Methods of treating left ventrÍcular diastolic dysfunction (LVDD) and heart failure with preserved ejection fraction (HFPEF)

Abstract

This invention relates to a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject afflicted with HFpEF, the method comprising administering to the subject an aldosterone synthase inhibitor once per day in an amount effective to treat HFpEF in the subject. This invention also relates to a method of treating left ventricular diastolic dysfunction (LVDD) in a subject afflicted with LVDD, the method comprising administering to the subject an aldosterone synthase inhibitor once per day in an amount effective to treat LVDD in the subject.

Description

Docket: 92506-A-PCT / GJG / DHMETHODS OF TREATING LEFT VENTRICULAR DIASTOLIC DYSFUNCTION (LVDD) AND HEART FAILURE WITH PRESERVED EJECTION FRACTION (HFPEF)

[0001] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.BACKGROUND OF THE INVENTION

[0002] Left ventricular diastolic dysfunction (LVDD) is a condition characterized by impaired relaxation and compliance of the left ventricle (LV). While chronic hypertension causes elevated LV end-diastolic pressure and LV hypertrophy, contributing to diastolic dysfunction, neurohormonal activation and genetic factors may also influence its onset (Castiglione 2023).

[0003] Heart failure with preserved ejection fraction (HFpEF) is a form of heart failure in which the “ejection fraction” — the percentage of the volume of blood the left ventricle of the heart pumps out with each contraction — is normal. In those afflicted with HFpEF, the left ventricle of the heart is stiffened and has impaired relaxation after pumping blood out of the heart.

[0004] Diverse mechanisms contribute to the development of HFpEF and LVDD, many of which are under-investigated and remain obscure.

[0005] There is no cure for HFpEF and antihypertensive therapies have inconsistent effects on LVDD and HFpEF. Moreover, subjects may be afflicted with hypertension without being afflicted with LVDD and HFpEF, and subjects may be afflicted with LVDD and HFpEF without being afflicted with hypertension. Thus, the effectiveness of new therapies in preventing or managing LVDD and HFpEF cannot be predicted based solely on the effectiveness of these therapies in treating hypertension.

[0006] There is therefore a need for new treatments for LVDD and HFpEF.BRIEF SUMMARY OF THE INVENTION

[0007] This invention relates to a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject afflicted with HFpEF, the method comprising administering to thesubject an aldosterone synthase inhibitor once per day in an amount effective to treat HFpEF in the subject.

[0008] This invention also relates to a method of treating left ventricular diastolic dysfunction (LVDD) in a subject afflicted with LVDD, the method comprising administering to the subject an aldosterone synthase inhibitor once per day in an amount effective to treat LVDD in the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1: Example 1 Study Timeline.

[0010] Figure 2: Graph of body weight over time of study groups in Example 1, showing that lorundrostat had no effect on body weight. ZSF1 -obese rate body weight was in line with the literature.

[0011] Figure 3: Aldosterone levels of study groups at baseline and Week 6 in Example 1.

[0012] Figure 4: Left Ventricular Diastolic Diameter (LVEDD) of Example 1 study groups overtime. ZSF1 -obese control rats demonstrated increased LVEDD, while ZSF1 -obese rats treated with lorundrostat (75mg / kg) exhibited stabilized LVEDD.

[0013] Figure 5: Left Ventricular End Systolic Diameter (LVESD) of Example 1 study groups overtime. ZSF1 -obese control rats demonstrated increased LVESD, while ZSF1 -obese rats treated with lorundrostat (75mg / kg) exhibited stabilized LVESD.

[0014] Figure 6: Left Ventricular End Diastolic Volume (LVEDV) of Example 1 study groups overtime. ZSF1 -obese control rats demonstrated increased LVEDV, while ZSF1 -obese rats treated with lorundrostat (75 mg / kg) exhibited stabilized LVEDV.

[0015] Figure 7: Left Ventricular End Systolic Volume (LVESV) of Example 1 study groups over time. ZSFl-obese control rats demonstrated increase LVESV, while ZSFl-obese rats treated with lorundrostat (75 mg / kg) exhibited stabilized LVESV.

