Givinostat hydrochloride monohydrate for use in treating arrhythmogenic cardiomyopathy

Abstract

The present invention relates to Givinostat hydrochloride monohydrate for use in the treatment of arrhythmogenic cardiomyopathy.

Description

[0001] Ref.: 24NV64IWO

[0002] Title

[0003] Givinostat hydrochloride monohydrate for use in treating arrhythmogenic cardiomyopathy

[0004] *****

[0005] Description

[0006] The present invention relates to Givinostat hydrochloride monohydrate for use in the treatment of arrhythmogenic cardiomyopathy.

[0007] State of the Art

[0008] Givinostat is a hydroxamic acid that acts as an inhibitor of Histone Deacetylase

[0009] (HDAC) and exerts its action on the homonymous class I and II enzymes.

[0010] The chemical name of Givinostat is [6-(diethylaminomethyl)naphthalen-2- yl]methyl[4(hydroxycarbamoyl) phenyl]carbamate; Givinostat is also known as

[0011] ITF2357 and it is normally used in the form of hydrochloride salt, in particular the hydrochloride monohydrate salt.

[0012] The chemical structure of Givinostat hydrochloride monohydrate is shown below:

[0013] ■ HCI

[0014] US9421184 discloses a method for treating muscular dystrophy by administering

[0015] Givinostat hydrochloride, preferably Givinostat hydrochloride monohydrate.

[0016] US9867799 discloses a method for treating muscular dystrophy by administering

[0017] Givinostat hydrochloride, preferably Givinostat hydrochloride monohydrate, together with an anti-inflammatory agent, such as a steroid. Ref.: 24NV64IWO

[0018] US2018311160 discloses an aqueous suspension comprising Givinostat and / or pharmaceutically acceptable salts and / or derivatives thereof, at least a wetting agent and / or at least a density-imparting agent.

[0019] Givinostat has been used in clinical trials for Muscular Dystrophies (Duchenne and Becker Muscular Dystrophies), for inflammatory diseases and for blood cancers (polycythemia vera, myelomas and lymphomas), including a Phase III study in Duchenne Muscular Dystrophy.

[0020] In March 2024 Givinostat was approved by the FDA for the treatment of young DMD patients. In June 2025 Givinostat was also approved by the EMA.

[0021] In the heart context, Givinostat exerted an effective role in reducing the activation of both primary adult rat ventricular fibroblasts and human ventricular fibroblasts as well as their production of extracellular matrix (ECM) components. Additionally, Givinostat was shown to block ECM remodeling and reduce the stiffness of the ventricle in a murine model of diastolic dysfunction (1 ).

[0022] Arrhythmogenic cardiomyopathy (ACM) is a genetic cardiac disease phenotypically characterized by structural and functional alterations of the cardiac tissue, with a prevalence estimated at ~1 :5.000 individuals (2). The disease exhibits variable expressivity and reduced, age-related penetrance. Clinical symptoms typically emerge in the third to fourth decades of life, and include syncope and ventricular tachycardia, with sudden cardiac death (SCD) often being its sole manifestation. The pathological hallmark of ACM is fibrofatty replacement of myocardial tissue, associated with ventricular atrophy (2). Histologically, the disease progresses from the subepicardial tissue towards the endocardium, resulting in a thinned, transmural lesion. The subform involving only the right ventricle is the most common, although left and biventricular forms are well-documented (3). Most pathogenic variants Ref.: 24NV64IWO resulting in ACM are identified in three genes, PKP2, DSP, and DSG2, which encode desmosomal proteins (2). Unfortunately, there are no curative treatments for ACM, and therapeutic options for patients focus on attenuating symptoms, slowing disease progression, and preventing SCD (4). Currently, new frontiers of research are focused on the development of gene therapy approaches aimed at the reestablishment of functional genes.

[0023] Indeed, PKP2 restoration carried out via AAV-administration of the wild-type form of the gene in two different ACM murine models revealed positive effects, arresting the disease or preventing pathogenic manifestations (5,6,7). Although ACM was first described nearly thirty years ago, the pathogenic mechanisms leading to its development are still only partially known. Several studies using different animal models point to the involvement of the Wnt / [3-catenin signaling in combination with Hippo pathway (8). However, it is not clear whether additional pathways are involved in the disease.

[0024] The accumulation of fibrofatty tissue progressively replacing the myocardium is recognized as a major pathological process associated with the development of lifethreatening ventricular arrhythmias and increased risk of sudden cardiac death in ACM. Cardiomyocytes have been for a long period considered as the main cell population involved in ACM since most of the pathogenic variations affect proteins involved in their mechano-functional junctions; however, contribution of other cell types to ACM pathogenesis is now well recognized. Indeed, it has been proposed that reparative processes triggered by cardiomyocyte death, rely on the activation of tissue-resident cardiac mesenchymal progenitors characterized by the expression of Sca-1 and PDGFRa (9). These cells, referred as fibro-adipogenic progenitors (FAP), were originally identified in skeletal muscles where they positively influence muscle Ref.: 24NV64IWO regeneration and homeostasis in physiological conditions (10,11 ). Nonetheless, FAP are able to differentiate into both fibroblasts and adipocytes in turn promoting fibrosis and fat deposition in case of chronic pathogenic conditions like muscular dystrophy (12).

[0025] Therefore, being able to inhibit the differentiation of these precursors may represent a promising tool to slow ACM progression.

[0026] Definitions

[0027] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference; thus, the inclusion of such definitions herein should not be construed to represent a substantial difference over what is generally understood in the art.

[0028] The term “physiologically acceptable excipient” herein refers to a substance devoid of any pharmacological effect of its own and which does not produce adverse reactions when administered to a mammal, preferably a human. Physiologically acceptable excipients are well known in the art and are disclosed, for instance in the Handbook of Pharmaceutical Excipients, sixth edition 2009, herein incorporated by reference.

[0029] The terms “comprising”, “having”, “including” and “containing” are to be construed open-ended terms (i.e. meaning “including, but not limited to”) and are to be considered as providing support also for terms as “consist essentially of”, “consisting essentially of”, “consist of” or “consisting of”.

