WO2025076457A1 - Hdac6-selective inhibitor for use in the treatment of obesity, cardiovascular and metabolic diseases and disorders - Google Patents

Abstract

Provided herein are methods of treating diseases or conditions responsive to HDAC6 inhibition, e.g., metabolic diseases and disorders, heart failure with preserved ejection fraction (HFpEF), and dilated cardiomyopathy, by orally administering to a subject a daily dose of about 10 mg to about 300 mg of Compound 1 having the formula: or a pharmaceutically acceptable salt thereof.

Description

HDAC6-SELECTIVE INHIBITOR FOR USE IN THE TREATMENT OF OBESITY, CARDIOVASCULAR AND METABOLIC DISEASES AND DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Application No. 63/588,664, filed October 6, 2023, which is incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure relates to methods of treating diseases and disorders responsive to HDAC6 inhibition, e.g., metabolic diseases and disorders, heart failure with preserved ejection fraction (HFpEF), and dilated cardiomyopathy, by orally administering to a subject a once-daily dose of about 10 mg to about 300 mg of Compound 1 or a pharmaceutically acceptable salt thereof.

BACKGROUND

[0003] Compound 1 is a highly specific small molecule inhibitor of histone deacetylase 6 (HDAC6) having the following structural formula:

[0004] Preclinical studies have demonstrated that Compound 1 is potentially useful for treating cardiac diseases and disorders, such as heart failure with preserved ejection fraction (HFpEF) and dilated cardiomyopathy, as well as metabolic diseases and disorders, including diabetes mellitus.

[0005] In view of its potential to address serious medical needs across of variety of indications, Compound 1 is being further evaluated in clinical trials.

SUMMARY

[0006] In some aspects, the present disclosure relates to methods of treating a disease or condition responsive to HDAC6 inhibition in a patient in need thereof, comprising orally administering to the patient once daily about 10 mg to about 300 mg of Compound 1 having the formula:

pharmaceutically acceptable salt thereof.

[0007] In some embodiments of the present methods, the disease or condition responsive to HDAC6 inhibition is a metabolic disease or disorder. In some embodiments, the metabolic disease or disorder is a metabolic disease or disorder associated with obesity, such as diet- induced obesity. In some embodiments, the metabolic disease or disorder is not diet-induced. In some embodiments, the metabolic disease or disorder is diabetes, pre-diabetes, diabetic cardiomyopathy, metabolic syndrome, hypertension, hypertriglyceridemia, or dyslipidemia. In some embodiments, the metabolic disease or disorder is diabetes. In some embodiments, the metabolic disease or disorder is diabetes mellitus. In some embodiments, the metabolic disease or disorder is pre-diabetes. In some embodiments, the metabolic disease or disorder is diabetic cardiomyopathy. In some embodiments, the metabolic disease or disorder is metabolic syndrome. In some embodiments, the metabolic disease or disorder is hypertriglyceridemia. In some embodiments, the metabolic disease or disorder is hypertension. In some embodiments, the metabolic disease or disorder is dyslipidemia.

[0008] In some embodiments of the present methods, the disease or condition responsive to HDAC6 inhibition is heart failure with preserved ejection fraction (HFpEF).

[0009] In some embodiments of the present methods, the disease or condition responsive to HDAC6 inhibition is dilated cardiomyopathy.

[0010] In some embodiments of the present methods, about 10 mg to about 200 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg to about 200 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg to about 100 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg, about 50 mg, or about 100 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg, about 75 mg, or about 200 mg of Compound 1 is administered to the patient. In some embodiments, about 10 mg, about 25 mg or about 75 mg of Compound 1 is administered to the patient. In some embodiments, about 10 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg of Compound 1 is administered to the patient. In some embodiments, about 50 mg of Compound 1 is administered to the patient. In some embodiments, about 75 mg of Compound 1 is administered to the patient. In some embodiments, about 100 mg of Compound 1 is administered to the patient. In some embodiments, about 125 mg of Compound 1 is administered to the patient. In some embodiments, about 150 mg of Compound 1 is administered to the patient. In some embodiments, about 200 mg of Compound 1 is administered to the patient. In some embodiments, about 250 mg of Compound 1 is administered to the patient. In some embodiments, about 300 mg of Compound 1 is administered to the patient.

[0011] In some embodiments of the present methods, the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 80 ng/mL to about 1700 ng/mL following administration of about 25 mg to about 300 mg of Compound 1. In some embodiments, the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 80 ng/mL to about 720 ng/mL following administration of about 25 mg to about 100 mg of Compound 1.

[0012] In some embodiments of the present methods, the administration provides an area under the curve (AUC) of Compound 1 of about 640 h*ng/mL to about 20400 h*ng/mL following administration of about 25 mg to about 300 mg of Compound 1. In some embodiments, the administration provides an area under the curve (AUC) of Compound 1 of about 640 h*ng/mL to about 6500 h*ng/mL following administration of about 25 mg to about 100 mg of the compound.

[0013] In some embodiments of the present methods, the patient has heart failure or is at risk of heart failure. In some embodiments, the patient has Class I heart failure as classified by the New York Heart Association (NYHA) Functional Classification. In some embodiments of the present methods, the patient has Class II heart failure as classified by the New York Heart Association (NYHA) Functional Classification. In some embodiments, the patient has Class III heart failure as classified by the NYHA Functional Classification.

[0014] In some embodiments of the present methods, the patient has an ejection fraction greater than about 45%. In some embodiments of the present methods, the patient has an ejection fraction less than about 45%. [0015] In some embodiments of the present methods, the patient has an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of greater than about 400 pg/mL. In some embodiments, the patient has atrial fibrillation and an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of greater than about 900 pg/mL.

[0016] In some embodiments of the present methods, the patient has or is at risk for hypertension. In some embodiments, the patient has or is at risk for diabetes (e.g., diabetes mellitus). In some embodiments, the patient has or is at risk for diabetic cardiomyopathy. In some embodiments, the patient has or is at risk for metabolic syndrome. In some embodiments, the patient has or is at risk for hyperglyceridemia or dyslipidemia. In some embodiments, the patient is obese or is at risk for obesity. In some embodiments, the patient has or is at risk for coronary artery disease (CAD). In some embodiments, the patient has or is at risk for heart disease. In some embodiments, the patient has or is at risk for valvular heart disease. In some embodiments, the patient has or is at risk for atrial fibrillation. In some embodiments, the patient has or it at risk for metabolic disease. In some embodiments, the patient has a diastolic dysfunction of Grade II, III, or IV.

[0017] In some embodiments, the subject does not have a metabolic disease. In some embodiments, the subject does not have metabolic syndrome. In some embodiments, the subject does not have diabetes (e.g., does not have diabetes mellitus). In some embodiments, the subject does not have hypertension. In some embodiments, the subject is not obese.

[0018] In some embodiments, the patient is at least 50 years old. In some embodiments, the patient is at least 65 years old. In some embodiments, the patient is at least 70 years old.

[0019] In some embodiments of the present methods, treatment provides the patient improving from Class III to Class II in the NYHA Functional Classification. In some embodiments, treatment provides the patient improving from Class III to Class I in the NYHA Functional Classification. In some embodiments, treatment provides an improvement in the patient’s NYHA Functional Classification from Class II to Class I.

[0020] In some embodiments of the present methods, treatment provides a decrease of NT-proBNP levels in the blood of the patient of about 5% to about 50%.

[0021] In some embodiments of the present methods, treatment provides about a 5 mL/m² to about a 20 mL/m² decrease in left atrial (LA) volume index. [0022] In some embodiments of the present methods, treatment provides an improvement of least 5 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score. In some embodiments, treatment provides an improvement of least 10 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score. In some embodiments, treatment provides an improvement of least 15 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score. In some embodiments, treatment provides an improvement of least 20 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score.

[0023] In some embodiments of the present methods, treatment provides improved performance in a standardized 6-minute walk test. In some embodiments, treatments provides at least a 10 meter increase in a standardized 6-minute walk test. In some embodiments, treatment provides an improvement in exercise capacity as measured by cardiopulmonary exercise testing. In some embodiments, treatment provides an improvement in pulmonary venous oxygen tension (pVO2) of 1-10 mL/kg/min.

[0024] In some embodiments of the present methods, treatment results in a reduction in the relative risk of heart failure hospitalization of about 5% to about 40%. In some embodiments of the present methods, treatment results in a reduction in the relative risk of heart failure hospitalization of about 15% to about 40%. In some embodiments, treatment results in a reduction in the relative risk of cardiovascular disease (CVD) death of about 5% to about 40%. In some embodiments, treatment results in a reduction in the relative risk of cardiovascular disease (CVD) death of about 15% to about 40%. In some embodiments, treatment results in a reduction in the relative risk of cardiovascular disease (CVD) death of about 5% to about 25%. In some embodiments, treatment results in an increase in overall survival.

[0025] In some embodiments, treatment provides an increase in patient’s heart pump function as measured by echocardiogram (ECG). In some embodiments, treatment provides the patient exhibiting no atrial or ventricular arrythmias when comparing baseline ECG monitoring with 72-hour monitoring.

[0026] In some embodiments of the present methods, at least a 100% (2 -fold) increase in the patient’s mean acetylated tubulin level over time compared to baseline level is observed about 2-4 hours after a daily dose of Compound 1 is administered. In some embodiments, about a 100% (2-fold) increase in the patient’s mean acetylated tubulin level over time compared to baseline level is observed about 2-4 hours after a daily dose of Compound 1 is administered. [0027] In some embodiments of the present methods, about a 50% to about a 75% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments, at least about a 50% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments, at least about a 75% increase in the patient’ s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound.

[0028] In some embodiments, treatment provides about a 10 mg/dL to about a 50 mg/dL reduction in fasting plasma glucose.

[0029] In some embodiments, treatment provides about a 0.3% to about a 1.5% reduction in Ale level.

[0030] In some embodiments, treatment provides about 1 kg to about a 5 kg drop in body weight.

[0031] In some embodiments of the present methods, Compound 1 is administered as an adjunctive to a sodium-glucose Cotransporter-2 (SGLT2) inhibitor. In some embodiments, the SGLT2 inhibitor is canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, or bexagliflozin.

[0032] In some embodiments, the present disclosure provides methods of treating metabolic disease (e.g., diabetes, such as diabetes mellitus or metabolic syndrome) or treating obesity in a patient in need thereof, comprising orally administering to the patient a once daily dose of about 10 mg to about 300 mg of Compound 1 having the formula:

pharmaceutically acceptable salt thereof.

[0033] In some embodiments, the present disclosure provides methods of treating heart failure with preserved ejection fraction (HFpEF) in a patient in need thereof, comprising orally administering to the patient a once daily dose of about 10 mg to about 300 mg of Compound 1 having the formula:

pharmaceutically acceptable salt thereof.

[0034] In some embodiments, the present disclosure provides methods of treating dilated cardiomyopathy in a patient in need thereof, comprising orally administering to the patient a once daily dose of about 10 mg to about 300 mg of Compound 1 having the formula:

pharmaceutically acceptable salt thereof.

[0035] In some embodiments, the dilated cardiomyopathy is familial dilated cardiomyopathy.

[0036] In some embodiments, the dilated cardiomyopathy is dilated cardiomyopathy due to one or more BLC2-Associated Athanogene 3 (BAG3) mutations.

[0037] In some embodiments, the subject having dilated cardiomyopathy has a deleterious mutation in the BAG3 gene. In some embodiments, the subject has BAG3^E455K mutation. In some embodiments, the subject having dilated cardiomyopathy has a deleterious mutation in the CSPR3 gene encoding MLP.

[0038] In some embodiments, the dilated cardiomyopathy is dilated cardiomyopathy due to one or more muscle LIM protein (MLP) mutations.

[0039] In some embodiments, the method of treating dilated cardiomyopathy restores the ejection fraction of the subject to at least about the ejection fraction of a subject without dilated cardiomyopathy. In some embodiments, the method of treating dilated cardiomyopathy restores the ejection fraction of the subject to at least about 20%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

[0040] In some embodiments, the method of treating dilated cardiomyopathy increases the ejection fraction of the subject compared to the subject’s ejection fraction before treatment. In some embodiments, the method of treating cardiomyopathy increase the ejection fraction of the subject to by at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 40%.

[0041] In some aspects, the disclosure provides a method of treating or preventing diastolic dysfunction (e.g., associated with HFpEF) in a subject in need thereof, comprising administering Compound 1 to the subject.

[0042] In some embodiments, the method treats or prevents at least one symptom of HFpEF. In some embodiments, the method reduces left ventricular (LV) mass. In some embodiments, the method reduces LV wall thickness. In some embodiments, the method improves LV relaxation. In some embodiments, the method improves LV filling pressure. In some embodiments, the method prevents heart failure in the subject.

[0043] In some embodiments, the methods described herein reduce cardiac fibrosis (e.g., associated with HFpEF). In some embodiments, administration of Compound 1 is effective to reduce cardiac fibroblast activation in a cell (e.g., in cell culture or in vivo). In some embodiments, administration of Compound 1 is effective to reduce expression of genes associated with fibrosis (e.g., in cells of a subject after administration of Compound 1 to the subject).

[0044] In some embodiments, the methods of the present disclosure prevent heart failure in the subject. In some embodiments, the methods reduce left ventricular internal diameter at diastole (LVIDd) in the subject. In some embodiments, the methods reduce left ventricular internal diameter at systole (LVIDs) in the subject.

[0045] In some embodiments, administration of Compound 1 is effective to reduce TGF-beta receptor signaling (e.g., in cells of a subject after administration of Compound 1 to the subject). In some embodiments, the methods described herein reduce cardiac muscle hypertrophy (e.g., associated with HFpEF).

[0046] In some embodiments, the methods described herein reduce mitochondrial dysfunction. In some embodiments, administration of Compound 1 is effective to increase expression of genes associated with oxidative phosphorylation and/or mitochondrial complex I (e.g., in cells of a subject after administration of Compound 1 to the subject). In some embodiments, administration of Compound 1 is effective to increase mitochondrial membrane potential in a cell (e.g., in vitro or in vivo). In some embodiments, administration of Compound 1 is effective to increase spare respiratory capacity in a cell (e.g., in cell culture or in vivo). [0047] In some aspects, the present disclosure provides methods of treating a disease or condition responsive to HDAC6 inhibition in a patient in need thereof, comprising orally administering to a patient a composition comprising 10 mg to 300 mg of Compound 1 or pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the composition is administered once daily.

BRIEF DESCRIPTION OF DRAWINGS

[0048] FIGs. 1A-1L show that HDAC6 inhibition with Compound 1 improves glucose tolerance and insulin resistance in diet induced obese mouse model.

[0049] FIG. 1A to FIG. IF show results of intraperitoneal glucose-tolerance tests (GTT) performed by injection of glucose (2 g/kg in saline) after 6 hours fasting. Tail blood glucose levels (mg/dl) were measured with a glucometer before (0 min) and at 15, 30, 45, 60 and 120 min after glucose administration. Diet Induced Obese (DIO) mice at 16 weeks of age developed severe glucose intolerance compared to controls. FIG. 1A shows GTT results in control and DIO mice. Based on glucose AUC level, DIO mice were evenly randomized into four treatment groups to receive Vehicle (n=9) or Compound 1 at three dosages 3, 10, 30mg/kg (n=10 each). Control mice were also divided to dose orally with vehicle (n=10) or 30 mg/kg Compound 1 (n=10). To assess the acute response of Compound 1 on glucose metabolism, GTT was performed at 6h after 1^st dose. FIG. IB shows GTT results before dose of Compound 1. FIG. 1C and FIG. ID show GTT results after a single dose of Compound 1. A single dose of Compound 1 at all three dosages significantly reduced glucose level. FIG. IE and FIG. IF show GTT results after Compound 1 treatment for 2 weeks. Compound 1 treatment for 2 weeks led to a pronounced improvements in glucose tolerance in a dose dependent manner.

[0050] FIG. 1G and FIG. 1H show results of intraperitoneal insulin-tolerance tests (ITT) performed by injection of insulin (lU/kg) after 6 hours fasting. Tail blood glucose levels (mg/dl) were measured with a glucometer before (0 min) and at 15, 30, 45, 60 and 120 min after insulin administration. HDAC6 inhibitor (Compound 1) treatment for 4 weeks improved insulin resistance in DIO mice. 10 and 30mg/kg were significantly reduced glucose AUC (ITT) with comparable activities, 3 mg/kg showed a trend of reduction.

[0051] FIG. II shows effects of Compound 1 on blood glucose in non-fasting mice. Tail blood samples were collected in the morning and measured with a glucometer. Compound 1 treatment for 6wks led to a dose-dependent reduction of non-fasting glucose, consistent with the data of glucose tolerant test after fasting.

[0052] FIG. 1J and FIG. IK show that treatment with Compound 1 caused a dose dependent reduction of body wight in DIO mice. FIG. IL shows that no differences in food consumption were observed between groups. Notably, control mice dosed with Compound 1 30mg/kg for 6wks did not show changes on blood glucose levels and body weights. Bars and error bars show means and SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0053] FIG. 2A, FIG. 2B and FIG. 2C show HD AC 6 inhibition with Compound 1 inhibits inflammatory genes in adipose tissue in diet induce obese mouse model. White adipose tissue (epididymal) was dissected from DIO mice after 6 hours of single dose 30mg/kg Compound 1. Realtime q-PCR data showed that Compound 1 remarkably inhibited upregulation of pro- inflammatory genes- IL-6 (FIG. 2A), IL-10 (FIG. 2B) and TGFbl (FIG. 2C) in white adipose tissue.

