenic desmoplakin (DSP) variants elicit a unique cardiomyopathy with early and excessive cardiac fibrosis. Applicants generated iPSC-derived MSCs (iPSC-MSCs) from healthy donors and 2 unrelated ACM patients with pathogenic heterozygous DSP variants to study how DSP mutations affect their fibrogenic potentials. It was found that DSP-mutant MSCs had -50% wild-type (WT) and no mutant DSP proteins (a DSP-deficient and haploinsufficiency phenotype). Importantly, TGF[31 induced excessive accumulation of VIMs / fibrillar collagens from decreased autophagic degradations, and overactivated fibrotic genes [COL J Al, COL3A1, and fibronectin (FN)\ in DSP-mutant MSCs (Heart Rhythm 2021 ; 18(8): S196).

[0107] Trichostatin A (TSA), a class I HDAC inhibitor, can increase JUP expression in fibrosarcoma lines and decrease adipogenesis. However, systemic inhibition of PPARymay lead to side effects. Our preliminary data, such as presented in FIG. 4, shows that HD AC3 -specific inhibitors can inhibit CM apoptosis and pathological cardiac fibrosis / adipogenesis. Exemplary HD AC3 -specific inhibitors compound 11 (Cpd 11) and RGFP966 (RGFP), the chemical structures of which are presented in FIG. 5, intersect with the iPPRE of ACM pathogenic pathways. The following experimentation was conducted to determine whether 2 structurally different HDAC3- specific inhibitors can effectively prevent 5F-protocol-induced ACM CM pathology, such as apoptosis.

[0108] RGFP966 (available commercially) and compound 11 (synthesized by Drs. Alan Kozikowski and Duncan Wardrop at University of Illinois-Chicago) were tested for their ability to reduce ACM CM apoptosis, abnormal fibrosis and adipogenesis in an in vitro iPSC-based ACM model. Both RGFP966 (ICso=6O-8O nM) and compound 11 (ICso=12O nM) effectively reduced ACM CM apoptosis, pathological fibrosis and adipogenesis at 0.5-1 pM, as shown in FIGS. 6A and 6B, respectively. RGFP966 has been tested in mice and has been shown to be safe without systemic side effects J Ocul Pharmacol Ther. 2018;34:260-273), but compound 11 appeared to be safer than RGFP966 at concentrations slightly above 1 pM, as shown in FIG. 6.

[0109] Specifically, the inhibitory effects of RGFP966 and compound 11 were evaluated respectively on TGF[31-induced fibrogenic responses in DSP-mutant MSCs. We found that both drugs inhibited pathological fibrosis but compound 11 had a better therapeutic window than RGFP966 (FIG. 6A). The iPSC-MSCs with a plakophilin-2 mutation tended to have higher adipogenic potentials. We also tested RGFP966 and compound 11 on 3F-induced pathological adipogenesis in vitro. FIG. 6B shows that both drugs effectively inhibited pathologicaladipogenesis. Specifically, we found that 0.5-1 pM RGFP966 or compound 1 1 effectively reduced pathological cardiac fibrosis (FIG. 6A), pathological cardiac adipogenesis (FIG. 6B), and ACM CM apoptosis (FIG. 6C).

[0110] FIG. 7 shows that the HD AC3 -specific inhibitor RGFP966 prevented cardiac dysfunctions in JUP KO mice in vivo. RGFP decreased ACM iPSC-CM apoptosis (FIG. 7A) in vitro (also see FIG. 4). RGFP also decreased PPARy and apoptotic gene levels (Caspase 3 and BAX / BCL2 ratios, FIG. 7B-C) as well as preserved cardiac ejection fraction (EF) in JUP KO mice (Fig. 7D) after 4 weeks of RGFP treatment.

[0111] Example 6: Evaluation RGFP966 and compound 11 in ACM mouse models

[0112] To confirm the fibrosis- / adipogenesis-suppressing efficacy of exemplary HDAC3- selective inhibitors in mouse models of ACM, RGFP966 and compound 11 will be further evaluated in the aforementioned two mouse ACM models. The compounds will also be tested on a DSP haplo-insufficient ACM mouse models (Sci Transl Med, 2020; 12, eaay8329) for their efficacy in inhibiting pathological fibrosis, reducing abnormal cardiac adipogenesis, preserving cardiac function and decreasing lethal ventricular arrhythmia in vivo.