[0030] The terms “consists essentially of”, “consisting essentially of” are to be construed as semi-closed terms, meaning that no other ingredients which materially affects the Ref.: 24NV64IWO basic and novel characteristics of the invention are included (optional excipients may thus be included).

[0031] The terms “consists of”, “consisting of” are to be construed as closed terms.

[0032] Figures

[0033] Figure 1. Transgenic mice show increased abundances of cFAPs

[0034] (A) Flow cytometry plots showing the gating strategy used to isolate cFAPs (cardiac fibroadipogenic progenitors) from murine hearts. Positive gates were set by analyzing signals from FMO (fluorescent minus one) samples. Live non-myocyte cardiac cells were sorted to isolate cells positive for PDGFRa but negative for CD31 and CD45. Positivity for Seal was used to discriminate between resting (PDGFRa+ Sca1 +) and committed (PDGFRa + Seal -) cFAPs. (B, C) Abundances of resting and committed cFAPs defined as percentage on total live cells. Data are presented with mean and SEM; n=11 ; parametric paired Student’s t-test. *P<0.05; **P<0.01 ; ***P<0.001.

[0035] Figure 2. Givinostat reduces cFAP proliferation

[0036] (A) Representative images of Edll staining (B) Cell proliferation in presence or absence of 50nM Givinostat (Giv) determined by Edll staining as percentage of Edll positive cells on total cells. n=6. Data are presented with mean and SEM; One-Way ANOVA with Tukey correction for multiple comparisons. Outcomes of pairwise comparisons are shown only for those comparisons with biological meaning, ns: non- significative *P<0.05; **P<0.01 ; ***P<0.001.

[0037] Figures 3A-D and 3E-I. Givinostat inhibits adipogenic differentiation

[0038] (A) Schematic representation of the workflow for adipogenic differentiation. (B) Representative bright-field microscope images of cells after 9 days of culturing in different conditions. (C) Adipogenic potential evaluated as number of adipocytes per cm2obtained exposing cFAPs to adipogenic stimulation in presence or absence of Ref.: 24NV64IWO

[0039] 50nM Givinostat. n=10. Data are presented with mean and SEM; One-Way ANOVA with Tukey correction for multiple comparisons. Outcomes of pairwise comparisons are shown only for those comparisons with biological relevance. Logarithmic transformation was applied to raw data prior to statistical test to ensure a Gaussian distribution. (D - E) Gene ontology (GO) terms associated to differentially expressed genes in treated versus untreated cells isolated from a transgenic ACM murine model (Tg-hQ mice). On the right: heat map with the genes associated to the most significant term of the corresponding GO. Each column represents data from a single biological replicate. The color scale bar represents the Z-score of gene expression. 2A, 4A, 6A and 8A: cells exposed to adipogenic differentiation. 2B, 4B, 6B and 8B: cells exposed to adipogenic differentiation and treated with 50nM Givinostat. BP: Biological Process. (F - I) Validation in Real Time qPCR of relevant adipogenic marker genes. Cebpa: CCAAT Enhancer Binding Protein Alpha; Pparg: peroxisome proliferator activated receptor gamma; Adipoq: Adiponectin; Plin: perilipin. Data are presented with mean and SEM; n=5; Mann-Whitney test, ns: non-significative; *P<0.05; **P<0.01 ; ***P<0.001 .PM: Proliferation Medium; AM I: Adipogenic Medium I; AM II: Adipogenic Medium II; Giv: Givinostat.

[0040] Figure 4. Givinostat alters the transcriptional profile of cells induced to adipogenic differentiation

[0041] Heat map of differentially expressed genes upon treatment with Givinostat in WT (A) of Tg-hQ (B) cells. Each column represents data from a single biological replicate. The color scale bar represents the Z-score of gene expression. 1A, 3A, 5A and 7A: WT cells exposed to adipogenic differentiation; 1 B, 3B, 5B and 7B: WT cells exposed to adipogenic differentiation and treated with 50nM Givinostat; 2A, 4A, 6A and 8A: Tg-hQ cells exposed to adipogenic differentiation; 2B, 4B, 6B and 8B: Tg-hQ cells Ref.: 24NV64IWO exposed to adipogenic differentiation and treated with 50nM Givinostat AM: Adipogenic Media; Giv: Givinostat.

[0042] Figure 5. Givinostat deregulated key processes connected to lipid metabolism.

[0043] Gene ontology (GO) terms associated to differentially expressed genes in treated versus untreated cells isolated from WT mice. On the right: heat map with the genes associated to the most significant term of the corresponding GO. Each column represents data from a single biological replicate. The color scale bar represents the Z-score of gene expression. 1A, 3A, 5A and 7A: cells exposed to adipogenic differentiation. 1 B, 3B, 5B and 7B: cells exposed to adipogenic differentiation and treated with 50nM Givinostat. BP: Biological Process.

[0044] Figure 6. Givinostat reduces the expression of known adipogenic markers.

[0045] Quantification of expression levels of known adipogenic markers investigated by RNA-Seq. Data are presented a FPKM (Fragments Per Kilobase Million) with mean and SEM; Cebpa: CCAAT / enhancer-binding proteins a; Pparg: peroxisome proliferator-activated receptors; Adipoq: Adiponectin; Plin: perilipin n=4; parametric paired Student’s t-test. *P<0.05; **P<0.01 ; ***P<0.001.

[0046] Figure 7. Givinostat inhibits fibrotic differentiation.