[0054] FIGs. 3A-3H show mice fed on HFD in combination with moderate TAC develop a cardio-metabolic heart failure phenotype that recapitulates systemic and cardiovascular features of HFpEF in human.

[0055] FIG. 3A show a schematic overview of HFpEF model induction by concomitant metabolic and moderate pressure overload stress in wildtype mice for 12 wks. n=5 mice in control group fed on regular diet without mTAC surgery, n=15 mice in HFpEF model development group induction with HFD/mTAC.

[0056] FIG. 3B and FIG. 3C show, respectively, HFD/mTAC induced continuous body weight increase and glucose intolerance compared to the control mice.

[0057] FIG. 3D shows left ventricular ejection fraction (LVEF) was preserved in mice with HFD/mTAC evaluated by echocardiography.

[0058] Significant concentric left ventricular (LV) hypertrophy was present in HFD/mTAC animals, as indicated by increases in LV mass (FIG. 3E) and LV diastolic wall thickness (FIG. 3F)

[0059] LV diastolic dysfunction with increased left ventricular filling pressure was developed in HFD/mTAC mice as evidenced by decreased e’ velocity (FIG. 3H) and increased ratio of E/e’ (FIG. 3G), measured by noninvasive Doppler imaging. Bars and error bars show means and SEM. *P<0.05, **P<0.01, ***p<0.001 Vs. control group.

[0060] FIGs. 4A-4O show oral dosing of Compound 1 improved glucose tolerance and diastolic dysfunction in HFD/mTAC mice. After the HFpEF phenotypes were established, animals were randomized to dose orally with 30 mg/kg Compound 1 (n=8) or vehicle (n=7) once per day for six weeks, respectively. n=3 mice in control group dosed with vehicle. Treatment with Compound 1 led to markedly improved glucose tolerance (FIG. 4A), with no difference of body weight change (FIG. 4B) compared to vehicle dosed animals. Echocardiographic evaluation (representative echocardiography-derived M-mode tracings, see FIG. 4D) revealed that Compound 1 treatment unaltered ejection fraction (FIG. 4C), however significantly reduced left ventricular mass (FIG. 4E) and LV wall thickness (FIG. 4F).

[0061] In addition, noninvasive Doppler imaging (representative pulsed-wave Doppler (FIG. 4G) and tissue Doppler (FIG. 4J) tracings) and invasive catheterization analysis showed that Compound 1 treatment sustained improved LV relaxation and LV filling pressures as shown by decreased prolongation of isovolumetric relaxation time (FIG. 4H), lower E/A (FIG. 41) and E/e’ ratios (FIG. 4K), improved e’ velocity (FIG. 4L), and reduced end diastolic pressure (FIG. 4M). Each of these efficacy parameters were normalized to control levels.

[0062] HFD/mTAC mice treated with Compound 1 showed a trending decrease in heart weight (FIG. 4N) and lung weight (FIG. 40), indicating improved LV hypertrophy and pulmonary congestion respectively, consistent with reduced filling pressure. Bars and error bars show means and SEM. *P<0.05, **P<0.01, ****p<0.0001.

[0063] Compound 1 inhibits upregulation genes commonly associated with HFpEF disease (FIGs. 5A-5H) Realtime q-PCR data showed that Compound 1 significantly inhibited upregulation of genes associated with fibrosis Postn (FIG. 5A), Collal (FIG. 5B), Col3al (FIG. 5C) and Col5a2 (FIG. 5D); cardiac stress, Nppb (FIG. 5E) and Myh6 (FIG. 5F); and inflammation Tnfa (FIG. 5G) and Caspl (FIG. 5H) in heart tissue of HFD/mTAC mice, consistent with the improvements of LV structure and heart function. Bars and error bars show means and SEM.

[0064] FIGs. 6A-6L show that wild-type (WT) mice on high-fat diet coupled with inhibition of constitutive nitric oxide synthases with N[w]-nitro-l-arginine methyl ester (HFD/L-NAME) for eight weeks develop obesity, hypertension and diastolic dysfunction, recapitulating HFpEF phenotypes in humans.

[0065] FIG. 6A shows a schematic overview of HFpEF model induction by concomitant metabolic and hypertensive stress in wildtype mice elicited by a combination of high fat diet and inhibition of constitutive nitric oxide synthase using N“-nitrol-arginine methyl ester (L- NAME). n=7 mice in control group fed on regular diet, n=26 mice in HFpEF model development group induction with HFD/L-NAME.

[0066] HFD/L-NAME treatment for 8 weeks significantly induced body weight increase (FIG. 6B), hypertension (FIG. 6C) and glucose intolerance (FIG. 6D) compared to the control mice. Echocardiographic evaluation revealed persistent preservation of the left ventricular ejection fraction (LVEF) (FIG. 6E). Significant concentric left ventricular (LV) hypertrophy was present in HFD/L-NAME mice, as indicated by increases in LV mass (FIG. 6F) and LV wall thickness at diastole (FIG. 6G), without LV chamber dilatation (FIG. 6H). In addition, mice concomitantly exposed to HFD/L-NAME exhibited signs of LV diastolic dysfunction with impaired relaxation and increased left ventricular filling pressure, as evidenced by prolonged IVRT (FIG. 61), decreased e’ velocity (FIG. 6J), and increased ratios of E/e’(FIG. 6K), E/A (FIG. 6L), measured by noninvasive Doppler imaging. Bars and error bars show means and SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 Vs. control group.

[0067] FIGs. 7A-7S shows Compound 1 treatment for 9 weeks improves glucose tolerance and diastolic dysfunction in HFD/L-NAME model.

[0068] FIG. 7A and FIG. 7B show results of Glucose tolerance test (GTT) performed after 5 weeks of dosing. Treatment with Compound 1 markedly improved glucose tolerance in HFD/L-NAME mice, but no changes in control animals.

[0069] FIG. 7C shows plasma insulin level during GTT at the indicated time points (0 and 30 minutes after glucose injection) as measured by a sensitive mouse insulin detection kit (Cat. 80-INSMS-E01, ALPCO). Compound 1 treatment led to decreased insulin secretion, suggesting that the improved glucose tolerance might be due to improved insulin action/sensitivity.

[0070] FIG. 7D and FIG. 7E show, respectively, that treatment with Compound 1 caused a significant reduction of body wight, but no difference of food consumption in mice fed with HFD/L-NAME. [0071] FIG. 7F shows that Compound 1 did not affect systolic blood pressure in HFD/L- NAME mice measured by non-invasive tail cuff method.

[0072] Echocardiography shows that Compound 1 treatment preserved ejection fraction (FIG. 7G), but significantly reduced left ventricular mass (FIG. 7H) and LV wall thickness (FIG. 71).

[0073] Noninvasive Doppler imaging and terminal invasive catheterization analysis revealed that treatment with Compound 1 for 9wks decreased prolongation of isovolumetric relaxation time (FIG. 7J), E/A (FIG. 7K) and E/e’ ratios (FIG. 7L), increased e’ velocity (FIG. 7M), and reduced end diastolic pressure (FIG. 7N), indicating the improved LV relaxation and filling pressure. In addition, HFD/L-NAME mice treated with Compound 1 showed a trending decrease in lung weight (FIG. 70), suggesting an improved pulmonary congestion, consistent with the reduction of filling pressure.

[0074] Notably, no adverse effects related to Compound 1 have been observed. Control animals dosed with Compound 1 has no changes on each of LV structural and functional parameters, as well as ECG signals- QT, QRS, R amplitude and PR intervals (FIG. 7P, FIG. 7Q, FIG. 7R and FIG. 7S, respectively), further supports an overall favorable safety profile of the compound. Bars and error bars show means and SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0075] FIG. 8 shows a heatmap of selected genes from significantly altered functional gene sets. Only genes with significant expression differences in each (GO) and WikiPathways (WP) database gene set are shown. Heatmap shows correction of key genes associated with cardiac muscle, fibrosis and mitochondrial function.

[0076] FIG. 9 shows significantly altered gene sets from Gene Ontology (GO) and WikiPathways (WP) databases. Selected categories were based on the normalized enrichment scores (NES) and FDR < 0.25. For each condition, bars on the left indicate data for vehicle- treated HFpEF mice relative to healthy controls, and bars on the right indicate data for HFpEF mice treated with Compound 1 relative to vehicle. TGFP: transforming growth factor beta, PDGFR: platelet-derived growth factor receptors, OXPHOS: oxidative phosphorylation.

[0077] FIGs. 10A-10J show correlation of mitochondrial genes and diastolic function. In FIGs. 10A-10F, the expression level of genes associated with different subunits of mitochondrial respiratory electron transport chain (NADH:Ubiquinone Oxidoreductase subunits) on the x-axis are plotted against markers of diastolic function (LVPWd and MV E/E’) on the y-axis. In particular, Ndufal3 (FIG. 10A), Ndufa5 (FIG. 10B), Ndufs7 (FIG. IOC), Ndufal (FIG. 10D), and Ndufa8 (FIG. 10E) on the x-axis are plotted against the marker of diastolic function, LVPWd, on the y-axis. Ndufal 3 (FIG. 10F), Ndufa5 (FIG. 10G), Ndufs7 (FIG. 10H), Ndufal (FIG. 101), and Ndufa8 (FIG. 10J) on the x-axis are plotted against the marker of diastolic function, MV E/E’ , on the y-axis. For better visualization, the expression levels of genes were plotted on logarithmic scale (log2 TPM). Values are Pearson correlation coefficients. Black circles indicate data for vehicle treated healthy animals. Dark gray and light gray circles show HFpEF mice treated with vehicle or Compound 1 respectively. FIG. 10K shows increased mitochondrial membrane potential, and FIG. 10L shows enhanced reserve respiratory capacity, in human iPSC-derived CMs treated with Compound 1 (3 pM) (relative to DMSO control). Oxygen consumption rate values were normalized to nuclear count. Data represent mean values ± SEM.

[0078] FIGs. 11A-11E show expression levels of genes associated with fibrosis Colla2 (FIG. HA), Col3al (FIG. 11B), Fbnl (FIG. 11C), Postn (FIG. 11D), and Cilp (FIG. HE) on the x-axis plotted against diastolic function parameter, LVPWd, on the y-axis. FIGs. 11F-11G show expression levels of genes associated with fibrosis Colla2 (FIG. HF), Col3al (FIG. HG), Fbnl (FIG. HH), Postn (FIG. HI), and Cilp (FIG. HJ) on the x-axis plotted against diastolic function parameter, IVRT, on the y-axis.

[0079] FIGs. 12A-12B show that HDAC6 inhibitors prevent fibroblast activation from TGF- P in human cardiac fibroblasts. FIG. 12A shows immunostaining of alpha-SMA (smooth muscle actin) in human cardiac fibroblasts in control (first panel from the top), treated with vehicle in the presence of TGF-beta (second panel from the top) or 1 uM Compound 1 in the presence of TGF-beta (third panel from the top). 10 ng/ml TGF-beta was used. Scale bars are 50 pM. FIG. 12B shows that Compound 1 (1 uM) effectively reduced TGF-beta induced human cardiac fibroblast activation as measured by alpha-SMA+ cells. Alpha-SMA staining count was determined by blinded analyses. Each point represents 9 images per well. Data represent mean values ± SEM.

[0080] FIG. 13 provides an overview of the Phase 1 clinical trial evaluating the safety of single ascending and multiple ascending doses of Compound 1 in healthy participants.

[0081] FIG. 14 provides a graph of the mean plasma concentration of Compound 1 over time in a single ascending dose (SAD) study after administration of 1 mg (Cohort 1), 5 mg (Cohort 2), 25 mg (Cohort 3), 100 mg (Cohort 4), 300 mg (Cohort 5), and 700 mg (Cohort 6) of Compound 1 to healthy participants.

[0082] FIG. 15 provides graphs of the mean plasma concentration of Compound 1 over time in a multiple ascending dose (MAD) study at day 1 and day 14 after daily administration of 25 mg (left panel), 100 mg (middle panel), and 300 mg (right panel) of Compound 1 to healthy participants.

[0083] FIG. 16A provides a graph of mean acetylated tubulin levels over time at day 1 of the SAD study after administration of 1 mg (Cohort 1), 5 mg (Cohort 2), 25 mg (Cohort 3), 100 mg (Cohort 4), 300 mg (Cohort 5), and 700 mg (Cohort 6) of Compound 1 to healthy participants.

[0084] FIG. 16B provides a graph of mean acetylated tubulin levels over time at day 14 in the MAD study after administration of 1 mg (Cohort 1), 5 mg (Cohort 2), 25 mg (Cohort 3), 100 mg (Cohort 4), 300 mg (Cohort 5), and 700 mg (Cohort 6) of Compound 1 to healthy participants.

[0085] FIG. 17 provides a graph showing the correlation between the Compound 1 AUC and acetylated tubulin AUC at various doses in the SAD and MAD studies.

[0086] FIG. 18 provides a graph showing mean acetylated histone relative to predose levels in the MAD study.

[0087] FIG. 19 shows a potential study design for a Phase 2 clinical trial for demonstrating proof-of-concept, identifying an effective dose, and demonstrating efficacy of Compound 1.

[0088] FIGs. 20A-20J show inhibiting HDAC6 with Compound 1 protects heart function in MLP^KO mice.

DETAILED DESCRIPTION

Overview

[0089] The present disclosure relates generally to the demonstration that Compound 1 administered to a patient in need at a dose of from about 10 mg to 300 mg is useful for treating a variety of diseases and conditions responsive to HDAC6 inhibition, including the cardiac, metabolic, fibrotic, and inflammatory diseases and disorders disclosed herein. Definitions

[0090] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0091] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of +/- 15 %, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0092] Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0093] The term “a” or “an” refers to one or more of that entity, i.e. can refer to plural referents. As such, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0094] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0095] As used herein, the term “HDAC6” refers to the enzyme that in humans is encoded by the HDAC6 gene. HDAC6 belongs to the class lib enzyme and contains two catalytic domains, a ubiquitin binding domain and a cytoplasmic retention domain (Haberland et al., 2009). HDAC6 is predominately a cytoplasmic enzyme and its best-characterized substrates include tubulin, HSP90 and cortactin (Brindisi et al., 2020). Pharmacological inhibition of HDAC6 blocks its deacetylase activity, thus resulting in hyperacetylation of its substrates, most notably tubulin (Hubbert et al., 2002).

[0096] As used herein, the term “HDAC6 inhibitor” refers to a compound that inhibits at least one enzymatic activity of HDAC6.

[0097] An HDAC6 inhibitor may be a “selective” HDAC6 inhibitor. The term “selective” as used herein refers to selectivity against other HDACs, known in the art as “isozymes.” In some embodiments, the selectivity ratio of HDAC6 over HDAC1 is from about 5 to about 30,0000, e.g., about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 15,000, about 20,000, about 25,000, or about 30,000, including all values and ranges therebetween.

[0098] For example, a HDAC6 inhibitor may be at least 100-fold selective against HDAC6 compared to all other isozymes of HD AC. In some cases, selectivity may be determined by reference of another HD AC inhibitor, such as a pan-HDAC inhibitor — that is an inhibitor that inhibits HDACs other than HDAC6 in addition to HDAC6. Givinostat is an example of a panHD AC6 inhibitor. In some embodiments, a selective HDAC6 inhibitor inhibits HDACs other than HDAC6 at least 100-fold less effectively than givinostat. HDAC6-selective inhibitors may have reduced cytotoxicity due to the cytoplasmic nature of HDAC6 substrates and reduced effects on nuclear targets (including H3K9 and c-MYC) and on global transcription (Nebbioso et al., 2017). [0099] As used herein, the term “treating” refers to acting upon a disease, disorder, or condition with an agent to reduce or ameliorate harmful or any other undesired effects of the disease, disorder, condition and/or their symptoms.

[0100] As used herein, the term “preventing” refers to reducing the incidence or risk of developing, or delaying the development of, harmful or any other undesired effects of the disease, disorder, condition and/or symptoms

[0101] “Administration,” “administering” and the like, refer to administration to a subject by a medical professional or by self-administration by the subject, as well as to indirect administration, which may be the act of prescribing a composition of the invention. Typically, an effective amount is administered, which amount can be determined by one of skill in the art. Any method of administration may be used. Administration to a subject can be achieved by, for example, oral administration, in liquid or solid form, e.g. in capsule or tablet form; intravascular injection; intramyocardial delivery; or other suitable forms of administration.

[0102] As used herein, the term “effective amount” and the like refers to an amount that is sufficient to induce a desired physiologic outcome (e.g., increased cardiac function, decreased mortality, or decreased risk/incidence of hospitalization, increased exercise capacity, or reduced expression of one or more biomarkers associated with heart failure — such as BNP). An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period which the individual dosage unit is to be used, the bioavailability of the composition, the route of administration, etc. It is understood, however, that specific amounts of the compositions for any particular subject depends upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the composition combination, severity of the particular disease being treated and form of administration.