[0113] Example 7: Administration & Treatment

[0114] A therapeutically effective amount of an HD AC3 -specific inhibitor is administered to a subject having arrhythmogenic cardiomyopathy. The HDAC3-specific inhibitor is administered to the subject via epicardial injection or intra-venous injection. In some examples, the HDAC3- specific inhibitor is administered to the subject as a pharmaceutical composition containing at least one pharmaceutically acceptable excipient. For exemplary purposes, the HDAC3-specific inhibitor is compound 11 or an analog thereof. Treatment efficacy, such as improving LV EF, decreasing heart failure, reducing PVC burdens / ventricular arrhythmia, and improving exercisetolerance, is determined according to methods available to one of skill in the art, such as described by Zhu et al., Front Cardiovasc Med. 2022; 9:926378. In one example, cardiac fibrosis, such as a reduction thereof following administration of the HD AC3 -specific inhibitor, can be determined via evaluation of degrees of late gadolinium enhancement (LGE) by cardiac MRI or histopathological evaluation of biopsied heart tissue.EQUIVALENTS AND SCOPE

[0115] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present invention is not intended to be limited to the above, but rather is as set forth in the appended claims.

[0116] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0117] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses and descriptive terms, from one or more of the listed claims are introduced into another claim. Forexample, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0118] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps.

[0119] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranged can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0120] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5% or up to 1 % of a given value. Alternatively, the term can mean within an order of magnitude, for example within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims,unless otherwise stated the term “about” means within an acceptable error range for the particular value should be assumed.

[0121] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the method of the invention can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

Claims

CLAIMSI claim1. Use of a histone deacetylase 3 (HDAC3)-specific inhibitor for the manufacture of a medicament for a treatment of cardiac fibrosis.

2. The method of claim 1, wherein the HD AC3 -specific inhibitor is RGFP966 ((2E)- N-(2-Amino-4-fluoropheny l)-3 - [(2E)- 1 -(3 -phenyl-2-propen- 1 -yl )- 1 H-pyrazol-4-yl] -2- propenamide, (E)-N-(2-amino-4-fluorophenyl)-3-(l-cinnamyl-lH-pyrazol-4-yl)acrylamide)), compound 11, BRD3308 (4-(acetylamino)-N-(2-amino-4-fluorophenyl)-benzamide), compound I, compound II, compound III, compound IV, compound V, compound VI, compound VII , an analog thereof, or a pharmaceutically acceptable salt thereof.

3. The method of any one of claims 1-2, wherein the HDAC3-specific inhibitor is RGFP966, compound 11, BRD3308, an analog thereof, or a pharmaceutically acceptable salt thereof.

4. The method of any one of claims 1 -2, wherein the cardiac disease is arrhythmogenic cardiomyopathy (ACM).

5. The method of any one of claims 1-2, wherein treating the cardiac disease comprises inhibiting cardiac fibrosis, fibro-fatty replacement of cardiomyocytes, cardiomyocyte apoptosis, or a combination thereof.

6. A method of treating cardiac fibrosis in a subject having a cardiac disease comprising administering a therapeutically effective amount of a histone deacetylase 3 (HDAC3)- specific inhibitor to the subject.

7. The method of claim 6, wherein the cardiac disease is arrhythmogenic cardiomyopathy (ACM).

8. The method of claim 6, wherein the HDAC3-specific inhibitor is RGFP966 ((2E)- N-(2-Amino-4-fluorophenyl)-3-[(2E)-l-(3-phenyl-2-propen-l-yl)-lH-pyrazol-4-yl]-2- propenamide, (E)-N-(2-amino-4-fluorophenyl)-3-(l-cinnamyl-lH-pyrazol-4-yl)acrylamide)), compound 11, BRD3308 (4-(acetylamino)-N-(2-amino-4-fluorophenyl)-benzamide), compound I, compound II, compound III, compound IV, compound V, compound VI, compound VII , an analog thereof, or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the HDAC3-specific inhibitor is RGFP966, compound 11, BRD3308, an analog thereof, or a pharmaceutically acceptable salt thereof.

10. The method of claim 6, comprising intravenously or locally administering a pharmaceutical composition comprising the HDAC3-specific inhibitor to the epicardial space of the subject.

11. The method of claim 6, wherein treating the cardiac disease comprises inhibiting cardiac fibrosis, fibro-fatty replacement of cardiomyocytes, cardiomyocyte apoptosis, or a combination thereof.

12. The method of claim 6, wherein the subject is human.

Images