[0047] (A) Schematic representation of the workflow for fibrotic differentiation. (B) Gene ontology (GO) terms associated to differentially expressed genes in treated versus untreated cells isolated from Tg-hQ mice. On the right: heat map with the genes associated to the most significant term of the corresponding GO. Each column represents data from a single biological replicate. The color scale bar represents the Z-score of gene expression. 2C, 4C, 6C and 8C: cells exposed to fibrogenic differentiation. 2D, 4D, 6D and 8D: cells exposed to fibrogenic differentiation and treated with 50nM Givinostat. MF: Molecular Function. (C - D) Validation in Real Ref.: 24NV64IWO

[0048] Time qPCR of relevant fibrogenic markers. Col1a1 : Collagen type 1 ; Postn: Periostin. Data are presented with mean and SEM; n=5; Mann-Whitney test. *P<0.05; **P<0.01 ; ***P<0.001 .PM: Proliferation Medium; FM: Fibrogenic Medium; Giv: Givinostat.

[0049] Figure 8. Givinostat alters the transcriptional profile of cells induced to fibrotic differentiation

[0050] Heat map of differentially expressed genes upon treatment with Givinostat in WT (A) of Tg-hQ (B) cells. Each column represents data from a single biological replicate. The color scale bar represents the Z-score of gene expression. 1 C, 3C, 5C and 7C: WT cells exposed to fibrogenic differentiation; 1 D, 3D, 5D and 7D: WT cells exposed to fibrogenic differentiation and treated with 50nM Givinostat; 2C, 4C, 6C and 8C: Tg- hQ cells exposed to fibrogenic differentiation; 2D, 4D, 6D and 8D: Tg-hQ cells exposed to fibrogenic differentiation and treated with 50nM Givinostat; FM: Fibrogenic Media; Giv: Givinostat.

[0051] Figure 9. Givinostat affects the expression ECM-related genes in WT cells.

[0052] Gene ontology (GO) terms associated to differentially expressed genes in treated versus untreated cells isolated from WT mice. On the right: heat map with the genes associated to the most significant term of the corresponding GO. Each column represents data from a single biological replicate. The color scale bar represents the Z-score of gene expression. 1 C, 3C, 5C and 7C: cells exposed to fibrogenic differentiation. 1 D, 3D, 5D and 7D: cells exposed to fibrogenic differentiation and treated with 50nM Givinostat. MF: Molecular Function.

[0053] Figure 10. Givinostat reduces the expression of known fibrotic markers. Ref.: 24NV64IWO

[0054] Expression of known fibrotic markers investigated by RNA-Seq. Data are presented a FPKM (Fragments Per Kilobase Million) with mean and SEM; n=4; parametric paired Student’s t-test. *P<0.05; **P<0.01 ; ***P<0.001.

[0055] Figure 11. Givinostat reduces collagenl production by TGF- -stimulated cFAPs.

[0056] Examples of western blot analysis and corresponding quantification of the amount of collagen 1 produced when cFAPs from Tg.hQ mice were exposed to TGF-[3 and eventually treated with Givinostat (Giv). Vinculin (Vin) was used as normalizer. Mann- Whitney test, ns: non-significative *P<0.05; **P<0.01 ; ***P<0.001.

[0057] Description of the invention

[0058] The present inventors have now found that Givinostat hydrochloride monohydrate is able to reduce cFAP proliferation and to inhibit cFAP adipogenic and fibrogenic differentiation in vitro.

[0059] The present invention relates Givinostat hydrochloride monohydrate for use in treating arrhythmogenic cardiomyopathy.

[0060] According to a preferred embodiment of the invention, Givinostat hydrochloride monohydrate is administered on a daily basis. Preferably, it is administered twice daily.

[0061] Givinostat hydrochloride monohydrate is administered in an amount ranging from 10 mg to 70 mg twice daily based on body weight, preferably from 13 mg to 54 mg twice daily.

[0062] According to a preferred embodiment of the invention, Givinostat hydrochloride monohydrate is in crystal form. Ref.: 24NV64IWO

[0063] According to a preferred embodiment of the invention, Givinostat hydrochloride monohydrate is administered in the form of a pharmaceutical composition comprising the same together with at least one physiologically acceptable excipient.

[0064] According to a preferred embodiment of the invention, the pharmaceutical composition of the invention contains from 10 to 300 mg of Givinostat hydrochloride monohydrate, preferably from 25 to 200 mg, per unit dosage form.

[0065] For the purposes of the present invention Givinostat hydrochloride monohydrate can be effectively administered alone or it can be administered in combination with at least one additional active agent selected from the group consisting of beta blockers (e.g. metoprolol) and antiarrhythmic agents (e.g. sotalol, flecainide or amiodarone).

[0066] According to a preferred embodiment of the invention, metoprolol can be administered once daily 50-200 mg (typically 100 mg).

[0067] According to a preferred embodiment of the invention, sotalol can be administered twice daily 80 mg or three times daily 80 mg; or flecainide once daily 150-200 mg.

[0068] The combination therapy according to the present invention includes administration of a single pharmaceutical dosage formulation comprising Givinostat hydrochloride monohydrate and the at least one additional active agent, as well as the administration of Givinostat hydrochloride monohydrate and the at least one additional active agent each in their own separate pharmaceutical dosage formulations.

[0069] Where separate dosage formulations are used, Givinostat hydrochloride monohydrate and the at least one additional active agent can be administered at essentially the same time, i.e., concurrently; or at separately staggered times, i.e., sequentially. The combination therapy according to the present invention is understood to include all these regimens. Ref.: 24NV64IWO

[0070] According to an embodiment of the invention, the pharmaceutical composition may be administered by oral, sublingual, rectal, intravascular, intravenous, or subcutaneous route; preferably by oral route.

[0071] According to an embodiment of the invention, the pharmaceutical composition is in a solid or a liquid form.

[0072] More in details, the solid form may be selected from the group consisting of powder, tablet, granulate, aggregate, compressed pill, coated pill, hard gelatin capsule, and gelatin capsule; the liquid form may be a suspension or a syrup.

[0073] According to an embodiment of the invention, the suspension may comprise at least a wetting agent and / or at least a density-imparting agent and / or at least a buffering agent and / or at least a suspending agent.

[0074] According to a preferred embodiment of the invention, the at least a wetting agent is a polyoxyethylene sorbitan fatty acid ester, poloxamer or a mixture thereof; the at least a density-imparting agent is sorbitol, sucrose or a mixture thereof; the at least a buffering agent is a phosphate buffer, citrate buffer, or tartrate buffer; the at least a suspending agent is tragacanth gum or xanthan gum.