[0103] As used herein, the terms “subject” or “patient” refers to a human subject or human patient. Specific examples of “subjects” and “patients” include, but are not limited to, individuals with a metabolic disease (e.g., obesity), and individuals with metabolic disease- related characteristics or symptoms. Specific examples of “subjects” and “patients” also include, but are not limited to, individuals with a cardiac disease or disorder, and individuals with cardiac disorder-related characteristics or symptoms. [0104] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0105] The term “pharmaceutically acceptable salts” include those obtained by reacting the active compound functioning as a base, with an inorganic or organic acid to form a salt, for example, salts of hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, camphorsulfonic acid, oxalic acid, maleic acid, succinic acid, citric acid, formic acid, hydrobromic acid, benzoic acid, tartaric acid, fumaric acid, salicylic acid, mandelic acid, carbonic acid, etc. Those skilled in the art will further recognize that acid addition salts may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods.

[0106] As used herein, the term “restores” refers to increasing the level of biochemical or physiological parameter to a level observed in the subject prior to development of disease or condition, or to the level observed in a subject not having the disease or condition.

[0107] As used herein, the term “reduces” refers to decreasing the level of biochemical or physiological parameter.

[0108] As used herein, the term “metabolic disease” refers to a condition causes by either excessive nutrient intake or by the body’s failure to properly metabolize nutrients. Metabolic disease includes but is not limited to obesity. It is a cluster of conditions that occur together, associated with heart diseases, type 2 diabetes and stroke. These conditions include hypertension, hyperglycemia, excess body fat and abnormal cholesterol or triglyceride levels.

[0109] The term “obesity” refers to a condition of having too much body fat. Obesity may increase the risk of diabetes, heart disease, stroke, and arthritis. Weight that is higher than what is considered healthy for a given height is described as overweight or obesity. Body Mass Index (BMI) is a screening tool for overweight and obesity. BMI is a person’s weight in kilograms divided by the square of height in meters. A calculator for BMI is available at www.cdc.gov at obesity/adult/defining.html. In various embodiments, the subject may have a BMI of 25, 30, 35, 40 or higher, such as a BMI of 25-30, 25-30, or 25-40. In some cases, the subject may have severe obesity (also known as class 3 obesity), defined as a BMI of 40 or greater. Obesity is a chronic disease, also a risk factor for other diseases, such as heart disease, hypertension, stroke, diabetes etc.

[0110] In some embodiments, the methods of the disclosure decrease, prevent, or ameliorate one or more symptoms of metabolic disease. Symptoms of metabolic disease include glucose intolerance, insulin resistance, high glucose level, and inflammation in adipose tissue.

[OHl] As used herein, the term “heart failure” refers to a condition in which the heart cannot pump enough blood to meet the body’s need.

[0112] “Heart failure (HF) is a complex clinical syndrome that can result from any structural or functional cardiovascular disorder causing systemic perfusion inadequate to meet the body’s metabolic demands without excessively increasing left ventricular filling pressures. It is characterized by specific symptoms, such as dyspnea and fatigue, and signs, such as fluid retention. As used herein, “chronic heart failure”, “congestive heart failure”, and “CHF” refer, interchangeably, to an ongoing or persistent forms of heart failure. Common risk factors for CHF include old age, diabetes, high blood pressure and being overweight. CHF is broadly classified according to the systolic function of the left ventricle as HF with reduced or preserved ejection fraction (HFrEF and HFpEF). The term “heart failure” does not mean that the heart has stopped or is failing completely, but that it is weaker than is normal in a healthy person. In some cases, the condition can be mild, causing symptoms that may only be noticeable when exercising, in others, the condition may be more severe, causing symptoms that may be lifethreatening, even while at rest. The most common symptoms of chronic heart failure include shortness of breath, tiredness, swelling of the legs and ankles, chest pain and a cough. In some embodiments, the methods of the disclosure decrease, prevent, or ameliorate one or more symptoms of CHF (e.g., HFpEF) in a subject suffering from or at risk for CHF (e.g., HFpEF). In some embodiments, the disclosure provides methods of treating CHF and conditions that can lead to CHF.

[0113] As used herein “acute heart failure” and “decompensated heart failure” refer, interchangeably, to a syndrome of the worsening of signs and symptoms reflecting an inability of the heart to pump blood at a rate commensurate to the needs of the body at normal filling pressure. AHF typically develops gradually over the course of days to weeks and then decompensates requiring urgent or emergent therapy due to the severity of these signs or symptoms. AHF may be the result of a primary disturbance in the systolic or diastolic function of the heart or of abnormal venous or arterial vasoconstriction, but generally represents an interaction of multiple factors, including volume overload. The majority of patients with AHF have decompensation of chronic heart failure (CHF) and consequently much of the discussion of the pathophysiology, presentation, and diagnosis of CHF is directly relevant to an understanding of AHF. In other cases, AHF results from an insult to the heart or an event that impairs heart function, such as an acute myocardial infarction, severe hypertension, damage to a heart valve, abnormal heart rhythms, inflammation or infection of the heart, toxins and medications. In some embodiments, the methods of the disclosure decrease, prevent, or ameliorate one or more symptoms of AHF in a subject suffering from or at risk for AHF. In some embodiments, the disclosure provides methods of treating AHF and conditions that can lead to AHF. AHF may be the result of ischemia associated with myocardial infarction.

[0114] In some embodiments, the methods of the disclosure decrease, prevent, or ameliorate one or more symptoms of heart failure in a subject suffering from or at risk for heart failure associated with HFpEF. The terms “heart failure with preserved ejection fraction” or “diastolic heart failure” are used, interchangeably, refers generally to a form of heart failure is characterized by signs and symptoms of heart failure and a left ventricular ejection fraction (LVEF) greater than 50%. The terms may also encompass heart failure associated with intermediate reductions in LVEF (40% to 49%). In some embodiments, HFpEF comprises HFpEF associated with CHF. In some embodiments, HFpEF comprises HFpEF associated with AHF.

[0115] HFpEF is more common among older patients and women. Typical symptoms include fatigue, weakness, dyspnea, orthopnea, paroxysmal nocturnal dyspnea, edemal. Signs of HFpEF (e.g., HFpEF associated with CHF) may include S3 heart sound, displaced apical pulse, and jugular venous distension. Echocardiographic findings of normal ejection fraction with impaired diastolic function confirm the diagnosis. Measurement of natriuretic peptides (e.g., BNP or NT-proBNP) is useful in the evaluation of patients with suspected heart failure with preserved ejection fraction in the ambulatory setting.

[0116] As used herein, the term “cardiomyopathy” refers to any disease or dysfunction of the myocardium (heart muscle) in which the heart is abnormally enlarged, thickened and/or stiffened. As a result, the heart muscle’s ability to pump blood is usually weakened. The etiology of the disease or disorder may be, for example, inflammatory, metabolic, toxic, infiltrative, fibroplastic, hematological, genetic, or unknown in origin. There are two general types of cardiomyopathies: ischemic (resulting from a lack of oxygen) and non-ischemic. [0117] As used herein, the term “dilated cardiomyopathy” or “DCM” refers to condition in which the heart muscle becomes weakened and enlarged. As a result, the heart cannot pump enough blood to the rest of the body. The most common causes of dilated cardiomyopathy are heart disease caused by a narrowing or blockage in the coronary arteries; poorly controlled high blood pressure; alcohol or drug abuse; diabetes, thyroid disease, or hepatitis; drug side effects; abnormal heart rhythm; autoimmune illnesses; genetic causes; infection; heart valves that are either too narrow or too leaky; pregnancy; exposure to heavy metals such as lead, arsenic, cobalt, or mercury. DCM can affect anyone at any age. However, it is most common in adult men. DCM includes idiopathic DCM. In some embodiments, the DCM is familial DCM.

[0118] As used herein, the term “deleterious mutation” refers to a mutation that decreases the function of a gene. Deleterious mutations may include missense mutations, deletions or insertions in coding regions, non-coding mutations that influence gene expression or gene splicing, or others. Deleterious mutations include partial or total deletion of a gene. As used herein, the term may refer to homozygous or heterozygous mutations in a gene, provided the mutation manifests a phenotypic effect upon the carrier.

[0119] As used herein, the term “left ventricular internal diameter at diastole” or “LVIDd” refers to left ventricular size at diastole.

[0120] As used herein, the term “left ventricular internal diameter at systole” or “LVIDs” refers to left ventricular size at systole.

[0121] As used herein, the term “left ventricular mass” refers to the weight of the left ventricle.

[0122] As used herein, the term “ejection fraction” refers to the amount of blood being bumped out of the left ventricle each time it contracts, expressed as a percentage to the total amount of blood in left ventricle.

[0123] The detailed description of the disclosure is divided into various sections only for the reader’ s convenience and disclosure found in any section may be combined with that in another section. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. Compound 1

[0124] Compound l is a highly specific small molecule inhibitor of HDAC6 in the fluoroalkyloxadiazole family having the structural formula:

The synthesis and evaluation of Compound 1 as an HDAC6 selective inhibitor is provided in WO2021/127643, which is incorporated herein by reference in its entirety. Preclinical studies, including animal efficacy models (see WO2022/235842 and WO2022/226388, which are each incorporated herein in their entirety) have demonstrated that Compound 1 is effective in treating a variety of diseases and disorders associated with HDAC6 activity or responsive to HDAC6 inhibition.

Methods of Treatment

[0125] In some aspects, the present disclosure provides methods of treating a disease or condition responsive to HDAC6 inhibition in a patient in need thereof, comprising orally administering to the patient about 10 mg to about 300 mg of Compound 1 having the formula:

or a pharmaceutically acceptable salt thereof.

Diseases and Disorders

[0126] In some embodiments of the present methods, the disease or condition responsive to HDAC6 inhibition is a metabolic, cardiac, fibrotic, or inflammatory disease or disorder.

[0127] In some embodiments of the present methods, the disease or condition responsive to HDAC6 inhibition is a metabolic disease or disorder. Accordingly, in some embodiments, the present disclosure provides methods of treating metabolic disease in a patient in need thereof, comprising orally administering to the patient a once daily dose of about 10 mg to about 300 mg of Compound 1 having the formula:

pharmaceutically acceptable salt thereof.

[0128] In some embodiments, the metabolic disease or disorder is a metabolic disease or disorder associated with obesity, such as diet-induced obesity. In some embodiments, the metabolic disease or disorder is not diet-induced. In some embodiments, the metabolic disease or disorder is diabetes, pre-diabetes, diabetic cardiomyopathy, metabolic syndrome, hypertension, hypertriglyceridemia, or dyslipidemia. In some embodiments, the metabolic disease or disorder is diabetes. In some embodiments, the metabolic disease or disorder is diabetes mellitus. In some embodiments, the metabolic disease or disorder is pre-diabetes. In some embodiments, the metabolic disease or disorder is diabetic cardiomyopathy. In some embodiments, the metabolic disease or disorder is metabolic syndrome. In some embodiments, the metabolic disease or disorder is hypertriglyceridemia. In some embodiments, the metabolic disease or disorder is hypertension. In some embodiments, the metabolic disease or disorder is dyslipidemia.

[0129] In some embodiments of the present methods, the disease or condition responsive to HDAC6 inhibition is heart failure with preserved ejection fraction (HFpEF). Accordingly, in some embodiments, the present disclosure provides methods of treating heart failure with preserved ejection fraction (HFpEF) in a patient in need thereof, comprising orally administering to the patient a once daily dose of about 10 mg to about 300 mg of Compound 1 having the formula:

pharmaceutically acceptable salt thereof.

[0130] In some embodiments, the methods of treating HFpEF result in the treatment or prevention of at least one symptom of HFpEF. In some embodiments, the methods of treating HFpEF the reduce left ventricular (LV) mass. In some embodiments, the methods of treating HFpEF reduce LV wall thickness. In some embodiments, the methods of treating HFpEF improve LV relaxation. In some embodiments, the methods of treating HFpEF improve LV filling pressure. In some embodiments, the methods of treating HFpEF prevent heart failure in the subject.

[0131] In some embodiments of the present methods, the disease or condition responsive to HDAC6 inhibition is dilated cardiomyopathy. In some embodiments, the present disclosure therefore provides methods of treating dilated cardiomyopathy in a patient in need thereof, comprising orally administering to the patient a once daily dose of about 10 mg to about 300 mg of Compound 1 having the formula:

pharmaceutically acceptable salt thereof.

[0132] In some embodiments, the dilated cardiomyopathy is familial dilated cardiomyopathy. In some embodiments, the dilated cardiomyopathy is dilated cardiomyopathy due to one or more BLC2-Associated Athanogene 3 (BAG3) mutations. In some embodiments, the dilated cardiomyopathy is dilated cardiomyopathy due to one or more muscle LIM protein (MLP) mutations.

[0133] In some embodiments, the subject having dilated cardiomyopathy has a deleterious mutation in the BAG3 gene. In some embodiments, the subject has BAG3^E455K mutation. In some embodiments, the subject having dilated cardiomyopathy has a deleterious mutation in the CSPR3 gene encoding MLP.

[0134] In some embodiments, the method of treating dilated cardiomyopathy restores the ejection fraction of the subject to at least about the ejection fraction of a subject without dilated cardiomyopathy. In some embodiments, the method of treating dilated cardiomyopathy restores the ejection fraction of the subject to at least about 20%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

[0135] In some embodiments, the method of treating dilated cardiomyopathy increases the ejection fraction of the subject compared to the subject’s ejection fraction before treatment. In some embodiments, the method of treating cardiomyopathy increase the ejection fraction of the subject to by at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 40%.

[0136] In some embodiments of the present methods, the disease or condition responsive to HDAC6 inhibition is a fibrotic disease or disorder. In some embodiments, the present disclosure therefore provides methods of treating a fibrotic disease in a patient in need thereof, comprising orally administering to the patient a once daily dose of about 10 mg to about 300 mg of Compound 1 having the formula:

pharmaceutically acceptable salt thereof.

[0137] In some embodiments, the fibrotic disease is idiopathic pulmonary fibrosis, scleroderma, non-alcoholic steatohepatitis (NASH), systemic sclerosis (SSC), myelofibrosis, kidney fibrosis, liver fibrosis, and heart fibrosis.

[0138] In some embodiments of the present methods, the disease or condition responsive to HDAC6 inhibition is an inflammatory disease or disorder. In some embodiments, the present disclosure therefore provides methods of treating a fibrotic disease in a patient in need thereof, comprising orally administering to the patient a once daily dose of about 10 mg to about 300 mg of Compound 1 having the formula:

pharmaceutically acceptable salt thereof.

[0139] In some embodiments, the inflammatory disease is inflammatory bowel disease (e.g., Crohn’s disease and ulcerative colitis), lupus, chronic obstructive pulmonary disease (COPD), asthma, psoriatic arthritis, rheumatoid arthritis (RA), or psoriasis.

[0140] In some aspects, the disclosure provides a method of treating or preventing diastolic dysfunction (e.g., associated with HFpEF) in a subject in need thereof, comprising administering a HDAC6 inhibitor to the subject. [0141] In some embodiments, the methods disclosed herein reduce cardiac fibrosis (e.g., associated with HFpEF). In some embodiments, administration of Compound 1 is effective to reduce cardiac fibroblast activation in a cell (e.g., in cell culture or in vivo). In some embodiments, administration of Compound 1 is effective to reduce expression of genes associated with fibrosis (e.g., in cells of a subject after administration of Compound 1 to the subject).

[0142] In some embodiments, the methods of the present disclosure prevent heart failure in the subject. In some embodiments, the methods reduce left ventricular internal diameter at diastole (LVIDd) in the subject. In some embodiments, the methods reduce left ventricular internal diameter at systole (LVIDs) in the subject.

[0143] In some embodiments, administration of Compound 1 is effective to reduce TGF-beta receptor signaling (e.g., in cells of a subject after administration of Compound 1 to the subject). In some embodiments, the methods described herein reduce cardiac muscle hypertrophy (e.g., associated with HFpEF).

[0144] In some embodiments, the methods described herein reduce mitochondrial dysfunction. In some embodiments, administration of Compound 1 is effective to increase expression of genes associated with oxidative phosphorylation and/or mitochondrial complex I (e.g., in cells of a subject after administration of Compound 1 to the subject). In some embodiments, administration of Compound 1 is effective to increase mitochondrial membrane potential in a cell (e.g., in vitro or in vivo). In some embodiments, administration of Compound 1 is effective to increase spare respiratory capacity in a cell (e.g., in cell culture or in vivo).

[0145] In other aspects, the present disclosure provides methods of treating a disease or condition responsive to HDAC6 inhibition in a patient in need thereof, comprising orally administering to a patient a composition comprising 10 mg to 300 mg of Compound 1 or pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the composition is administered once daily. In some embodiments, the composition is administered twice daily. The compositions disclosed herein are useful from treating the present methods.

Dosing and Pharmacokinetics

[0146] In some embodiments, Compound 1 is administered once-a-day (qd). In some embodiments, Compound 1 is administered twice-a-day (bid). In some embodiments, Compound 1 is administered three times a day (tid). In some embodiments, Compound 1 is administered every other day. In some embodiments, Compound 1 is administered once a week.