[0075] According to a more preferred embodiment of the invention, the suspension essentially comprises Givinostat hydrochloride monohydrate, a polyoxyethylene sorbitan fatty acid ester, sorbitol, a tartrate buffer and tragacanth gum.

[0076] The following examples are intended to be illustrative of the invention rather than limiting the scope thereof.

[0077] Experimental section

[0078] 1. Methods

[0079] 1.1 Animals Ref.: 24NV64IWO

[0080] The study was carried out using a transgenic strain (Tg-hQ) previously generated in our laboratory and characterized by cardiomyocyte-specific overexpression of a mutated human DSG2 gene, carrying the p.Q558* nonsense mutation (13). Non- transgenic animals from the same original C57BL / 6N strain were used as control. Mice were euthanized by cervical dislocation at the age of 10 months. Hearts were perfused with heparin in PBS (10 U / mL), the atria were removed and only the ventricular part was processed.

[0081] All animal procedures performed were conform to the guidelines from Directive 2010 / 63 / EU of the European Parliament on the protection of animals used for scientific purposes and to the current NIH guidelines for the Care and Use of Laboratory Animals. All experimental protocols were approved by the internal Animal Research Ethical Committee according to the Italian Ministry of Health regulation (approval number 68 / 2011 ).

[0082] 1.2 Isolation of FAPs from cardiac tissue cFAPs were isolated from 10 month-old control (WT) and Tg-hQ mice. Freshly dissected hearts were digested for 30 min at 37°C in a volume of 500 pL of 2mg / mL Collagenase II (Sigma) with 2.5 mM CaCl2, followed by a second digestion for 1 hour at 37°C in a solution of 1 ,5 U / mL Collagenase D (Sigma) and2.4 U / mL Dispase II (Sigma) in 5 mM CaCh The digested material was suspended in sorting buffer containing 0,5% w / v BSA, 2mM di EDTA in PBS, dissolved by pipetting and passed through 70 pm and 40 pm cell strainers. The cell suspension was centrifuged, and the resulting pellet was resuspended in red blood cell lysis buffer (0,15 mM NH4CI; 8,4 mM KHCO3; 1 ,2 mM EDTA) for 1 minute and then washed with sorting buffer. The cell pellet obtained after centrifugation was processed for fluorescence-activated Ref.: 24NV64IWO cell sorting. To reduce variability due to the isolation process, in each cell preparation one control and one Tg-hQ mouse were processed and analyzed in parallel.

[0083] 1.3 Fluorescence-activated cell sorting (FACS)

[0084] Cells were labelled for FACS with a cocktail containing anti-CD31- PE-Cy7 (Thermo Fisher Scientific), anti-CD45-eFluor450 (Thermo Fisher Scientific), anti-PDGFRa- APC (Thermo Fisher Scientific) and anti-Sca1-FITC (Thermo Fisher Scientific). In addition, Viobility™ 405 / 520 Fixable Dye (Miltenyi Biotec) was used to distinguish live from dead cells. A small volume of cell suspension was used to generate each FMO (Fluorescence Minus One) control. Stained cells were resuspended in sorting buffer and sorted using a FACSAria™ lllu cell sorter. Resting and committed cFAPs were identified as CD45- CD31 - Sca1 + PDGFRa+ and CD45- CD31- Seal- PDGFRa+ respectively. Sorted cells were collected in FBS-pretreated tubes containing DMEM GlutaMAX (Gibco) supplemented with 20% FBS, penicillin (100 U / mL) and streptomycin (100 ug / mL).

[0085] 1.4 Cell cultures and differentiation

[0086] Resting cFAPs were expanded in proliferation medium (DMEM GlutaMAX high glucose, 20% FBS, 10% Horse Serum, penicillin 100 U / mL, streptomycin 100 ug / mL) with 5ng / mL FGF[3 (Immunotools Gmbh, Germany). To promote adipogenic differentiation, two different media were employed. After initial three days in proliferation medium, cells were exposed for three days to the first adipogenic medium (DMEM GlutaMAX high glucose, 100 U / mL penicillin, 100 U / mL streptomycin, 10% di FBS, 0,25 mM Dexamethasone (Sigma), 0,5 mM di 3-isobutil-1 - metilxantine (Sigma) and 10 pg / ml insulin (Sigma); thereafter indicated as AM I), followed by three days with the second adipogenic medium (DMEM GlutaMAX high glucose, 100 U / mL penicillin, 100 U / mL streptomycin, 10% FBS, 10 pg / ml insulin; Ref.: 24NV64IWO thereafter indicated as AM II). For fibroblast differentiation, cells were cultured in proliferation medium for three days and subsequently for six more days with the addition of 5 ng / mL TGF[3 (Peptrotech). Unless otherwise specified, all media and supplements were from Gibco. Where indicated, Givinostat (provided by Italfarmaco, Milan, Italy) was added to the different media at a final concentration of 50 nM. Equivalent volumes of the vehicle (DMSO) were added to the untreated control conditions.

[0087] 1.5 Evaluation of cell proliferation

[0088] The proliferation rate of cultured cells was assessed via Click-iT® EdU Imaging Kit (Invitrogen) following the manufacturer’s protocol. Briefly, cFAPs, cultured in proliferation medium either with or without 50nM Givinostat, were exposed to 10 pM EdU for 16hrs. Incubation with the reaction cocktail was performed for 30 min at room temperature. Afterward, cells were rinsed off in PBS and mounted on microscope slides with FluoShield mounting medium (Invitrogen). Images were acquired using a Leica DM6 B microscope and analyzed with Imaged; at least 10 randomly chosen fields per coverslip were quantified for each experimental condition.

[0089] 1.6 RNA isolation

[0090] Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions and resuspended in RNAse-free water. Concentration of extracted RNA was determined using a NanoDrop™ spectrophotometer (Thermo Scientific).

[0091] 1.7 Bulk RNA-Sequencing

[0092] RNA-Sequencing was performed on four biological replicates of fibrogenic and adipogenic differentiated cells with or without treatment with Givinostat. Cells at the end of the differentiation protocol were collected in Trizol reagent (Invitrogen) and Ref.: 24NV64IWO sent to BGI genomics technical service (China) where RNA was extracted, enriched for mRNA, and fragmented. cDNA was synthetized and ligated to adaptors. Samples were sequenced using the BGISEQ sequencing platform with paired-end reads of length 100. Reads of low quality were filtered before data analysis. Clean reads were then aligned to Mus musculus reference genome GCF_000001635.26_GRCm38.p6 using HISAT and to reference genes using Bowtie2. The average mapping ratio with the reference genome was 96.90%, and the average mapping ratio with genes was 83.21 %; 19.222 genes were identified. Genes without at least 10 counts in 3 samples for each condition of a given comparison were filtered out. Differentially expressed genes were determined using the edgeR R package with TMM normalization. Enrichment analysis was performed using the clusterProfiler R package.

[0093] 1.8 Retrotrascription and Real-time PCR

[0094] RNA was retrotranscribed to cDNA using PrimeScript™ Reverse Transcriptase (Takara), following the manufacturer’s protocol.

[0095] Real-time quantitative PCR was performed using Powerllp™ SYBR™ Green master mix (Applied Biosystems™ by Thermo Fisher Scientific) on a CFX384 Touch Real- Time PCR System (BioRad) for 40 cycles. All amplification reactions were carried out in triplicate. Primers are listed in Table S1. Run data were analyzed using the BioRad CFX Manager™ software (BioRad), with further calculations performed using Excel (Microsoft). The TATA Binding Protein (TPB) gene was used as a housekeeping gene, and relative quantifications between experimental conditions were obtained using the 2-AACt method. Analyses were performed separately for cFAPs obtained from WT or Tg-hQ mice. Graphs were generated using Prism 9 (GraphPad).

[0096] 1.9 Protein extraction and Western Blot Ref.: 24NV64IWO

[0097] Cells were lysed with complete RIPA buffer containing 1X protease inhibitor (Roche) and 1X phosphatase inhibitor (Roche). Total protein concentration was quantified using the Pierce BCA protein assay kit (Thermo Scientific) following the manufacturer’s instructions.

[0098] Protein extracts were separated by SDS-PAGE on precast 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and blotted onto nitrocellulose membranes using the Trans-Blot Turbo system (Bio-Rad). Membranes were blocked with 5% milk in Trisbuffered saline (TBS) containing 0.05% Tween and subsequently incubated overnight with primary antibodies diluted in 2.5% milk TBS-0.05% Tween. Antibodies used for western blotting are provided in Table S2. Appropriate horseradish peroxidase-linked IgG (Jackson Immuno Research) were incubated for 1 hour at room temperature. Bands were detected by chemiluminescence using the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). Membranes were imaged with the Alliance 9.7 Imaging System (UVItec Limited) and quantitative densitometry was performed with the ImageJ software.

[0099] 1.10 Statistical analysis

[0100] Values in the graphs are presented as mean with standard error of the mean. Statistical analyses were performed with Prism 9 (GraphPad) using the appropriate tests depending on the experimental design as specified in figure legends. If necessary, logarithmic transformation was applied to not-normally distributed data prior to statistical analysis.

[0101] 1.11 Data availability

[0102] Transcriptom ic data generated in this study have been deposited in the Gene Expression Omnibus under the accession code PRJNA1039596.

[0103] 2. Results Ref.: 24NV64IWO

[0104] 2.1 Transgenic mice show increased abundances of cFAPs number cFAPs from hearts of 10 month-old Tg-hQ mice and control littermates were isolated via enzymatic digestion followed by fluorescence-activated cell sorting (FACS). Cell suspensions were labelled with antibodies recognizing the pan-hematopoietic marker CD45, the endothelial marker CD31 , the mesenchymal progenitor marker PDGFRa, and the sternness marker Seal . cFAPs were selected as CD45- CD31- PDGFRa+; Seal expression was used to discriminate between resting (Sca1 +) and committed (Seal-) cells. The sorting strategy is summarized in Figure 1A. The present inventors consistently observed a larger amount of both resting and committed cFAPs in the hearts of Tg-hQ mice compared to WT, with the difference being more pronounced in the case of Sca1 + cells (Figure 1B-C). All the experiments described henceforth were carried out using exclusively the resting cFAP fraction.

[0105] 2.2 Givinostat reduces cFAP proliferation in vitro

[0106] In order to evaluate the effect of Givinostat on cFAPs behavior, the present inventors firstly performed overnight Edll labelling to determine whether the drug affects cell proliferation. Freshly isolated cells were cultured in proliferation medium either with or without 50 nM Givinostat. While there was no difference in the proliferation rate of cFAPs derived from WT and Tg-hQ hearts, Givinostat resulted in approximately a 40% reduction of Edll-positive nuclei in both kind of cells, indicating a significant decrease in the number of cycling cells (Figure 2A, 2B). Notably, no increase in apoptotic cells, as assessed by TUNEL assay, was observed in Givinostat treated condition compared to the untreated control, thus excluding potential toxic effects induced by the drug (data not shown).

[0107] 2.3 Givinostat inhibits cFAP adipogenic differentiation in vitro Ref.: 24NV64IWO cFAPs from WT or Tg-hQ hearts were expanded for 3 days in proliferation medium and then induced to differentiate into adipocytes by the sequential use of two adipogenic media (AM I and AM II), as described in the Methods section. Two experimental conditions were set exposing the cells either to 50 nM Givinostat (AM + Giv) or to the vehicle (AM) (Figure 3A). Regardless from their origin, cFAPs did not undergo spontaneous differentiation into adipocytes when cultured in standard proliferation medium (PM). When provided with adipogenic stimuli, cFAPs differentiated into adipocytes, although with variable yields among different replicates (Figure 3B, C). No differences were observed in the average number of adipocytes obtained from WT and Tg-hQ cultures (Figure 3C). However, when exposed to 50 nM Givinostat, the adipogenic potential of cFAPs was dramatically reduced, resulting in approximatively 70% fewer adipocytes per square cm. This effect was observed equally in Tg-hQ and WT-derived cFAPs (Figure 3C).