[0147] In some embodiments of the present methods, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 205 mg, about 210 mg, about 215 mg, about 220 mg, about 225 mg, about 230 mg, about 235 mg, about 240 mg, about 245 mg, about 250 mg, about 255 mg, about 260 mg, about 265 mg, about 270 mg, about 275 mg, about 280 mg, about 285 mg, about 290 mg, about 295 mg, or about 300 mg of Compound 1 is administered to the patient, including all ranges and values therebetween. In some embodiments, about 10 mg to about 200 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg to about 200 mg of Compound 1 is administered to the patient. In some embodiments, about 10 mg to about 150 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg to about 150 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg to about 100 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg, about 50 mg, or about 100 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg, about

75 mg, or about 200 mg of Compound 1 is administered to the patient. In some embodiments, about 10 mg, about 25 mg or about 75 mg of Compound 1 is administered to the patient. In some embodiments, about 10 mg of Compound 1 is administered to the patient. In some embodiments, about 15 mg of Compound 1 is administered to the patient. In some embodiments, about 20 mg of Compound 1 is administered to the patient. In some embodiments, about 25 mg of Compound 1 is administered to the patient. In some embodiments, about 30 mg of Compound 1 is administered to the patient. In some embodiments, about 35 mg of Compound 1 is administered to the patient. In some embodiments, about 40 mg of Compound 1 is administered to the patient. In some embodiments, about 45 mg of Compound 1 is administered to the patient. In some embodiments, about 50 mg of Compound 1 is administered to the patient. In some embodiments, about 55 mg of Compound 1 is administered to the patient, In some embodiments, about 60 mg of Compound 1 IS administered to the patient,

some embodiments, about 65 mg of Compound 1 is administered to the patient, In some embodiments, about 70 mg of Compound 1 IS administered to the patient, In some embodiments, about 75 mg of Compound 1 is administered to the patient, In some embodiments, about 80 mg of Compound 1 IS administered to the patient,

some embodiments, about 85 mg of Compound

IS administered to the patient, In some embodiments, about 90 mg of Compound 1 IS administered to the patient, In some embodiments, about 95 mg of Compound 1 is administered to the patient, In some embodiments, about 100 mg of Compound 1 is administered to the patient, In some embodiments, about 105 mg of Compound 1 is administered to the patient, In some embodiments, about 110 mg of Compound 1 IS administered to the patient,

some embodiments, about 115 mg

Compound 1 is administered to the patient, In some embodiments, about 120 mg of Compound 1 IS administered to the patient, In some embodiments, about 125 mg of Compound 1 is administered to the patient, In some embodiments, about 130 mg of Compound 1 IS administered to the patient, In some embodiments, about 135 mg of Compound 1 is administered to the patient, In some embodiments, about 140 mg of Compound 1 IS administered to the patient,

some embodiments, about 145 mg of Compound 1 is administered to the patient, In some embodiments, about 150 mg of Compound 1 IS administered to the patient, In some embodiments, about 155 mg of Compound 1 is administered to the patient, In some embodiments, about 160 mg of Compound 1 IS administered to the patient,

some embodiments, about 165 mg of Compound 1 is administered to the patient, In some embodiments, about 170 mg of Compound 1 IS administered to the patient, In some embodiments, about 175 mg of Compound 1 is administered to the patient, In some embodiments, about 200 mg of Compound 1 IS administered to the patient, In some embodiments, about 250 mg of Compound 1 is administered to the patient. In some embodiments, about 300 mg of Compound 1 is administered to the patient.

[0148] In some embodiments of the present methods, the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 80 ng/mL to about 1700 ng/mL following administration of about 25 mg to about 300 mg of Compound 1. [0149] In some embodiments, the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 80 ng/mL to about 720 ng/mL following administration of about 25 mg to about 100 mg of Compound 1.

[0150] In some embodiments, the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 80 ng/mL to about 160 ng/mL following administration of about 25 mg of Compound 1.

[0151] In some embodiments, the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 425 ng/mL to about 665 ng/mL following administration of about 100 mg of Compound 1.

[0152] In some embodiments, the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 1050 ng/mL to about 1700 ng/mL following administration of about 300 mg of Compound 1.

[0153] In some embodiments of the present methods, the administration provides an area under the curve (AUC) of Compound 1 of about 640 h*ng/mL to about 20400 h*ng/mL following administration of about 25 mg to about 300 mg of Compound 1.

[0154] In some embodiments, the administration provides an area under the curve (AUC) of Compound 1 of about 640 h*ng/mL to about 6500 h*ng/mL following administration of about 25 mg to about 100 mg of the compound.

[0155] In some embodiments, the administration provides an area under the curve (AUC) of Compound 1 of about 640 h*ng/mL to about 1070 h*ng/mL following administration of about 25 mg of Compound 1.

[0156] In some embodiments, the administration provides an area under the curve (AUC) of Compound 1 of about 4075 h*ng/mL to about 6500 h*ng/mL following administration of about 100 mg of Compound 1.

[0157] In some embodiments, the administration provides an area under the curve (AUC) of Compound 1 of about 13000 h*ng/mL to about 20400 h*ng/mL following administration of about 300 mg of Compound 1.

[0158] In some embodiments, the administration provides a half-life (ti/2) of Compound 1 between about 7.5 h and 13.5 following administration of about 25 mg to about 300 mg of Compound 1. Pharmacodynamics

[0159] In some embodiments of the present methods, at least an 85%, at least a 90%, at least a 95%, at least a 100%, or at least a 105% increase in the patient’s mean acetylated tubulin level over time compared to baseline level is observed about 2-4 hours after a daily dose of Compound 1 is administered. In some embodiments of the present methods, at least a 100% (2 -fold) increase in the patient’s mean acetylated tubulin level over time compared to baseline level is observed about 2-4 hours after a daily dose of Compound 1 is administered. In some embodiments, about an 85%, about a 90%, about a 95%, about a 100%, or about a 105% increase in the patient’s mean acetylated tubulin level over time compared to baseline level is observed about 2-4 hours after a daily dose of Compound 1 is administered. In some embodiments, about a 100% (2-fold) increase in the patient’s mean acetylated tubulin level over time compared to baseline level is observed about 2-4 hours after a daily dose of Compound 1 is administered.

[0160] In some embodiments of the present methods, about a 50% to about an 85% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments of the present methods, about a 50% to about a 80% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments of the present methods, about a 50% to about a 75% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments of the present methods, about a 50% to about a 70% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments, at least about a 50% increase in the patient’ s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments, at least about a 55% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments, at least about a 60% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments, at least about a 65% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments, at least about a 70% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound. In some embodiments, at least about a 75% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound.

[0161] In some embodiments, treatment provides about a 10 mg/dL to about a 50 mg/dL reduction in fasting plasma glucose, e.g., about a 10 mg/dL, about a 15 mg/dL, about a 20 mg/dL, about a 25 mg/dL, about a 30 mg/dL, about a 35 mg/dL, about a 40 mg/dL, about a 45 mg/dL, or about a 50 mg/dL reduction in fasting plasma glucose, including all ranges and values therebetween.

[0162] In some embodiments, treatment provides about a 0.3% to about a 1.5% reduction in Ale level, e.g., about a 0.3%, about a 0.4%, about a 0.5%, about a 0.6%, about a 0.7%, about a 0.8%, about 0.9%, about a 1.0%, about a 1.1%, about a 1.2%, or about a 1.3% reduction in Ale level, including all ranges and values therebetween.

Patient Population

[0163] In some embodiments of the present methods, the patient has heart failure or is at risk of heart failure. In some embodiments, the patient has Class I heart failure as classified by the New York Heart Association (NYHA) Functional Classification. In some embodiments of the present methods, the patient has Class II heart failure as classified by the New York Heart Association (NYHA) Functional Classification. In some embodiments, the patient has Class III heart failure as classified by the NYHA Functional Classification.

[0164] In some embodiments of the present methods, the patient has or is at risk for hypertension. In some embodiments, the patient has or is at risk for diabetes (e.g., diabetes mellitus). In some embodiments, the patient has or is at risk for diabetic cardiomyopathy. In some embodiments, the patient has or is at risk for metabolic syndrome. In some embodiments, the patient has or is at risk for hyperglyceridemia or dyslipidemia. In some embodiments, the patient is obese or is at risk for obesity. In some embodiments, the patient has or is at risk for coronary artery disease (CAD). In some embodiments, the patient has or is at risk for heart disease. In some embodiments, the patient has or is at risk for valvular heart disease. In some embodiments, the patient has or is at risk for atrial fibrillation. In some embodiments, the patient has or it at risk for metabolic disease. In some embodiments, the patient has a diastolic dysfunction of Grade II, III, or IV. In some embodiments, the patient has a diastolic dysfunction of Grade II. In some embodiments, the patient has a diastolic dysfunction of Grade III. In some embodiments, the patient has a diastolic dysfunction of Grade IV.

[0165] In some embodiments, the subject does not have a metabolic disease. In some embodiments, the subject does not have metabolic syndrome. In some embodiments, the subject does not have diabetes (e.g., does not have diabetes mellitus). In some embodiments, the subject does not have hypertension. In some embodiments, the subject is not obese.

[0166] In some embodiments of the present methods, the patient has heart failure or is at risk of heart failure. In some embodiments, the patient has Class I heart failure as classified by the New York Heart Association (NYHA) Functional Classification. In some embodiments of the present methods, the patient has Class II heart failure as classified by the New York Heart Association (NYHA) Functional Classification. In some embodiments, the patient has Class III heart failure as classified by the NYHA Functional Classification.

[0167] In some embodiments of the present methods, the patient has an ejection fraction greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, or greater than about 50%. In some embodiments, the patient has an ejection fraction greater than about 40%. In some embodiments, the patient has an ejection fraction greater than about 45%. In some embodiments, the patient has an ejection fraction greater than about 50%. In some embodiments, the patient has an ejection fraction less than about 50%. In some embodiments, the patient has an ejection fraction less than about 45%. In some embodiments, the patient has an ejection fraction less than about 30%.

[0168] In some embodiments of the present methods, the patient has an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of greater than about 300 pg/mL. In some embodiments, the patient has an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of greater than about 400 pg/mL. In some embodiments, the patient has an N- terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of greater than about 500 pg/mL. In some embodiments, the patient has atrial fibrillation and an N-terminal pro b- type natriuretic peptide (NT-proBNP) level in the blood of greater than about 750 pg/mL. In some embodiments, the patient has atrial fibrillation and an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of greater than about 825 pg/mL. In some embodiments, the patient has atrial fibrillation and an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of greater than about 900 pg/mL. In some embodiments, the patient has an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of about 400 pg/mL to about 1500 pg/mL. In some embodiments, the patient has an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of about 400 pg/mL to about 1200 pg/mL. In some embodiments, the patient has an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of about 400 pg/mL to about 1000 pg/mL. In some embodiments, the patient has an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of about 400 pg/mL to about 750 pg/mL.

[0169] In some embodiments, the patient is at least 18 years old. In some embodiments, the patient is at least 30 years old. In some embodiments, the patient is at least 40 years old. In some embodiments, the patient is at least 50 years old. In some embodiments, the patient is at least 60 years old. In some embodiments, the patient is at least 65 years old. In some embodiments, the patient is at least 70 years old. In some embodiments, the patient is a male. In some embodiments, the patient is a female.

[0170] In some embodiments of the present methods, Compound 1 is administered to the patient as an adjunctive therapy to a sodium-glucose Cotransporter-2 (SGLT2) inhibitor. In some embodiments, the SGLT2 inhibitor is canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, or bexagliflozin. In some embodiments, the SGLT2 inhibitor is empagliflozin.

Outcomes and Clinical Endpoints

[0171] In some embodiments of the present methods, treatment provides a decrease of NT-proBNP levels in the blood of the patient of about 5% to about 50%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%, including all ranges and values therebetween. In some embodiments of the present methods, treatment provides a decrease of NT-proBNP levels in the blood of the patient of about 5% to about 25%. In some embodiments of the present methods, treatment provides a decrease of NT-proBNP levels in the blood of the patient of about 25% to about 50%. In some embodiments of the present methods, treatment provides a decrease of NT-proBNP levels in the blood of the patient of about 15% to about 30%.

[0172] In some embodiments of the present methods, treatment provides the patient improving from Class III to Class II in the NYHA Functional Classification. In some embodiments, treatment provides the patient improving from Class III to Class I in the NYHA Functional Classification. In some embodiments, treatment provides an improvement in the patient’s NYHA Functional Classification from Class II to Class I.

[0173] In some embodiments of the present methods, treatment provides about a 5 mL/m² to about a 20 mL/m² decrease in left atrial (LA) volume index, e.g., about a 5 mL/m² decrease in LA volume index, about a 10 mL/m² decrease in LA volume index, about a 15 mL/m² decrease in LA volume index, or about a 20 mL/m² decrease in LA volume index, including all ranges and values therebetween. In some embodiments, treatment provides about a 5 mL/m² to about a 15 mL/m² decrease in left atrial (LA) volume index. In some embodiments, treatment provides about a 10 mL/m² to about a 20 mL/m² decrease in left atrial (LA) volume index.

[0174] In some embodiments of the present methods, treatment provides an improvement of least 5 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score. In some embodiments, treatment provides an improvement of least 10 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score. In some embodiments, treatment provides an improvement of least 20 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score.

[0175] In some embodiments, treatment provides improved performance in a standardized 6- minute walk test. In some embodiments, treatments provides an improvement of 5 meters, 10 meters, 15 meters, 20 meters, 25 meters, or 30 meters in a standardized 6-minute walk test.

[0176] In some embodiments, treatment provides an improvement in exercise capacity as measured by cardiopulmonary exercise testing.

[0177] In some embodiments, treatment provides an improvement in pulmonary venous oxygen tension (pVO2) of 1-10 mL/kg/min, e.g., 1 mL/kg/min, 2 mL/kg/min, 3 mL/kg/min, 4 mL/kg/min, 5 mL/kg/min, 6 mL/kg/min, 7 mL/kg/min, 8 mL/kg/min, 9 mL/kg/min, or 10 mL/kg/min, including all ranges and values therebetween.

[0178] In some embodiments, treatment results in a reduction in the relative risk of heart failure hospitalization of about 5% to about 50%. In some embodiments, treatment results in a reduction in the relative risk of heart failure hospitalization of about 5% to about 40%. In some embodiments, treatment results in a reduction in the relative risk of heart failure hospitalization of about 5% to about 20%. In some embodiments, treatment results in a reduction in the relative risk of heart failure hospitalization of about 15% to about 30%. In some embodiments, treatment results in a reduction in the relative risk of heart failure hospitalization of about 20% to about 30%. In some embodiments, treatment results in a reduction in the relative risk of heart failure hospitalization of about 30% to about 40%.

[0179] In some embodiments, treatment results in a reduction in the relative risk of cardiovascular disease (CVD) death of about 5% to about 40%. In some embodiments, treatment results in a reduction in the relative risk of cardiovascular disease (CVD) death of about 5% to about 25%. In some embodiments, treatment results in a reduction in the relative risk of cardiovascular disease (CVD) death of about 10% to about 25%. In some embodiments, treatment results in a reduction in the relative risk of cardiovascular disease (CVD) death of about 5% to about 15%. In some embodiments, treatment results in a reduction in the relative risk of cardiovascular disease (CVD) death of about 15% to about 25%.

[0180] In some embodiments, treatment results in an increase in overall survival.

[0181] In some embodiments, treatment provides the patient exhibiting an increase in heart pump function as measured by echocardiogram (ECG).

[0182] In some embodiments, treatment provides the patient exhibiting no atrial or ventricular arrythmias when comparing baseline ECG monitoring with 72-hour monitoring.

Pharmaceutical Compositions and Kits

[0183] In some embodiments, the present disclosure provides pharmaceutical compositions comprising Compound 1 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients that are useful in treating a disease or condition responsive to HDAC6 inhibition. The pharmaceutically acceptable excipients and adjuvants are added to the composition or formulation for a variety of purposes. In some embodiments, suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose and polyvinylpyrrolidone. In some embodiments, the pharmaceutically acceptable carrier includes a pharmaceutically acceptable binder, and/or diluent. Non-limiting examples of binders include hydroxypropylmethyl cellulose, polyvinylpyrrolidone, other cellulosic materials, and starch. Diluents include, but are not limited to, lactose and other sugars, starch, dicalcium phosphate, and cellulosic materials. [0184] In some embodiments, the present disclosure provides a kit comprising Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof, and instructions for use in a method of treating a disease or condition disclosed herein.

[0185] In some embodiments, the present disclosure provides a kit comprising Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof, and instructions for use in a method for treating HFpEF.

[0186] In some embodiments, the present disclosure provides a kit comprising Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof, and instructions for use in a method for treating dilated cardiomyopathy.

[0187] In some embodiments, the present disclosure provides a kit comprising Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof, and instructions for use in a method for treating a metabolic disease or disorder disclosed herein.

[0188] In some embodiments, the present disclosure provides a kit comprising Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof, and instructions for use in a method for treating an inflammatory disease or disorder disclosed herein.

[0189] In some embodiments, the present disclosure provides a kit comprising Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof, and instructions for use in a method for treating fibrotic disease or disorder disclosed herein.

Methods of Administration

[0190] As disclosed herein, Compound 1, a pharmaceutical salt thereof, or a composition thereof is suitable for oral administration to the patient in need. In some embodiments, the composition is a solid dosage form, such as a tablet or capsule.

[0191] Various dosing schedules of Compound 1 described herein (and compositions comprising Compound 1 or a pharmaceutically acceptable salt thereof) are contemplated including single administration or multiple administrations over a period of time, such as a plurality of weeks.

[0192] In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered twice daily, once daily, once every two days, once every three days, once a week, once in two weeks, once in three weeks or once a month. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered once daily (qd). In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered twice daily (bid).

[0193] Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof can be administered for any period of time that is effective for treating a disease or disorder disclosed herein. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a plurality of weeks. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period ranging from 4 weeks to 5 years or more. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period of at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 18 weeks, at least 24 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, at least 48 weeks, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period of at least 4 weeks. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period of at least 8 weeks. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period of at least 12 weeks. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period of at least 24 weeks. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period of at least 1 year. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period of at least 2 years, at least 3 years, at least 4 years, or at least 5 years or more. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof can be administered for an indeterminate period of time, e.g., when the disease or disorder is chronic.

[0194] The appropriate dosage of an HDAC6 inhibitor described herein for use in the methods described herein will depend on the type of inhibitor used, the condition of the subject (e.g., age, body weight, health), the responsiveness of the subject, other medications used by the subject, and other factors to be considered at the discretion of the medical practitioner performing the treatment. Combination Treatments

[0195] In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof can be administered to a patient in combination with another medication or therapy. In some embodiments, the other medication is standard-of-care (SoC) treatment.

[0196] In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof can be administered to a subject in combination with one or more additional therapies, where the therapy is a cardioprotective therapy, an anti-hypertensive therapy, a therapy for a heart condition (e.g., heart failure), a therapy for HFpEF, or a therapy for metabolic disease. The additional therapy can be any such therapy known in the art. In some embodiments of metabolic disease treatments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subject in combination with another metabolic disease therapy. In some embodiments of HFpEF treatments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subject in combination with another HFpEF therapy.

[0197] In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subject in combination with an anti -hypertensive therapy. For example, the following anti-hypertensive agents can be used in combination with HDAC6 inhibitors: thiazide diuretics (e.g., Capozide, Dyazide, Hyzaar, Lopressor HCT, Maxzide, Prinzide, Clorpres, Tenoretic, or Thalitone), calcium channel blockers (e.g., Amlodipine, Diltiazem, Felodipine, Isradipine, Nicardipine, Nifedipine, Nisoldipine, or Verapamil), ACE inhibitors (e.g., Benazepril, Captopril, Enalapril, Fosinopril, Lisinopril, Moexipril, Perindopril, Quinapril, Ramipril, or Trandolapril), angiotensin II receptor antagonists (ARB) (e.g., Azilsartan, Candesartan, Eprosartan, Irbesartan, Losartan, Olmesartan, Telmisartan, or Valsartan), SGLT2 inhibitors (e.g., empagliflozin), and beta blockers (e.g., Acebutolol, Atenolol, Bisoprolol, Metoprolol, Nadolol, Nebivolol, or Propranolol).

[0198] In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subject in combination with an ACE inhibitor. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subject in combination with a beta blocker. [0199] In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subject in combination with a sodium-glucose cotransporter 2 (SLGT2) inhibitor (e.g., canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, or bexagliflozin). In some embodiments, Compound 1 is Compound 1 is administered as an adjunctive therapy to the sodium-glucose Cotransporter-2 (SGLT2) inhibitor.

[0200] In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subject in combination with a thiazide diuretic. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subject in combination with a calcium channel blocker. In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subject in combination with an angiotensin II receptor antagonist (ARB).

[0201] In some embodiments, Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof is administered to a subj ect before, at the same time, or after the additional therapy. In some embodiments, the subject being treated in accordance with the methods described herein has not received an anti-hypertensive therapy. In some embodiments, the subject being treated in accordance with the methods described herein has not received metabolic disease therapy. In some embodiments, the subject being treated in accordance with the methods described herein has not received HFpEF therapy. In some embodiments, the subject being treated in accordance with the methods described herein has not received a cardioprotective therapy and/or a heart condition (e.g., heart failure) therapy.

[0202] In some embodiments provided herein are kits comprising Compound 1, a pharmaceutically acceptable salt thereof, or a composition thereof and one or more additional agents (e.g., an additional agent for the treatment of metabolic syndrome, HFpEF or an antihypertensive agent). In some embodiments, provided herein are kits comprising (i) an HDAC6 inhibitor (e.g., in a therapeutically effective amount), and (ii) one or more additional agents, such as a thiazide diuretic, a calcium channel blocker, an ACE inhibitor, an angiotensin II receptor antagonist (ARB), an SGLT2 inhibitor (e.g., canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, or bexagliflozin), or a beta blocker(e.g., in a therapeutically effective amount). NUMBERED EMBODIMENTS OF THE DISCLOSURE

1. A method of treating or preventing a disease or disorder associated with HDAC6 activity or responsive to HDAC6 inhibition in a patient in need thereof, comprising administering to the patient about 10 mg to about 300 mg of Compound 1 having the formula:

or a pharmaceutically acceptable salt thereof.

2. The method of embodiment 1, wherein the disease or disorder is a metabolic disease or disorder.

3. The method of embodiment 1 or 2, wherein the disease or disorder is a metabolic disease or disorder associated with obesity, optionally diet-induced obesity.

4. The method of embodiment 2 or 3, wherein the metabolic disease or disorder is diabetes, pre-diabetes, diabetic cardiomyopathy, metabolic syndrome, hypertension, hypertriglyceridemia, or dyslipidemia.

5. The method of any one of embodiments 1-4, wherein the disease or disorder is obesity.

6. The method of any one of embodiments 1-4, wherein the disease or disorder is diabetes.

7. The method of any one of embodiments 1-6, wherein the patient is at risk for obesity.

8. The method of any one of embodiments 1-7, wherein the patient has or is at risk for hypertension. 9. The method of any one of embodiments 1-8, wherein the patient has or is at risk for metabolic syndrome.

10. The method of any one of embodiments 1-9, wherein the patient has or is at risk for diabetes mellitus.

11. The method of any one of embodiments 1-10, wherein the patient has or is at risk for diabetic cardiomyopathy.

12. The method of any one of embodiments 1-11, wherein the patient has or is at risk for hyperglyceridemia or dyslipidemia.

13. The method of any one of embodiments 1-12, wherein the method treats or prevents at least one symptom of metabolic disease.

14. The method of any one of embodiments 1-13, wherein the method improves glucose tolerance.

15. The method of any one of embodiments 1-14, wherein the method improves insulin resistance.

16. The method of any one of embodiments 1-15, wherein the method reduced glucose level.

17. The method of any one of embodiments 1-16, wherein the method inhibits inflammatory genes in adipose tissue.

18. The method of any one of embodiments 1-17, wherein the method prevents heart failure in the patient.

19. The method of embodiment 1, wherein the disease or disorder is heart failure with preserved ejection fraction (HFpEF). 20. The method of embodiment 19, wherein the patient has or is at risk for hypertension.

21. The method of any one of embodiment 19 or 20, wherein the patient has or is at risk for diabetes mellitus.

22. The method of any one of embodiments 19-21, wherein the patient has or is at risk for coronary artery disease (CAD).

23. The method of any one of embodiments 19-22, wherein the patient has or is at risk for valvular heart disease.

24. The method of any one of embodiments 19-23, wherein the patient has or is at risk for atrial fibrillation.

25. The method of any one of embodiments 19-24, wherein the method treats or prevents at least one symptom of HFpEF.

26. The method of any one of embodiments 19-25, wherein the method reduces left ventricular (LV) mass.

27. The method of any one of embodiments 19-26, wherein the method reduces LV wall thickness.

28. The method of any one of embodiments 19-27, wherein the method improves LV relaxation.

29. The method of any one of embodiments 19-28, wherein the method improves LV filling pressure.

30. The method of any one of embodiments 19-29, wherein the method prevents heart failure in the patient.

31. The method of embodiment 1, wherein the disease or disorder is dilated cardiomyopathy. 32. The method of embodiment 31, wherein the dilated cardiomyopathy is familial dilated cardiomyopathy.

33. The method of embodiment 31 or 32, wherein the dilated cardiomyopathy is dilated cardiomyopathy due to one or more BLC2-Associated Athanogene 3 (BAG3) mutations.

34. The method of any one of embodiments 31-33, wherein the patient has a deleterious mutation in the BAG3 gene.

35. The method of any one of embodiments 31-34, wherein the dilated cardiomyopathy is dilated cardiomyopathy due to one or more muscle LIM protein (MLP) mutations.

36. The method of any one of embodiments 31-35, wherein the patient has a deleterious mutation in the CSPR3 gene encoding MLP.

37. The method of any one of embodiments 31-36, wherein the method restores the ejection fraction of the patient to at least about the ejection fraction of a patient without dilated cardiomyopathy.

38. The method of embodiment 37, wherein the method restores the ejection fraction of the patient to at least about 20%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

39. The method of any one of embodiments 31-38, wherein the method increases the ejection fraction of the patient compared to the patient’s ejection fraction before treatment.

40. The method of any embodiment 39, wherein the method increase the ejection fraction of the patient by at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 40%.

41. The method of any one of embodiments 31-40, wherein the method reduces HDAC6 activity in the heart of the patient. 42. The method of any one of embodiments 31-41, wherein the method prevents heart failure in the patient.

43. The method of any one of embodiments 31-42, wherein the method reduces left ventricular internal diameter at diastole (LVIDd) in the patient.

44. The method of any one of embodiments 31-43, wherein the method reduces left ventricular internal diameter at systole (LVIDs) in the patient.

45. The method of any one of embodiments 31-44, wherein the method reduces left ventricular mass in the patient.

46. The method of any one of embodiments 1-45, wherein the patient is at least 18 years old

47. The method of any one of embodiments 1-46, wherein the patient is at least 50 years old.

48. The method of any one of embodiments 1-47, wherein about 25 mg to about 200 mg of Compound 1 is administered to the patient.

49. The method of any one of embodiments 1-47, wherein about 25 mg to about 100 mg of Compound 1 is administered to the patient.

50. The method of any one of embodiments 1-47, wherein about 25 mg, about 50 mg, or about 100 mg of Compound 1 is administered to the patient.

51. The method of any one of embodiments 1-47, wherein about 25 mg, about 75 mg, or about 200 mg of Compound 1 is administered to the patient

52. The method of any one of embodiments 1-47, wherein about 10 mg, about 25 mg or about 75 mg of Compound 1 is administered to the patient. 53. The method of any one of embodiments 1-47, wherein about 10 mg of Compound 1 is administered to the patient.

54. The method of any one of embodiments 1-47, wherein about 25 mg of Compound 1 is administered to the patient.

55. The method of any one of embodiments 1-47, wherein about 50 mg of Compound 1 is administered to the patient.

56. The method of any one of embodiments 1-47, wherein about 75 mg of Compound 1 is administered to the patient.

57. The method of any one of embodiments 1-47, wherein about 100 mg of Compound 1 is administered to the patient.

58. The method of any one of embodiments 1-47, wherein about 125 mg of Compound 1 is administered to the patient.

59. The method of any one of embodiments 1-47, wherein about 150 mg of Compound 1 is administered to the patient.

60. The method of any one of embodiments 1-47, wherein about 200 mg of Compound 1 is administered to the patient.

61. The method of any one of embodiments 1-47, wherein about 300 mg of Compound 1 is administered to the patient.

62. The method of any one of embodiments 61, wherein Compound 1 is administered once- a-day.

63. The method of any one of embodiments 1-62, wherein Compound 1 is administered at a fixed dose. 64. The method of any one of embodiments 1-63, comprising administering to the patient a composition comprising Compound 1 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients.

65. The method of any one of embodiments 1 -64, wherein Compound 1 , a pharmaceutically acceptable salt thereof, or composition thereof is administered orally.

66. The method of any one of embodiments 1-65, wherein Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered once daily (qd).

67. The method of any one of embodiments 1-66, wherein Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a plurality of weeks.

68. The method of any one of embodiments 1-67, wherein Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period ranging from 4 weeks to 5 years or more.

69. The method of any one of embodiments 1-67, wherein Compound 1, a pharmaceutically acceptable salt thereof, or composition thereof is administered for a period of at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 18 weeks, at least 24 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, at least 48 weeks, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

70. The method of any one of embodiments 64-69, wherein the composition is a solid dosage form.

71. The method of embodiment 70, wherein the solid dosage form is a tablet or capsule.

72. The method of any one of embodiments 1-71, wherein the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 80 ng/mL to about 1700 ng/mL following administration of about 25 mg to about 300 mg of Compound 1. 73. The method of any one of embodiments 1-71, wherein the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 80 ng/mL to about 720 ng/mL following administration of about 25 mg to about 100 mg of Compound 1.

74. The method of any one of embodiments 1-73, wherein the administration provides an area under the curve (AUC) of Compound 1 of about 600 h*ng/mL to about 16300 h*ng/mL.

75. The method of any one of embodiments 1-73, wherein the administration provides an area under the curve (AUC) of Compound 1 of about 600 h*ng/mL to about 5200 h*ng/mL.

76. The method of any one of embodiments 1-75, wherein the administration provides an area under the curve (AUC) of Compound 1 of about 640 h*ng/mL to about 20400 h*ng/mL following administration of about 25 mg to about 300 mg of Compound 1.

77. The method of any one of embodiments 1-75, wherein the administration provides an area under the curve (AUC) of Compound 1 of about 640 h*ng/mL to about 6500 h*ng/mL following administration of about 25 mg to about 100 mg of the compound.

78. The method of any one of embodiments 1-77, wherein the administration provides a half-life (ti/2) of Compound 1 between about 7.5 h and 13.5 h.

79. Use of Compound 1 or a pharmaceutically acceptable salt thereof in a method of treating a disease or condition responsive to HDAC6 inhibition in a patient in need thereof, wherein the method comprises orally administering to the patient once daily about 10 mg to about 300 mg of Compound 1 having the formula:

or a pharmaceutically acceptable salt thereof. 80. Use of Compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease or condition responsive to HDAC6 inhibition in a patient in need thereof.

81. The use of claim 79 or 80, wherein the disease or condition responsive to HDAC6 inhibition is heart failure with preserved ejection fraction (HFpEF).

EXAMPLES

[0203] The invention is further illustrated by the following examples. The examples below are non-limiting are merely representative of various aspects of the invention.

Example 1: Compound 1 Improves Glucose Tolerance and Insulin Resistance in a Diet- Induced Obesity Mouse Model

[0204] This example demonstrates that histone deacetylase 6 (HDAC6) selective inhibitor Compound 1 improves glucose tolerance and insulin resistance in a diet induced obese mouse model. The diet-induced obesity model closely mimics high-fat/high-density foods that contribute to obesity in humans. (Reviewed in Wang et al. Methods Mol Biol. 821 :421-433 (2012).)

[0205] FIGs. 1A-1L show that HDAC6 inhibition with Compound 1 (TYA-11631 or TYA ‘631) improves glucose tolerance and insulin resistance in a diet induced obese mouse model.

[0206] 16-weeks old male Diet-Induced Obese (DIO) C57BL/6J (Cat. 380050) mice and age/gender matched controls (Cat. 000664) were purchased from the Jackson Laboratory®. DIO mice (n=40) were fed a rodent diet with 60 kcal% fat (DI 2492) and control mice were fed a rodent diet with 10 kcal% fat (D12450B) starting from 6-weeks of age.

[0207] Intraperitoneal glucose-tol erance tests (GTT) were performed by injection of glucose (2 g/kg in saline) after 6 hours fasting. Tail blood glucose levels (mg/dl) were measured with a glucometer before (0 min) and at 15, 30, 45, 60 and 120 min after glucose administration. DIO mice at 16 weeks of age developed severe glucose intolerance compared to controls. (FIG. 1A, FIG. IB).