[0108] Bulk RNA sequencing in both treated and untreated cells was performed to assess the effect of Givinostat on adipogenic differentiation at the transcriptional level. Three differentially expressed genes (DEGs) (Tagap, Rnaset2b, Nwd1) were detected in the comparison between transcriptomes of treated WT and Tg-hQ cells, whereas four DEGs (Tagap, Rnaset2b, Rnaset2a, Scgblal) were identified in the comparison between untreated cFAPs, suggesting similar behavior of these cells regardless of their origin. Conversely, Givinostat markedly affected the transcriptome profile of treated cells compared to the untreated condition. In WT cells, 744 DEGs were found, most of which were down-regulated (566) rather than up-regulated (178). Among the 1002 DEGs identified in Tg-hQ treated cFAPs, 763 out of 1002 were down-regulated while 239 were up-regulated upon treatment (Figure 4A, 4B). Ref.: 24NV64IWO

[0109] Gene ontology (GO) analysis conducted separately for WT and Tg-hQ DEGs highlighted common alterations in biological processes associated with lipid metabolism; indeed, fatty acids and triglyceride metabolic processes were among the top 5 enriched terms (Figure 3D, Figure 5). Most genes involved in these processes appeared to be downregulated in presence of the drug. Givinostat also altered key adipogenic pathways, including the PPAR signaling pathway and the regulation of lipolysis in adipocytes (Figure 3E, Figure 5). Consistent with these findings, key regulators of adipogenic activation (Cebpa and Pparg) and maturation (Adipoq and Plin) were significantly down-regulated in treated cells compared to untreated controls (Figure 6). The reduced expression of these markers was further confirmed on an independent set of samples by RT-PCR using Tbp as housekeeping gene for normalization (Figure 3 F-l).

[0110] Taken together, these data reveal the anti-adipogenic action of Givinostat and highlight its potential to inhibit fatty accumulation in the heart by blocking cFAP differentiation toward adipocytes.

[0111] 2.4 Givinostat inhibits cFAP fibrogenic differentiation in vitro

[0112] Differentiation towards fibroblasts was induced in vitro by the addition of 5 ng / mL TGF[3 in the proliferation medium of both WT and Tg-hQ cells and was eventually combined with 50nM Givinostat treatment as schematized in Figure 7 A. Bulk RNA sequencing was performed to investigate the effect of the compound at the transcriptom ic level. Tg-hQ and WT cells cultured under the same conditions presented almost identical transcriptomes, with only three (Tagap, Rnaset2b, Zfp965) and four (Tagap, Rnaset2b, H2ac19, Comp) DEGs in untreated and treated cells, respectively. However, Givinostat treatment led to differential gene expression in both Tg-hQ and WT cells compared to the untreated counterpart. In Tg-hQ cells, 698 Ref.: 24NV64IWO differentially expressed genes were detected, with 257 genes up-regulated and 441 genes down-regulated. In WT cells, differential expression analysis identified 754 genes that were either up-regulated (291 genes) or down-regulated (463 genes) in the treated condition versus the untreated (Figure 8A, 8B).

[0113] GO analysis, conducted separately for Tg-hQ and WT cells, identified the extracellular matrix (ECM) constituent as the most enriched term in both cases (Figure 7B, Figure 9).

[0114] Transcript levels of two representative ECM components, Col1a1 and Postn, which were significantly down-regulated in the transcriptom ic data (Figure 10), were confirmed by RT-PCR on an independent set of samples (Figure 7C, 7D). Immunoblotting further demonstrated the reduced production of Collagen I at the protein level (Figure 11). Taken together, these data confirm the anti-fibrotic role of Givinostat.

[0115] Therefore, the treatment with Givinostat could represent a novel strategy to tackle cardiac fatty accumulation in ACM.

[0116] Additionally, in cells cultured in a pro-fibrotic environment and exposed to Givinostat the present inventors observed a drastic reduction in the expression of several genes encoding different types of collagen and other components of the extracellular matrix. This result corroborates anti-fibrotic role of Givinostat.

[0117] In summary, Givinostat was shown to have the potential of blocking fibro-fatty degeneration in ACM by acting on cFAPs, a cell population of cardiac stromal cells enriched in multipotent mesenchymal progenitors. The pharmacologic activity of HDACis in Duchenne muscular dystrophy was largely ascribed to their ability to affect FAP plasticity and FAP-derived secretory pathways during DMD progression (14,15). Ref.: 24NV64IWO

[0118] The present inventors now found that Givinostat had profound effects on cFAP plasticity that opens the way for its use as a therapeutic agent to treat ACM.

[0119] Therapeutic efficacy of Givinostat in murine models of arrhythmogenic cardiomyopathy (ACM)

[0120] Models

[0121] Two complementary mouse models can be used:

[0122] 1. PKP2 conditional knockout (PKP2-cKO). A cardiomyocyte-specific, tamoxifen-inducible PKP2 knockout line (aMHC-Cre-ER(T2) / Pkp2fl / fl) (16). After tamoxifen induction at five months of age, by 28 days post-induction (dpi), these mice develop biventricular dilated cardiomyopathy with extensive fibrosis. Right ventricular (RV) dysfunction is the earliest detectable phenotype (14 dpi), followed by RV-predom inant fibrosis and progression to end-stage biventricular failure.

[0123] 2. Dsg2 knock-in (KI-Dsg2-G105R). A knock-in line carrying the p.G105R mutation in Dsg2. At 9 months, mice display a mild phenotype with desmosomal disorganization, focal fibrosis, and chronic activation of inflammatory pathways. Immune profiling shows increased pro-inflammatory macrophages and reduced anti-inflammatory populations, indicating persistent immune imbalance contributing to ACM pathogenesis.