[0208] Based on glucose AUC level, DIO mice were evenly randomized into four treatment groups to receive Vehicle (n=9) or Compound 1 at three dosages 3, 10, 30mg/kg (n=10 each). Control mice were also divided to dose orally with vehicle (n=10) or 30 mg/kg Compound 1 (n=10). To assess the acute response of Compound 1 on glucose metabolism, GTT was performed at 6h after 1^st dose. Strikingly, single dose of Compound 1 at all three test dosages significantly reduced glucose level. (FIG. 1C, FIG. ID). Compound 1 treatment for 2 weeks led to pronounced improvements in glucose tolerance in dose-dependent manner. (FIG. IE, FIG. IF)

[0209] To evaluate the effects of Compound 1 on insulin resistance, intraperitoneal insulintolerance test (ITT) was performed by injection of insulin (lU/kg) after 6 hours fasting. Tail blood glucose levels (mg/dl) were measured with a glucometer before (0 min) and at 15, 30, 45, 60 and 120 min after insulin administration. HDAC6 inhibitor (Compound 1) treatment for 4 weeks improved insulin resistance in DIO mice. 10 and 30mg/kg were significantly reduced glucose AUC (ITT) with comparable activities, 3 mg/kg showed a trend of reduction. (. 1G, FIG. 1H)

[0210] Effects of Compound 1 on blood glucose were also assessed in non-fasting mice. Tail blood samples were collected in the morning and measured with a glucometer. Compound 1 treatment for 6wks led to a dose-dependent reduction of non-fasting glucose, consistent with the data of glucose tolerant test after fasting. (FIG. II)

[0211] Treatment with Compound 1 caused a dose dependent reduction of body weight in DIO mice (See FIG. 1J, FIG. IK, FIG. IL) No differences of food consumption were observed between groups. Notably, control mice dosed with Compound 1 30mg/kg for 6wks did not show changes on blood glucose levels and body weights. Bars and error bars show means and SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0212] FIGs. 2A-C show HD AC 6 inhibition with Compound 1 inhibits inflammatory genes in adipose tissue in a diet induced obese mouse model.

[0213] White adipose tissue (epididymal) was dissected from DIO mice after 6 hours of single dose 30mg/kg Compound 1. Realtime q-PCR data showed that Compound 1 remarkably inhibited upregulation of pro-inflammatory genes- IL-6 (FIG. 2A), IL-10 (FIG. 2B) and TGFbl (FIG. 2C) in white adipose tissue. Inhibition or ablation of these genes have been shown to protect mice from diet-induced obesity with improvement of glucose tolerance and insulin sensitivity (Han et al., PNAS 2020,' Rajbhandari et al., Cell 2018).

[0214] Taken together, these data suggest a potential molecular mechanism underlying the effects of an HDAC6 inhibitor e.g., Compound 1) on systemic glucose metabolism. Example 2: Compound 1 Improves Cardiac Structure and Heart Function in a HFpEF Mouse Model

[0215] This example demonstrates that histone deacetylase 6 (HDAC6) selective inhibitor Compound 1 improves cardiac structure and heart function in a mouse model of heart failure with preserved ejection fraction (HFpEF).

[0216] First, an animal model of HFpEF was established: Surgically applying moderate trans- aortic constriction (mTAC) in wild type C57BL6 mice fed on high fat diet (HFD) was found to induce a cardio-metabolic heart failure phenotype that recapitulates systemic and cardiovascular features of HFpEF in human (FIGs. 3A-3H).

[0217] HFpEF model animals were dosed orally with 30 mg/kg HDAC6 inhibitor (Compound 1) or vehicle once per day for six weeks. HDAC6 inhibitor effectively treated HFpEF. The HDAC6 inhibitor treatment regimen significantly reduced left ventricular (LV) mass (FIG. 4E) and LV wall thickness (FIG. 4F) measured by echocardiograph; and HDAC6 inhibitor treatment improved glucose tolerance (FIG. 4A). In addition, Compound 1 treatment sustained improved LV relaxation and LV filling pressures as shown by decreased prolongation of isovolumetric relaxation time (FIG. 4H); lower E/A (FIG. 41) and E/e’ ratios (FIG. 4L); improved e’ velocity (FIG. 4K); and reduced end diastolic pressure (FIG. 4M). Each of these efficacy parameters were normalized to control levels.

[0218] Furthermore, HFD/mTAC mice treated with Compound 1 showed a trending decrease in lung weight (FIG. 40), indicating improved pulmonary congestion, consistent with reduced filling pressure. No treatment related adverse events or toxicities were observed in the animals in the study.

[0219] At molecular level, the HDAC6 inhibitor significantly inhibited upregulation of genes commonly associated with fibrosis (Postn, Collal, Col3al, Col5a2), cardiac stress (Nppb and inflammation Tnfa) in heart tissue of HFD/mTAC mice (FIGs. 5A-5H).

[0220] The results demonstrate that selectively inhibiting HDAC6 reverses a number of adverse pathophysiological processes in HFpEF.

Example 3: Compound 1 Improves Cardiac Structure and Heart Function in a Second HFpEF Mouse Model

[0221] This example demonstrates that histone deacetylase 6 (HDAC6) selective inhibitor Compound 1 improves cardiac structure and heart function in a second mouse model of heart failure with preserved ejection fraction (HFpEF), which recapitulates the vast majority of the clinical features of the syndrome: high-fat diet (HFD) coupled with inhibition of constitutive nitric oxide synthases (NOS) with N[w]-nitro-l-arginine methyl ester (L-NAME). (Schiattarella et al. Nature S IS y. 351-356 (2019)).

[0222] First, the mouse model was validated. HFD/L-NAME treatment significantly induced body weight increase (FIG. 6B), hypertension (FIG. 6C) and glucose intolerance (FIG. 6D) compared to the control mice. Echocardiographic evaluation revealed persistent preservation of the left ventricular ejection fraction (LVEF) (FIG. 6E). Significant concentric left ventricular (LV) hypertrophy was present in HFD/L-NAME mice, as indicated by increases in LV mass (FIG. 6F) and LV wall thickness at diastole (FIG. 6G), without LV chamber dilatation (FIG. 6H). In addition, mice concomitantly exposed to HFD/L-NAME exhibited signs of LV diastolic dysfunction with impaired relaxation and increased left ventricular filling pressure, as evidenced by prolonged IVRT (FIG. 61), decreased e’ velocity (FIG. 6J), and increased ratios of E/e’(FIG. 6K), E/A (FIG. 6L), measured by noninvasive Doppler imaging.

[0223] Second, treatment with an HDAC6 inhibitor, Compound 1, was shown to improve glucose tolerance and diastolic dysfunction in the HFD/L-NAME model.

[0224] Animals were randomized to dose orally with 30 mg/kg Compound 1 (n=8) or vehicle (n=12) once per day for nine weeks, respectively. In control groups, 6 mice were received vehicle and 8 mice were dosed with 30 mg/kg Compound 1. As shown in FIG. 7A and FIG. 7B, Glucose tolerance test (GTT) was performed after 5 weeks of dosing.

[0225] Treatment with Compound 1 markedly improved glucose tolerance in HFD/L-NAME mice, but no changes in control animals. (FIG. 7B) Plasma insulin level during GTT at the indicated time points (0 and 30 minutes after glucose injection) was measured by a sensitive mouse insulin detection kit (Cat. 80-INSMS-E01, ALPCO). Compound 1 treatment led to decreased insulin secretion, suggesting that the improved glucose tolerance might be due to improved insulin action/sensitivity. (FIG. 7C) Treatment with Compound 1 caused a significant reduction of body wight, but no difference of food consumption in mice fed with HFD/L-NAME. (FIG. 7D, FIG. 7E) Compound 1 did not affect systolic blood pressure in HFD/L-NAME mice measured by non-invasive tail cuff method. (FIG. 7F) Echocardiography showed that Compound 1 treatment preserved ejection fraction (FIG. 7G), however significantly reduced left ventricular mass (FIG. 7H) and LV wall thickness (FIG. 71). [0226] Noninvasive Doppler imaging and terminal invasive catheterization analysis revealed that treatment with Compound 1 for 9wks decreased prolongation of isovolumetric relaxation time (FIG. 7J), E/A (FIG. 7K) and E/e’ ratios (FIG. 7L), increased e’ velocity (FIG. 7M), and reduced end diastolic pressure (FIG. 7N), indicating the improved LV relaxation and filling pressure. In addition, HFD/L-NAME mice treated with Compound 1 showed a trending decrease in lung weight (FIG. 70), suggesting an improved pulmonary congestion, consistent with the reduction of filling pressure.

[0227] Notably, no adverse effects related to Compound 1 have been observed. Control animals dosed with Compound 1 has no changes on each of LV structural and functional parameters, as well as ECG signals- QT, QRS, PR intervals and R amplitude (FIG. 7P, FIG. 7Q, FIG. 7R and FIG. 7S), further supports an overall favorable safety profile of the compound.

Example 4: Compound 1 Reduces Cardiac Fibrosis and Enhances Mitochondrial Function in a Mouse Model of HFpEF

[0228] This example demonstrates that histone deacetylase 6 (HDAC6) selective inhibitor Compound 1 reduces cardiac fibrosis and enhances mitochondrial function in a mouse model of HFpEF. In particular, this example demonstrates that Compound 1 (i) corrects dysregulated fibrosis and oxidative phosphorylation gene expression in HFpEF model, (ii) increases mitochondrial membrane potential and spare respiratory capacity in human iPSC-CMs, and (iii) prevents fibroblast activation from TGF-beta in primary human cardiac fibroblasts.

[0229] As described below, transcriptional analysis was performed on cardiac tissue from mice with HFpEF using RNA sequencing and qPCR. RNA-Seq data presented here show reduced expression of gene sets associated with hypertrophy, fibrosis, and PDGFR signaling in HFpEF mice treated with Compound 1. qPCR data further confirmed reduced expression of fibrotic genes which correlated with improved diastolic function. Additionally, RNA-Seq data presented here show that gene sets associated with mitochondrial energy production were enriched in Compound 1 treated HFpEF mice. The increased expression of mitochondrial genes correlated with improved diastolic function. Compound 1 was further tested to determine whether it has a direct effect on metabolism in an in-vitro model using human induced pluripotent stem cell-derived cardiomyocytes. The data presented here show enhanced reserve respiratory capacity, indicating improved ATP production in response to stress in Compound 1 treated human iPSC-derived CMs. Materials and Methods

[0230] HFpEF was induced surgically by moderate transaortic constriction in high fat diet-fed mice for 12 weeks (mTAC/HFD). After HFpEF phenotypes were established, mice received Compound 1 (30 mg/kg) or vehicle orally once daily for 6 weeks. Compound 1 treated mice showed improved cardiac function as measured by reduced left ventricular posterior wall thickness (LVPWd), isovolumic relaxation time (IVRT) and mitral valve E/e’ (MV E/e’).

Test System

[0231] Twelve-week-old male C57B1/6NJ mice were purchased from Jackson Laboratories. Mice were maintained under specific pathogen-free conditions and provided with sterile food and water ad libitum. Animals were housed in an animal research facility in accordance with the National Research Council of the National Academies guidelines for the care and use of laboratory animals. Animals were allowed to acclimate for 3 days prior to experiment.

[0232] Induced pluripotent stem cell derived cardiomyocytes (iPSC-derived cardiomyocyte): iCell Cardiomyocyte² were purchased from Cellular Dynamics (Madison, WI). Cells were cultured in a low-glucose and lipid rich medium to enhance cellular maturation for one week prior to treatment.

RNA Extraction and mRNA Sequencing (RNA-Seq) Method and Analysis

[0233] RNA was extracted from mouse heart tissues (control mice, vehicle-treated n=3, HFpEF mice, vehicle-treated, n=5, and HFpEF mice, Compound 1 -treated, n=6) using the polyA-tail-specific protocol from Illumina (San Diego, CA, Cat. #20020594). RNA library preparation and ribosomal RNA removal was performed on 100 ng RNA using the TruSeq Stranded mRNA kit (Illumina, San Diego, CA, Cat. #20020594) following the manufacturer’s instructions. Libraries were sequenced as 100 bp single end reads using Illumina NovaSeq SP with an average of 45.7 million reads per sample. Raw RNA-seq reads in FASTQ format were aligned directly to GENCODE (version M25) for reference transcript assembly (GRCm38.p6 and ensemble 101). Next, a script using tximport was used to generate an expression matrix normalized to transcripts per million (TPM). In this analysis, only genes detected in at least 90% of all samples were used. Protein-coding genes were determined using Ensembl release mus musculus annotations (GRCm38, Apr 2020) and extracted by biomaRt (version 2.46.3). Non-protein-coding and mitochondrial genes were omitted, followed by renormalization to TPM. The generated expression matrix (16,499 genes) was log2-transformed after adding 1 as the pseudo-count. [0234] To evaluate functional perturbations, pre-ranked Gene Set Enrichment Analysis was performed using GSEA developed by Broad institute (version 4.1.0). GSEA assesses whether differences in expression of gene sets between two phenotypes are statistically significant (Subramanian, 2005). Prior to analysis, a ranked list was calculated with each gene assigned a score and direction (“+” or “-”) based on the Estatistics values. Gene sets were only considered statistically significant if the false discovery rate (FDR) was less than 0.25 as determined by the multiple hypothesis testing correction method (Benjamini, 1995). The normalized enrichment score (NES), which reflects the degree to which a gene set is over-represented in the ranked list and normalized for gene set size, was used to select significantly altered gene sets. The Pearson correlation coefficient was used to calculate correlation between genes of interest and cardiac diastolic function parameters.

RNA Extraction and TaqMan qPCR Analysis Method

[0235] Fifteen to thirty milligrams (15-30 mg) of mouse cardiac tissue (control mice, vehicle- treated n=3, HFpEF mice, vehicle-treated, n=5, and HFpEF mice, Compound 1-treated, n=6) was placed directly into 500 pL of Tri-Reagent (Zymo research, Irvine, CA, Cat. #R2050-l- 200) and snap frozen at -80°C. Samples were thawed and homogenized by Bullet Blender Tissue Homogenizer (Storm Pro BT24M, Next Advance, Troy) for 15 minutes at 4°C. RNA was extracted using the Direct-Zol RNA Miniprep Plus Kit (Zymo research, Irvine, CA, Cat. #R2070) according to manufacturer instruction. Upon collecting RNA, the concentration was determined by NanoDrop (ThermoScientific, Waltham, MA,). cDNA was reverse transcribed from 750 ng of RNA through random hexamers using the SuperScriptlll kit (Invitrogen, Waltham, MA, Cat. #18080051). cDNA samples were diluted 6-fold in nuclease free water. Real-Time qPCR reactions were performed using the Standard TaqMan Universal PCR Master Mix (Applied Biosystems, Waltham, MA, Cat. #43-181-57) with the listed TaqMan probes (Life Technologies, San Diego, CA) in Table 1.

Table 1: TaqMan Probes Used in qPCR Analysis

[0236] Real-Time qPCR reactions were performed using the QuantStudio7 Flex Real-Time PCR Systems (Life Technologies, San Diego, CA) with thermal cycling parameters of a 2- minute UNG incubation at 50°C, a 10-minute polymerase activation at 95°C, and 40 PCR cycles consisting of a denaturation for 15 seconds at 95°C and a 1 minute anneal/extension at 60°C. Gene expression was normalized to Gapdh as a housekeeping gene. Four technical replicates were analysed for each sample. Relative gene expression was determined using the 2 ^AACT method. Statistical analysis was performed in Prism Version 9 (GraphPad Software, San Diego, CA) using an unpaired /-test.

Metabolic Measurements Using Seahorse Oximetry

[0237] The Seahorse Xfe96 Analyzer (Agilent, Santa Clara, CA), which measures oxygen consumption rate (OCR) of live cells in a multi-well plate, was used to assess metabolic activity of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-derived CMs). Seahorse XF96 V3 PS Cell Culture Microplates (Cat. #101085-004) were coated with 100 pL of Matrigel (Corning, Coming, NY, Cat. #356231) in phenol-free DMEM medium at a dilution of 1/100 overnight. The following day, the Matrigel was removed and 25 pL of Plating Medium (Cellular Dynamics, Madison, WI, Cat. #R1151) was added to each well of the Seahorse XF96 V3 PS Cell Culture Microplates. One vial of iCell Cardiomyocytes² (~6 million cells, Cellular Dynamics, Madison, WI, Cat. #01434) was thawed in a 37°C water bath for 2 minutes. Cells were seeded directly onto the Seahorse XF96 V3 PS Cell Culture Microplates for a final density of 15,000 cells per well in Plating Medium. The corner wells were excluded for background recordings. Cells were allowed to rest at room temperature for 10 minutes to allow for even distribution of seeding, then placed in a 37°C incubator. Five hours following cell seeding, the medium was changed to Maintenance Medium (Cellular Dynamics, Madison, WI, Cat. #R1151) for 3 days for recovery. Once beating monolayers were observed, the medium was changed to fatty acid enriched Maturation Medium (Feyen, 2020) for one week to increase metabolic maturity of iPSC-derived CMs. Cells were replenished with fresh medium every 3 days. The day of the assay, cells were incubated in starvation medium containing 2 mM glutamine in DMEM (Agilent, Santa Clara, CA, Cat. #103575-100) for one hour. Cells were then treated with DMSO (0.1%) or Compound 1 (3 pM) in Mercola Medium for 6 hours. Cells were washed and incubated for 1 hour prior to the assay with Seahorse XF DMEM Basal Medium supplemented with 2 mM glutamine, 2 mM pyruvate, and 3 mM glucose. The Seahorse Xfe96 cartridge was prepared according to manufacturer’s guidelines. Oxygen consumption rates (OCR) were measured followed by the Mito Stress Test (Agilent, Santa Clara, CA, Cat. #103015-100) with inhibitors injected in the following order: oligomycin (2.5 pM), FCCP (1 pM), rotenone and antimycin A (0.5 pM). OCR was normalized to total nuclear count as measured by Hoechst staining. Basal respiration was calculated as: (last rate measurement before first oligomycin injection) - (minimum rate measurement after rotenone/antimycin). Reserve respiratory capacity (RRC) was calculated as: (Maximal Respiration after the addition of FFCP) - (Basal Respiration). Statistical analysis was performed in Prism Version 9 (GraphPad Software, San Diego, CA) using an unpaired /-test.