[0124] The PKP2-cKO model represents a severe, rapidly progressing phenotype, whereas the KI-Dsg2AG105R model reflects a milder, late-onset disease with prominent inflammatory involvement.

[0125] Treatment Protocol

[0126] • PKP2-cKO mice: daily treatment for 28 days, initiated at tamoxifen induction. Ref.: 24NV64IWO

[0127] • KI-Dsg2-G105R mice: daily treatment for 3 months, starting at 6 months of age.

[0128] Givinostat will be administered by oral gavage, preferably at 10 mg / kg / day.

[0129] Task 1. In vivo cardiac phenotype evaluation

[0130] Objective: To assess cardiac function in treated and untreated ACM mice.

[0131] Methods:

[0132] • General monitoring: Mice will be weighed weekly to detect treatment-related systemic toxicity. A weight loss >10% relative to control averages, or abnormal behaviour, will prompt additional investigations, including renal and hepatic function assays (e.g., creatinine, ALT, ALP).

[0133] • Electrocardiography (ECG): Performed weekly (for PKP2-cKO mice) or biweekly (for KI-Dsg2-G105R mice) to monitor rhythm disturbances and detect potential drug-induced arrhythmias.

[0134] • Echocardiography and MRI: At treatment end, under isoflurane anaesthesia, both ventricles will be evaluated for morphological and functional changes. o Assessment of wall thickness, ejection fraction, and wall stiffness as indicators of disease progression. o Detailed quantification of left ventricular function, including systolic and diastolic parameters as well as load-independent indices of contractility, relaxation, and myocardial stiffness.

[0135] Task 2. Ex vivo cardiac phenotype evaluation

[0136] Objective: To characterize histological, ultrastructural, and molecular features of treated versus untreated hearts. Ref.: 24NV64IWO

[0137] Methods:

[0138] • Histology: o Hematoxylin and eosin (H&E) for overall morphology. o Sirius Red for fibrosis quantification. o Immunofluorescence for smooth muscle actin to identify myofibroblasts.

[0139] • Ultrastructural analysis: Transmission electron microscopy (TEM) for desmosome integrity and sarcomere organization.

[0140] • Molecular assays: o Western blot and qRT-PCR for collagen I expression. o Hydroxyproline assay for collagen cross-linking. o Transcriptom ic profiling if significant antif ibrotic effects are detected.

[0141] • Flow cytometry (FACS): Quantification of cardiac immune and stromal cell subsets (e.g., macrophages, fibro-adipogenic progenitors), with analysis of activation states relevant to fibrosis and inflammation.

[0142] Overall, these analyses will generate integrated functional, histological, and molecular datasets for comparative evaluation of treatment effects.

[0143] Ref.: 24NV64IWO

[0144] References

[0145] 1. Travers JG, Wennersten SA, Pena B, Bagchi RA, Smith HE, Hirsch RA, Vanderlinden LA, Lin YH, Dobrinskikh E, Demos-Davies KM, Cavasin MA, Mestroni L, Steinkuhler C, Lin CY, Houser SR, Woulfe KC, Lam MPY, McKinsey TA. HDAC Inhibition Reverses Preexisting Diastolic Dysfunction and Blocks Covert Extracellular Matrix Remodeling. Circulation 2021 ; 143(19): 1874-1890

[0146] 2. Corrado D, Link MS, Calkins H. Arrhythmogenic Right Ventricular Cardiomyopathy. New Engl J Med 2017;376:61-72.

[0147] 3. Rastegar N, Zimmerman SL, Te Riele ASJM, James C, Burt JR, Bhonsale A, Murray B, Tichnell C, Judge D, Calkins H, Tandri H, Bluemke DA, Kamel IR. Spectrum of Biventricular Involvement on CMR Among Carriers of ARVD / C- Associated Mutations. JACC Cardiovasc Imaging 2015;8:863-864.

[0148] 4. van der Voorn SM, Te Riele ASJM, Basso C, Calkins H, Remme CA, van Veen TAB. Arrhythmogenic cardiomyopathy: pathogenesis, pro-arrhythmic remodelling, and novel approaches for risk stratification and therapy. Cardiovasc Res 2020; 116:1571-1584

[0149] 5. Bradford WH, Zhang J, Gutierrez-Lara EJ, Liang Y, Do A, Wang T, Nguyen L, Mataraarachchi N, Wang J, Gu Y, Wang J, Gu Y, McCulloch A, Peterson KL, Sheikh F. Plakophilin 2 gene therapy prevents and rescues arrhythmogenic right ventricular cardiomyopathy in a mouse model harboring patient genetics. Nat. Cardiovasc. Res 2023; 2:1246-1261.

[0150] 6. Kyriakopoulou E, Versteeg D, de Ruiter H, Perini I, Seibertz F, Ddring Y, Zentilin L, Tsui H, van Kampen SJ, Tiburcy M, Meyer T, Voigt N, van Tintelen JP, Zimmermann WH, Giacca M, van Rooij E. Therapeutic efficacy of AAV-mediated Ref.: 24NV64IWO restoration of PKP2 in arrhythmogenic cardiomyopathy. Nat Cardiovasc Res 2023; 2:1262-1276.

[0151] 7. van Opbergen CJM, Narayanan B, Sacramento CB, Stiles KM, Mishra V, Frenk E, Ricks D, Chen G, Zhang M, Yarabe P, Schwartz J, Delmar M, Herzog CD, Cerrone M. AAV-Mediated Delivery of Plakophilin-2a Arrests Progression of Arrhythmogenic Right Ventricular Cardiomyopathy in Murine Hearts: Preclinical Evidence Supporting Gene Therapy in Humans. Circ Genom Precis Med 2024; 17(1 ):e004305

[0152] 8. Chen SN, Gurha P, Lombardi R, Ruggiero A, Willerson JT, Marian AJ. The hippo pathway is activated and is a causal mechanism for adipogenesis in arrhythmogenic cardiomyopathy. Circ Res 2014;114:454-468.

[0153] 9. Soliman H, Paylor B, Scott RW, Lemos DR, Chang C, Arostegui M, Low M, Lee C, Fiore D, Braghetta P, Pospichalova V, Barkauskas CE, Korinek V, Rampazzo A, MacLeod K, Underhill TM, Rossi FMV. Pathogenic Potential of Hic1 -Expressing Cardiac Stromal Progenitors. Cell Stem Cell 2020;26:459-461.

[0154] 10. Uezumi A, Fukada S, Yamamoto N, Takeda S, Tsuchida K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat Cell Biol 2010; 12: 143-152

[0155] 11. Wosczyna MN, Konishi CT, Perez Carbajal EE, Wang TT, Walsh RA, Gan Q, Wagner MW, Rando TA. Mesenchymal stromal cells are required for regeneration and homeostatic maintenance of skeletal muscle. Cell Rep 2019; 27: 2029-2035

[0156] 12. Lemos DR, Babaeijandaghi F, Low M, Chang C-K, Lee ST, Fiore D, Zhang R- H, Natarajan A, Nedospasov SA, Rossi FMV. Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro / adipogenic progenitors. Nat Med 2015; 21 : 786-794 Ref.: 24NV64IWO

[0157] 13. Calore M, Lorenzon A, Vitiello L, Poloni G, Khan MAF, Beffagna G, Dazzo E, Sacchetto C, Polishchuk R, Sabatelli P, Doliana R, Carnevale D, Lembo G, Bonaldo P, De Windt L, Braghetta P, Rampazzo A. A novel murine model for arrhythmogenic cardiomyopathy points to a pathogenic role of Wnt signalling and miRNA dysregulation. Cardiovasc Res. 2019;115:739-751.

[0158] 14. Sandona M, Consalvi S, Tucciarone L, De Bardi M, Scimeca M, Angelini DF, Buffa V, D'Amico A, Bertini ES, Cazzaniga S, Bettica P, Bouche M, Bongiovanni A, Puri PL, Saccone V. HDAC inhibitors tune miRNAs in extracellular vesicles of dystrophic muscle-resident mesenchymal cells. EMBO Rep. 2020 Sep 3;21 (9):e50863. doi: 10.15252 / embr.202050863. Epub 2020 Aug 5. PMID: 32754983; PMCID: PMC7507515.

[0159] 15. Mozzetta C, Sartorelli V, Steinkuhler C, Puri PL. HDAC inhibitors as pharmacological treatment for Duchenne muscular dystrophy: a discovery journey from bench to patients. Trends Mol Med. 2024 Mar;30(3):278-294. doi: 10.1016 / j.molmed.2024.01.007. Epub 2024 Feb 26.

[0160] 16. Cerrone M, Montanach J, Lin X, Zhao Y-T, Zhang M, Esperanza A-P et al. Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm. Nat Commun. 2017; 8:106

Claims

Ref.: 24NV64IWOClaims1. Givinostat hydrochloride monohydrate for use in treating arrhythmogenic cardiomyopathy.

2. Givinostat hydrochloride monohydrate for use according to claim 1 , characterized in that it is administered to a patient on a daily basis.

3. Givinostat hydrochloride monohydrate for use according to claim 1 or 2, characterized in that it is administered to a patient in an amount ranging from 10 mg to 70 mg twice daily based on body weight, preferably from 13 mg to 54 mg twice daily.

4. Givinostat hydrochloride monohydrate for use according to any one of the preceding claims, characterized in that it is administered to a patient together with at least one active agent selected from the group consisting of beta blockers and antiarrhythmic agents, preferably metoprolol, sotalol, flecainide or amiodarone.

5. Givinostat hydrochloride monohydrate for use according to claim 4, characterized in that it is administered together with said at least one active agent simultaneously, separately or sequentially.

6. Givinostat hydrochloride monohydrate for use according to any one of the preceding claims, wherein it is administered in the form of a pharmaceutical composition together with at least one physiologically acceptable excipient.

7. Givinostat hydrochloride monohydrate for use according to claim 6, wherein said pharmaceutical composition is administered by oral, sublingual, rectal, intravascular, intravenous, or subcutaneous route, preferably by oral route.

8. Givinostat hydrochloride monohydrate for use according to any one of claim 6 or 7, wherein said pharmaceutical composition is in a solid or a liquid form.Ref.: 24NV64IWO9. Givinostat hydrochloride monohydrate for use according to claim 8, wherein said solid form is selected from the group consisting of powder, tablet, granulate, aggregate, compressed pill, coated pill, hard gelatin capsule, and gelatin capsule.

10. Givinostat hydrochloride monohydrate for use according to claim 8, wherein said liquid form is a suspension or a syrup.

11. Givinostat hydrochloride monohydrate for use according to claim 10, wherein said suspension comprises at least a wetting agent and / or at least a densityimparting agent and / or at least a buffering agent and / or at least a suspending agent.

12. Givinostat hydrochloride monohydrate for use according to any one of claims10 or 11 , wherein said suspension comprises at least a wetting agent, at least a density-imparting agent, at least a buffering agent and at least a suspending agent.

13. Givinostat hydrochloride monohydrate for use according to any one of claims11 or 12, wherein said at least a wetting agent is a polyoxyethylene sorbitan fatty acid ester, poloxamer or a mixture thereof; said at least a densityimparting agent is sorbitol, sucrose or a mixture thereof; said at least a buffering agent is a phosphate buffer, citrate buffer, or tartrate buffer; said at least a suspending agent is tragacanth gum or xanthan gum.

14. Givinostat hydrochloride monohydrate for use according to any one of claims 10 to 13, wherein said suspension essentially comprises Givinostat hydrochloride monohydrate, a polyoxyethylene sorbitan fatty acid ester, sorbitol, a tartrate buffer and tragacanth gum.Ref.: 24NV64IWO15. Givinostat hydrochloride monohydrate for use according to any one of the preceding claims, wherein Givinostat hydrochloride monohydrate is in crystal form.

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