Results

Transcriptional Analysis of Compound 1 Effects on HFpEF Mouse Model by RNA-Seq [0238] Comprehensive transcriptional profiling of protein coding genes in heart tissue from control mice and HFpEF mice (mTAC/HFD) was performed using RNA sequencing. To evaluate functional perturbations, unbiased gene set enrichment analysis was performed on two comparisons: 1) vehicle-treated HFpEF mouse tissue relative to vehicle-treated control mouse tissue, 2) Compound 1 -treated versus vehicle-treated HFpEF mouse tissue.

[0239] Gene sets associated with muscle hypertrophy and contraction, fibrosis (transforming growth factor P receptor signaling, type I collagen synthesis, extracellular matrix structural constituent), and platelet-derived growth factor receptor (PDGFR) signaling were enriched in vehicle-treated HFpEF animals relative to control and were reversed in HFpEF mice treated with Compound 1 (FIG. 8 and Fig. 9). Gene sets associated with mitochondrial function including electron transport chain, oxidative phosphorylation, and complex I were significantly depleted in HFpEF mice treated with vehicle relative to control (FIG. 8 and FIG. 9). These mitochondrial gene sets were significantly enriched in HFpEF mice treated with Compound 1 (FIG. 8, FIG. 9 and Table 2)

Table 2:GSEA Categories Altered (FDR<0.25) in HFpEF Mice Treated with Vehicle or Compound 1

[0240] Pearson correlation coefficient analysis was performed between the expression of genes identified by GSEA analysis and different parameters of LV structure and cardiac diastolic function, including Left ventricular posterior wall thickness at end diastole (LVPWd), and isovolumic relaxation time (IVRT), or mitral valve E/e’ (MV E/e’). The expression levels of several genes associated with fibrosis (Colla2, Col3al, Fbnl, Postn, Clip) were significantly increased in vehicle-treated HFpEF animals with impaired diastolic function as shown by increased LVPWd and IVRT. Compound 1 treatment resulted in reduced expression of fibrosis-associated genes in HFpEF mice with improved diastolic function (FIGs. 11A-11J, Table 3 and Table 4).

Table 3: Echocardiogram Data and the Expression Level of Fibrotic Genes

Vehicle treated healthy animals were used as control.

Table 4: Pearson Correlation Coefficient (r Value) and Statistical Significance (p-Value)

Between Echocardiogram Data and Genes Associated with Fibrosis

[0241] The expression level of genes associated with different subunits of the mitochondrial respiratory electron transport chain, (NADH:Ubiquinone Oxidoreductase subunits; Ndufal3, NdufalS, Ndufa5, Ndufs7, Ndufal, Ndufa8) were significantly reduced in vehicle-treated HFpEF animals with impaired diastolic function as shown by increased LVPWd and MV E/e’. In response to Compound 1 treatment, the expression level of mitochondrial genes increased in HFpEF mice and was positively correlated with improved diastolic function (FIGs. 10A- 10F, Table5 and Table 6).

Table 5: Echocardiogram Data and the Expression Level of Mitochondrial Genes

Healthy animals treated with vehicle were used as control.

Table 6: Pearson Correlation Coefficient (r) And Statistical Significance (p-Value) Between Echocardiogram Data and Genes Associated with Mitochondria

Effects of Compound 1 on Fibrosis Genes in HFpEF Mouse Model by qPCR

[0242] Fibrotic markers periostin (encoded by the Postn gene), collagen 3 Al (encoded by the

Col3al gene), and collagen 1A1 (encoded by the Collal gene), showed a trend in increased expression in HFpEF mouse hearts as measured by qPCR. Compound 1 treatment significantly reduced the expression of fibrotic genes to near healthy control levels (Table 7).

Table 7: mRNA Expression Levels Measured by qPCR

Rep indicates replicate.

Effects of Compound 1 on iPSC-CM Metabolic Status

[0243] The metabolic status was compared of human iPSC-derived CMs treated with Compound 1 (3 pM) to vehicle-treated cells (DMSO). Measurements of basal respiration and reserve respiratory capacity were collected using Seahorse oximetry. While both groups had similar basal respiration rate, Compound 1 -treated human iPSC-derived CMs had higher membrane potential and reserve respiratory capacity, demonstrating greater ATP production, the cells’ ability to respond to energetic stress, and a direct effect on cardiomyocytes (FIGs.

10G and 10H and Table ) Table 8: Metabolic Parameters in Human iPSC-derived CMs.

[0244] Accordingly, Compound 1 treatment was shown to reduce pathogenic transcriptional signatures activated in a mouse model of HFpEF (gene sets include hypertrophy, fibrosis, and platelet-derived growth factor receptor signaling). Targeted gene expression analysis using qPCR confirmed reduced expression of fibrotic genes which correlated with improved diastolic function. Also, based on RNA-Seq data, Compound 1 enriched gene sets associated with mitochondrial energy production in HFpEF mice. The increased expression of mitochondrial genes correlated with an improved HFpEF phenotype. In order to functionally assess the metabolic effects of Compound 1 in vitro, iPSC-derived CMs were used. The data showed increased reserve respiratory capacity in Compound 1 treated human iPSC-derived CMs, indicating improved ATP production in response to metabolic demand.

[0245] Taken together, these results show that HDAC6 selective inhibitors reverse preexisting diastolic dysfunction through multiple pathways in the heart associated with fibrosis and mitochondrial dysfunction, which both contribute to HFpEF pathogenesis. These results also confirm that HDAC6 selective inhibitors have a direct benefit on the heart in HFpEF models, and that the improvements seen are due to multi-modal mechanisms in the heart and are not only as a result of improvement in systemic metabolism and inflammation.

Example 7: Biochemical Activity and Potency of Compound 1

[0246] Compound 1 was synthesized according to methods disclosed in PCT/US2020/066439, published as WO2021127643A1, which is incorporated herein by reference in its entirety. These compounds were tested for potency against HDAC6 and selectivity against HDAC1 in a biochemical assay. A biochemical assay was adopted using a luminescent HDAC-Glo VII assay (Promega) and measured the relative activity of HDAC6 and HDAC1 recombinant proteins. Compounds were first incubated in the presence of HDAC6 or HDAC1 separately, followed by addition of the luminescent substrate. The data was acquired using a plate reader and the biochemical ICso were calculated from the data accordingly. Data is tabulated in Table 9. From these studies, it was determined that the compounds of the present disclosure are selective inhibitors of HDAC6 over HDAC1, providing selectivity ratios from about 5 to about 30,0000.

Table 9. Characterization Data and HDAC6 Activity for Compound 1.

Example 8: Evaluation of Compound 1 in a Phase 1 Clinical Study

[0247] Study Overview: This Phase 1 clinical study was a 2-stage, first-in-human (FIH), randomized, double-blinded, placebo-controlled study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of Compound 1 following single (Stage 1) and multiple (Stage 2) ascending oral doses in healthy adult participants.

[0248] Participants: Compound 1 was evaluated in healthy adults (women and infertile men), 18-60 years of age.

[0249] Study Design: The study was conducted in two stages, with placebo control (Fig. 13).

[0250] Stage 1 : A single ascending dose (SAD) stage evaluated six dosing cohorts (1 mg, 5 mg, 25 mg, 100 mg, 300, and 700 mg of Compound 1 or matching placebo). Healthy adult participants received a single oral dose of Compound 1 or matched placebo under fasting conditions. A demonstration of target engagement (measured by tubulin acetylation in circulating PBMCs) was required to define the initial dose in the second stage of the study. Dosing in each SAD cohort was initiated with a sentinel pair (1 placebo, 1 Compound 1) at least 24 hours before dosing of remaining participants in that cohort. Dosing of the remaining participants was conducted after a safety evaluation of the sentinel pair. All participants in Stage 1 received a single dose of Compound 1 or a matched placebo, blinded and under fasting conditions. Stage 1 participants remained in the study starting at the time of dosing through at least 14 days postdose.

[0251] Stage 2: The multiple ascending dose (MAD) stage evaluated once-daily dosing at 25 mg, 100 mg or 300 mg for 14 consecutive days in 3 cohorts. Healthy adult participants received multiple oral doses of Compound 1 or matched placebo, blinded, for 14 days, under fasting conditions. Dose levels and regimens, either once daily (QD), twice daily (BID), every other day (QOD) or other, were determined based on interim pharmacokinetic analysis from previous cohorts. After initial dosing on Day 1, each subsequent morning dose was administered within ± 1 hour of the dosing time established on Day 1. For BID dosing the evening dose was administered approximately 12 hours following the morning dose. Stage 2 participants remained in the study starting at the time of their first dosing for at least 28 days.

[0252] Participants in each stage were administered Compound 1 orally as a suspension with a total of -240 mL of liquid vehicle.

[0253] Study Objectives

[0254] Primary Objectives (Stage 1): To evaluate the safety and tolerability of single oral ascending doses of Compound 1 in healthy participants. [0255] Secondary Objectives (Stage 1): To characterize the pharmacokinetics of Compound 1 following single oral ascending doses of Compound 1.

[0256] Exploratory Objectives (Stage 1):

• To evaluate the effects of single oral ascending doses of Compound 1 on ECG parameters, including concentration-QT (C-QT) analysis.

• To make a preliminary assessment of the pharmacodynamic activity of Compound 1 and to explore the potential relationships between drug exposure and the safety and pharmacodynamics of single oral ascending doses of Compound 1.

• To explore potential relationships between selected covariates and exposure to single oral ascending doses of Compound 1.

• To identify biomarkers that are associated with susceptibility to developing adverse events (AEs) or can provide evidence of Compound 1 activity (i.e., pharmacodynamic biomarkers) following single oral ascending doses of Compound 1.

[0257] Primary Objectives (Stage 2): To evaluate the safety and tolerability of multiple oral ascending doses of Compound 1 in healthy participants.

[0258] Secondary Objectives (Stage 2): To characterize the pharmacokinetics of Compound 1 following multiple oral ascending doses of Compound 1.

[0259] Exploratory Objections (Stage 2):

• To evaluate the effects of multiple oral ascending doses of Compound 1 on ECG parameters, including C-QT analysis.

• To make a preliminary assessment of the pharmacodynamic activity of Compound 1 and to explore the potential relationships between drug exposure and the safety and pharmacodynamics of multiple oral ascending doses of Compound 1.

• To explore potential relationships between selected covariates and exposure to multiple oral ascending doses of Compound 1.

• To identify biomarkers that are associated with susceptibility to developing AEs or can provide evidence of Compound 1 activity (i.e., pharmacodynamic biomarkers), or that may correlate with response to Compound 1 (i.e., predictive biomarkers), following multiple oral ascending doses of Compound 1. [0260] Subjects: A total of 48 participants (Compound = 36 | placebo = 12) enrolled in Stage 1 (SAD) and 24 participants (Compound 1 = 18 | placebo = 6) in Stage 2 (MAD) (Table 10).

Table 10. Baseline characteristics

[0261] Treatments Administered

[0262] Drug Product: The Compound 1 drug product was a unit dose of Compound 1 reconstituted in a liquid and administered as an oral rinse. The procedure for preparing the drug product included the following:

• On the day of dosing, the Compound 1 unit dose was reconstituted with 240 mL of a commercially available liquid vehicle (Ora-Sweet SF® or Ora-Blend SF®), mixed, and orally administered to the study participant.

• An additional volume of liquid vehicle was used as a rinse, and orally administered to the study participant to ensure the complete dose was administered. The total volume of liquid vehicle ingested by a study participant was approximately 8 oz.

[0263] Placebo: The matched placebo was a unit dose of Hypromellose Acetate Succinate- MF grade; (HPMCAS-MF). The matched placebo was prepared and administered to study participants in a manner identical to the Compound 1 drug product described above.

[0264] Safety and Tolerability Endpoints: Endpoints were measured as the number and severity of treatment-emergent adverse events (TEAEs) following a single oral dose of Compound 1 and placebo in Stage 1 and following multiple oral doses in Stage 2. Secondary and exploratory clinical endpoints including pharmacodynamic and safety biomarkers and pharmacokinetic measures are described in more detail below. [0265] Pharmacodynamic and Safety Biomarkers

[0266] Biomarkers were measured throughout Stage 1 and Stage 2.

[0267] Primary biomarkers analyzed included tubulin acetylation levels and histone acetylation levels.

[0268] Extended and exploratory biomarkers included Elb Ale, glucose, insulin levels, cardiac biomarkers (e.g., NT -proBNP and hs-cTnl levels), metabolic biomarkers (e.g., C-peptide, adiponectin, fructosamine, ketone bodies, lipid panel, and Lp[a]), inflammatory and fibrosis markers (e.g., Hs-CRP, IL-6, and Pro-C6), endocrine markers (e.g., testosterone, LH, FSH and prolactin (for males only), and additional pharmacodynamic biomarkers involved in the Compound 1 pathway. For stage 2 only, a glucose tolerance test and fat pad biopsy was also conducted.

[0269] Safety and Tolerability Results of Compound 1

• Compound 1 was well tolerated at the doses evaluated

• No serious adverse events or dose-limiting toxicities were reported

• Frequency of adverse events (AEs) in Compound 1 -treated participants did not increase with dose

• Findings in SAD and MAD stages of the study were largely similar

• Most frequent AEs observed were related to GI disturbance

• Frequency of GI AEs were similar between Compound 1- and placebo-treated participants, and across dose groups, suggesting that such findings were likely due to the vehicle used to administer Compound 1 or placebo

• Compound 1 did not result in any concerning patterns of cardiovascular AEs (including on telemetry, ECGs, and vital signs) across the dose ranges evaluated

• Compound 1 administration did not result in hematologic findings as have been reported with other HDAC6 inhibitors

• AEs of dizziness and blurred vision were considered not related to study drug

[0270] Pharmacokinetic Analyses: Pharmacokinetic parameters for Compound 1 were assessed in plasma at Stage 1 and Stage 2 and in urine at Stage 2 only. These parameters were analyzed using standard non-compartmental analysis methods. Measures from both stages were used for the analysis of dose proportionality, as well as dose selection and escalation. A more detailed summary of the pharmacokinetic measures analyzed is included below. [0271] Pharmacokinetic Measures

[0272] Plasma Stage 1 : Analytical methods included calculating area under the concentrationtime curve, from time 0 to 24 hours (AUC0-24), time 0 to the last observed non-zero concentration (AUCo-t), and time 0 extrapolated to infinity (AUCo-inf). Additional calculations included percent of AUCo-inf extrapolated (AUC%/extrap), apparent total plasma clearance after oral (extravascular) administration (CL/F), maximum observed concentration (Cmax), mean residence time extrapolated to infinity (MRTo-inf), lag time between drug administration and the onset of absorption (Tiag), time to reach Cmax (Tmax), apparent first-order terminal elimination rate constant (Xz) and half-life (tU), and apparent volume of distribution during the terminal elimination phase after oral (extravascular) administration (Vz/F).

[0273] Plasma Stage 2: Analytical methods varied by time point and dosing schedule at Day 1, Days 2-13, and Day 14. Calculations performed at Day 1 dosing included AUC0-12 (if BID dosing), AUCO-24 (if QOD dosing), Cmax and Tmax. Calculations performed between Days 2 and 13 included concentration at end of dosing interval (Ctrough) and at 4 hours on Days 2-6 (C4). Calculations performed at Day 14 included AUC during a dose interval (tau) after multiple doses (AUCtau), average observed plasma concentration (Cavg), apparent total plasma clearance after oral (extravascular) administration after multiple doses (CLss/F), Cmax after multiple doses (Cmax,ss), minimum observed concentration after multiple doses (Cmin,ss), Tmax after multiple doses (Tmax,ss), and time to reach Cmin after multiple doses (Tmin,ss), percent peak- to-trough fluctuation (%FLUC), mean residence time at steady state (MRTss), accumulation ratio calculated from AUCtau (RA, AUCtau, ss) and Cmax (RA, Cmax, ss) after multiple doses, and Az and tU.

[0274] Urine Stage 2: Analytical methods were performed on Days 1 and 14 for the highest dose tested in Stage 2. Calculations included the amount of unchanged drug excreted in the urine collection interval from tl to t2 (Aetl-t2), cumulative amount of unchanged drug excreted in the urine over the entire period of sample collection (CumAe), renal clearance (CL_r), and fraction of administered dose excreted unchanged in urine (Fe).

[0275] Dose proportionality was evaluated for Compound 1 AUC and Cmax parameters on Day 1 of Stage 1 and Days 1 and 14 of Stage 2 using the power model approach. Steady state analyses were performed in Stage 2. A PK/PD model was developed during the study to support dose selection and escalation. [0276] Pharmacokinetics Results (SAD): Plasma exposure generally increased proportionally with Compound 1 dose across the dose ranges evaluated (Fig. 14). At doses where the concentration-time profile was fully characterized (25 mg - 700 mg), terminal elimination half-life ranged from 8.13 hours (SD ± 2.95) to 14.6 hours (SD ± 6.04), suggesting that once-daily dosing was appropriate for MAD stage of the study.

[0277] Pharmacokinetics Results (MAD): Exposure increased with once-daily dosing at each dose evaluated (Fig. 15); reaching steady state by approximately Day 10. Mean (±SD) terminal half-life after last dose ranged from 9.27 hours (±1.5) to 13.7 hours (±3.35) suggesting that once-daily dosing remains appropriate for future clinical trials (Table 11).

Table 11. Summary of Compound 1 Pharmacokinetic Parameters (Day 14) from MAD

Stage of Phase 1 Study

Cmax, AUC, and accumulation ratio shown as geometric mean and geometric SD. Tmax shown as median values; half-life shown as arithmetic mean and SD.

[0278] Pharmacodynamics (MAD + SAD)

[0279] Background: HDAC6 deacetylates cytoplasmic proteins such as acetylated alpha tubulin, resulting in de-acetylation of lysine residues in the protein. When HDAC6 activity is inhibited by Compound 1, acetylated-lysine 40 (K40) tubulin accumulates in cells. As a pharmacodynamic biomarker of HDAC6 inhibition after Compound 1 treatment in vivo, acetyl- K40 tubulin levels were measured in peripheral blood mononuclear cell (PBMC) extracts using ligand binding assays developed to acetyl-tubulin and total tubulin on the Meso Scale Discovery (MSD) platform.

[0280] Assay: Phase 1 study participants were treated with Compound 1 or placebo under single- or repeat-dosing regimens. Peripheral blood was collected for pharmacodynamic analysis at designated times before and after dosing. PBMCs were isolated from the blood and cryopreserved until analysis.

[0281] For acetyl-tubulin analysis, PBMCs were thawed and lysed with MSD lysis buffer. Lysates were normalized to protein and run on the acetyl tubulin and total tubulin assays. Briefly, 96-well assay plates were coated overnight with anti-tubulin antibody as the capture antibody and blocked the following morning. Blanks, controls and study samples were loaded on the assay plate normalized to protein, with half of the wells plated for acetyl-tubulin and half of the wells for total tubulin analysis of the same samples. After overnight incubation, wells were incubated with either acetyl-tubulin antibody or a second total tubulin antibody for detection. Plates were incubated with the SULFO-TAG detection reagent and MSD Read Buffer and read on an MSD plate reader.

[0282] For normalization of data for each subject, acetyl-tubulin MSD signal for each time point was divided by the MSD signal for the Day 1 pre-dose sample, resulting in relative fold change from baseline. The same analysis was performed for total tubulin levels and assessed for fold change from baseline. In the single ascending dose and multi-ascending dose phases of the study, acetyl-tubulin levels increased with increasing Compound 1 dose.

[0283] To monitor the specificity of Compound 1 in vivo in the study subjects, blood was collected pre- and post-dose to measure acetylated histone levels, an indication of inhibition of nuclear HD AC isoforms. The assay procedures for acetyl histone H3 (H3K9) and total histone were similar to the tubulin assays, with the exception that preparation of lysates required a histone extraction kit in order to lyse nuclei more completely to access histone proteins. Data processing for fold change from baseline was identical to the tubulin procedures. No increases in histone H3 acetylation were detected at any of the doses in the Phase 1 study indicating no inhibition of other HD AC isoforms by Compound 1 in vivo.

[0284] Results: Acetylated tubulin levels in peripheral blood mononuclear cells (PBMCs) from Compound 1 treated participants were higher than those in placebo-treated participants starting at doses as low as 5 mg (Fig. 16A) indicating engagement with HDAC6 target. Maximal acetylated tubulin levels increased with Compound 1 dose (Fig. 16A) suggesting robust dose-dependent target engagement. Maximal acetylated tubulin levels on Day 14 were higher than those on Day 1 (Fig. 16B). Duration of effect at higher SAD doses and in MAD stage lasted 24-48 hours (Figs. 16A and 16B). In mouse HFpEF models, maximal efficacy was observed at 3 mg/kg; which corresponded to about half of maximal acetylated tubulin levels. In addition, in mouse models, acetylated tubulin levels above baseline were required for 6-8 hours post-dose to reach maximal efficacy; not throughout 24 hour dosing interval. Therefore, Compound 1 doses approximately in the low-to-mid range evaluated in this study (approximately 25 mg - 100 mg once daily) may meet or exceed plasma exposures and target engagement observed preclinically for maximal efficacy.

[0285] Increasing Compound 1 exposure in the SAD and MAD studies correlated with increasing pharmacodynamic effect (Fig. 17) as measured by mean tubulin acetylation.

[0286] No changes or trends in acetylated histone relative to pre-dose were observed in any SAD or MAD cohorts (Fig. 18).

[0287] Conclusions: Compound 1 is a highly selective, orally bioavailable HDAC6 inhibitor under development as a potential treatment for patients with HFpEF. Compound 1 is well tolerated in healthy adult participants after single (1 mg - 700 mg) and multiple once-daily doses (25/100/300 mg x 14 d). Adverse events observed were of similar frequency between Compound 1 treated and placebo groups. Frequency of AEs did not increase with Compound 1 dose. Compound 1 dosing did not change activity of other HDAC isoforms; distinguishing this agent from other HDAC6 inhibitors. Plasma exposure generally increased proportionally with Compound 1 dose. Robust HDAC6 inhibition (target engagement) was observed by increasing acetylated tubulin levels at Compound 1 doses as low as 5 mg. Plasma exposures and target engagement observed in this study met or exceeded levels observed preclinically at maximal (3mg/kg dose) or near maximal (0.3 mg/kg dose) efficacy in a mouse HFpEF model. Results of this study support further evaluation of Compound 1 in HFpEF patients with once- daily dosing (roughly in 25 mg - 100 mg dose range).

Example 9: Evaluation of Heart-Function Protection of Compound 1 in MLP^KO Mice

[0288] Schematic of drug treatment in MLP^KO mouse model is provided in FIG. 20A. Compound 1 was administered daily by oral gavage at 30 mg/kg starting when mice were 1.5 months of age.

[0289] As shown in FIG. 20B, immunostaining of iPSC-CMs treated with Compound 1 (5.5pM) results in hyper-Ac-Tubulin. Scale bar = 200 pm.

[0290] The evaluation of biochemical selectivity in FIG. 20C confirms that Compound 1 is highly selective (3500-fold) for HDAC6 over other HDACs.

[0291] Western blot of iPSC-CMs treated with Compound 1 stained with monoclonal anti-Ac- Lysine. Givinostat (Giv; pan-HDAC inhibitor control) showed both on-target (Ac-Tubulin stain) and off-target (Ac-Histone H3 and H4 stain) activity. Compound 1 only shows specific on-target activity with no detectable off-target activity (FIG. 20D).

[0292] (FIG. 20E) Daily dosing of Compound 1 protected heart function during the 9-week dosing period as measured by ejection faction. Error bars = SEM. *P < 0.05, **P < 0.01.

[0293] (FIG. 20F) Ejection fraction was tracked from the first day of dosing, and delta ejection fraction was measured. Mice treated with Compound 1 during the 9-week period show a 4.0% decline, whereas it dropped by 14.8% in the vehicle-treated arm. Error bars = SEM. *P < 0.05, **P < 0.01.

[0294] Ejection fraction (FIG. 20G) and delta ejection fraction (FIG. 20H) (compared to the pre-dose baseline) at 15 weeks of age and 9 weeks of dosing shows Compound 1 protects against declining heart function in MLP^KO mice. Error bars = SEM. **P < 0.01.

[0295] Left ventricular internal diameter at diastole (LVIDd) (FIG. 201) and systole (LVIDs) (FIG. 20J) were reduced by Compound 1 in MLP^KO mice. Error bars = SEM.

Example 10: Evaluation of Compound 1 in a Phase 2 Clinical Study

[0296] Fig. 19 provides a potential Phase 2 clinical trial design in patients with heart failure with preserved ejection fraction (HFpEF).

[0297] Study Goals

• Establish proof of concept (POC) using clinically approvable endpoints such as a six- minute walking distance test (6MWD) and Kansas City Cardiomyopathy Questionnaire (KCCQ)

• Determine a dose for a Phase 3 clinical trial

[0298] Study Design

• Part A - Dose ranging randomized study to select the appropriate dose (low, mid, or high) of Compound 1.

• Part B - Confirmatory POC comparing the Compound 1 dose from Part A to placebo. o Interim analysis includes an early stoppage point based on efficacy and a reestimation of sample size. INCORPORATION BY REFERENCE

[0299] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

What is claimed is:

1. A method of treating a disease or condition responsive to HDAC6 inhibition in a patient in need thereof, comprising orally administering to the patient once daily about 10 mg to about 300 mg of Compound 1 having the formula:

or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein about 25 mg to about 200 mg of Compound 1 is administered to the patient.

3. The method of claim 1 or 2, wherein about 25 mg to about 100 mg of Compound 1 is administered to the patient.

4. The method of claim 1 or 2, wherein about 25 mg, about 50 mg, or about 100 mg of Compound 1 is administered to the patient.

5. The method of claim 1 or 2, wherein about 25 mg, about 75 mg, or about 200 mg of Compound 1 is administered to the patient

6. The method of claim 1 or 2, wherein about 10 mg, about 25 mg or about 75 mg of Compound 1 is administered to the patient.

7. The method of claim 1 or 2, wherein about 10 mg of Compound 1 is administered to the patient.

8. The method of claim 1 or 2, wherein about 25 mg of Compound 1 is administered to the patient.

9. The method of claim 1 or 2, wherein about 50 mg of Compound 1 is administered to the patient.

10. The method of claim 1 or 2, wherein about 75 mg of Compound 1 is administered to the patient.

11. The method of claim 1 or 2, wherein about 100 mg of Compound 1 is administered to the patient.

12. The method of claim 1 or 2, wherein about 125 mg of Compound 1 is administered to the patient.

13. The method of claim 1 or 2, wherein about 150 mg of Compound 1 is administered to the patient.

14. The method of claim 1 or 2, wherein about 200 mg of Compound 1 is administered to the patient.

15. The method of claim 1 or 2, wherein about 250 mg of Compound 1 is administered to the patient.

16. The method of claim 1 or 2, wherein about 300 mg of Compound 1 is administered to the patient.

17. The method of any one of claims 1-16, wherein the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 80 ng/mL to about 1700 ng/mL following administration of about 25 mg to about 300 mg of Compound 1.

18. The method of any one of claims 1-16, wherein the administration provides a maximum blood plasma concentration (Cmax) of Compound 1 of about 80 ng/mL to about 720 ng/mL following administration of about 25 mg to about 100 mg of Compound 1.

19. The method of any one of claims 1-18, wherein the administration provides an area under the curve (AUC) of Compound 1 of about 640 h*ng/mL to about 20400 h*ng/mL following administration of about 25 mg to about 300 mg of Compound 1.

20. The method of any one of claims 1-18, wherein the administration provides an area under the curve (AUC) of Compound 1 of about 640 h*ng/mL to about 6500 h*ng/mL following administration of about 25 mg to about 100 mg of Compound 1.

21. The method of any one of claims 1-20, wherein the patient has heart failure or is at risk of heart failure.

22. The method of any one of claims 1-21, wherein the patient has Class I heart failure as classified by the New York Heart Association (NYHA) Functional Classification.

23. The method of any one of claims 1-22, wherein the patient has Class II heart failure as classified by the New York Heart Association (NYHA) Functional Classification.

24. The method of any one of claims 1-22, wherein the patient has Class III heart failure as classified by the NYHA Functional Classification.

25. The method of any one of claims 1-24, wherein the patient has an ejection fraction of greater than about 45%.

26. The method of any one of claims 1-25, wherein the patient has an N-terminal pro b- type natriuretic peptide (NT-proBNP) level in the blood of greater than about 400 pg/mL.

27. The method of any one of claims 1-25, wherein the patient has atrial fibrillation and an N-terminal pro b-type natriuretic peptide (NT-proBNP) level in the blood of greater than about 900 pg/mL.

28. The method of any one of claims 1-27, wherein the patient is obese or is at risk for obesity.

29. The method of any one of claims 1-28, wherein the patient has or is at risk for hypertension.

30. The method of any one of claims 1-29, wherein the patient has or is at risk for diabetes mellitus.

31. The method of any one of claims 1-30, wherein the patient has or is at risk for coronary artery disease (CAD).

32. The method of any one of claims 1-31, wherein the patient has or is at risk for heart disease.

33. The method of any one of claims 1-32, wherein the patient has or is at risk for valvular heart disease.

34. The method of any one of claims 1-33, wherein the patient has or is at risk for atrial fibrillation.

35. The method of any one of claims 1-34, wherein the patient has a diastolic dysfunction of Grade II, III, or IV.

36. The method of any one of claims 1-35, wherein Compound 1 is administered as an adjunctive to a sodium-glucose Cotransporter-2 (SGLT2) inhibitor.

37. The method of claim 36, wherein the SGLT2 inhibitor is canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, or bexagliflozin.

38. The method of any one of claims 1-37, wherein treatment provides a decrease of NT -proBNP levels in the blood of the patient of about 5% to about 50%.

39. The method of any one of claims 1-38, wherein treatment provides the patient improving from Class III to Class II in the NYHA Functional Classification.

40. The method of any one of claims 1-38, wherein treatment provides the patient improving from Class III to Class I in the NYHA Functional Classification.

41. The method of any one of claims 1-38, wherein treatment provides an improvement in the patient’s NYHA Functional Classification from Class II to Class I.

42. The method of any one of claims 1-41, wherein treatment provides about a 5 mL/m² to about a 20 mL/m² decrease in left atrial (LA) volume index.

43. The method of any one of claims 1-42, wherein treatment provides an improvement of least 5 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score.

44. The method of any one of claims 1-42, wherein treatment provides an improvement of least 10 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score.

45. The method of any one of claims 1-42, wherein treatment provides an improvement of least 20 points in the patient’s Kansas City Cardiomyopathy Questionnaire (KCCQ) score.

46. The method of any one of claims 1-45, wherein treatment provides improved performance in a standardized 6-minute walk test.

47. The method of any one of claims 1-46, wherein treatment provides an improvement in exercise capacity as measured by cardiopulmonary exercise testing.

48. The method of any one of claims 1-47, wherein treatment provides an improvement in pulmonary venous oxygen tension (pVO2) of 1-10 mL/kg/min.

49. The method of any one of claims 1-48, wherein treatment results in a reduction in the relative risk of heart failure hospitalization of about 5% to about 40%.

50. The method of any one of claims 1-49, wherein treatment results in a reduction in the relative risk of cardiovascular disease (CVD) death of about 5% to about 25%.

51. The method of any one of claims 1-50, wherein treatment results in an increase in overall survival.

52. The method of any one of claims 1-51, wherein treatment provides the patient exhibiting an increase in heart pump function as measured by echocardiogram (ECG).

53. The method of any one of claims 1-52, wherein treatment provides the patient exhibiting no atrial or ventricular arrythmias when comparing baseline ECG monitoring with 72-hour monitoring.

54. The method of any one of claims 1-53, wherein at least a 100% (2 -fold) increase in the patient’s mean acetylated tubulin level over time compared to baseline level is observed about 2-4 hours after a daily dose is administered.

55. The method of any one of claims 1-54, wherein about a 50% to about a 75% increase in the patient’s acetylated tubulin concentration over time (AUC) compared to baseline concentration is observed after administration of 25 mg to 300 mg of the compound.

56. The method of any one of claims 1-55, wherein treatment provides about a 10 mg/dL to about a 50 mg/dL reduction in fasting plasma glucose.

57. The method of any one of claims 1-56, wherein treatment provides about a 0.3% to about a 1.5% reduction in Ale level.

58. The method of any one of claims 1-57, wherein treatment provides about 1 kg to about a 5 kg drop in body weight.

59. The method of any one of claims 1-58, wherein the disease or condition responsive to HDAC6 inhibition is a metabolic disease or disorder.

60. The method of claim 59, wherein the metabolic disease or disorder is diabetes, prediabetes, diabetic cardiomyopathy, metabolic syndrome, hypertension, hypertriglyceridemia, or dyslipidemia.

61. The method of claim 59, wherein the metabolic disease or disorder is disease or disorder associated with obesity, optionally diet-induced obesity.

62. The method of any one of claims 1-58, wherein the disease or condition responsive to HDAC6 inhibition is heart failure with preserved ejection fraction (HFpEF).

63. The method of any one of claims 1-58, wherein the disease or condition responsive to HDAC6 inhibition is dilated cardiomyopathy.

64. The method of any one of claims 1-63, comprising administering to the patient a composition comprising Compound 1 or pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients.

65. The method of claim 64, wherein the composition is administered for a plurality of weeks.

66. The method of claim 64 or 65, wherein the composition is administered for a period ranging from 4 weeks to 5 years or more.

67. The method of any one of claims 64-66, wherein the composition is administered for a period of at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 18 weeks, at least 24 weeks, at least 30 weeks, at least 36 weeks, at least 42 weeks, at least 48 weeks, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

68. The method of any one of claims 64-67, wherein the composition is in the form of a solid dosage form.

69. The method of claim 68, wherein the solid dosage form is a tablet or capsule.