[0016] Figure 8: Mitral Valve E / E’ ratio (MV E / E’ ratio) of Example 1 study groups over time. ZSFl-obese control rats demonstrated increased MV E / E’ ratio. By contrast, lorundrostat (50 mg / kg and 75 mg / kg) prevented an increase in MV E / E’ ratio ZSFl-obese rats.

[0017] Figure 9: Mitral Valve E / A ratio (MV E / A ratio) of Example 1 study groups over time. ZSF1 obese control rats demonstrated increased MV E / A Ratio. By contrast, lorundrostat (50 and 75mg / kg) prevented an increase in MV E / A Ratio.

[0018] Figure 10: Fractional shortening (FS) of Example 1 study groups over time. Lorundrostat exhibited no effect on Fractional Shortening, with all study groups exhibiting FS within the normal range.

[0019] Figure 11: Ejection fraction (EF) of Example 1 study groups over time. Lorundrostat exhibited no effect on EF, with all study groups exhibiting EF within the normal range.

[0020] Figure 12: Left Ventricular Anterior Wall Thickness of Example 1 study groups over time. Lorundrostat exhibited no effect on Left Ventricular Anterior Wall Diastole (LVAWd) or Left Ventricular Anterior Wall Systole (LVAWs) relative to control.

[0021] Figure 13: Left Ventricular Posterior Wall Thickness of Example 1 study groups over time. Lorundrostat exhibited no effect on Left Ventricular Posterior Wall Diastole (LVPWd) or Left Ventricular Posterior Wall Systole (LVPWs) relative to control.

[0022] Figure 14: Blood glucose levels of Example 1 study groups over time. Lorundrostat stabilized blood glucose levels relative to the control group.

[0023] Figure 15: Levels of endothelial dysfunction related biomarkers (Endothelin-1, and Intercellular adhesion molecule-1) of Example 1 study groups at week 12. Endothelial dysfunction related biomarkers were increased in obese ZSF1 rats and dose dependent endothelial function improvement was observed in the study groups treated with lorundrostat.

[0024] Figure 16: Aldosterone concentration of Example 1 study groups at week 12.

[0025] Figure 17: Example 2 study timeline.

[0026] Figure 18: E / A ratio measured in rats of Example 2 at Week 12 (W12) and Week 18 (W18).

[0027] Figure 19: E7A’ ratio measured in rats of Example 2 at Week 12 (W12) and Week 18 (W18).

[0028] Figure 20: Isolvolumic relaxation time (IVRT) measured in rats of Example 2 at Week 12 (W12) and Week 18 (W18).

[0029] Figure 21: Summary of Study 1 and Study 2 serum analyte analysis.

[0030] Figure 22: Serum analysis of Study 1 and Study 2 subjects show that lorundrostat is associated with significant reductions in candidate causal risk biomarkers for heart failure. * how many circulating biomarkers reported in the study. #: MR: whether risk biomarkers are supported by Mendelian RandomizationDETAILED DESCRIPTION OF THE INVENTION

[0031] This invention relates to a method of treating left ventricular diastolic dysfunction (LVDD) in a subject afflicted with LVDD, the method comprising administering to the subject an aldosterone synthase inhibitor once per day in an amount effective to treat LVDD in the subject.

[0032] In embodiments, treating LVDD comprises:(a) increasing the subject’s E / A ratio relative to the subject’s E / A ratio prior to treatment with the aldosterone synthase inhibitor and / or relative to the subject’s E / A ratio in the absence of treatment with the aldosterone synthase inhibitor; (b) increasing the subject’s E’ / A’ ratio relative to the subject’s E’ / A’ ratio prior to treatment with the aldosterone synthase inhibitor and / or relative to the subject’s E’ / A’ ratio in the absence of treatment with the aldosterone synthase inhibitor; and / or(c) increasing the subject’s isovolumic relaxation time (IVRT) relative to the subject’s IVRT prior to treatment with the aldosterone synthase inhibitor and / or relative to the subject’s IVRT in the absence of treatment with the aldosterone synthase inhibitor.

[0033] In embodiments, the subject is afflicted with both LVDD and heart failure with preserved ejection fraction (HFpEF), preferably wherein the aldosterone synthase inhibitor is administered to the subject in an amount effective to treat both LVDD and HFpEF in the subject.

[0034] In embodiments, the subject is afflicted with LVDD, HFpEF, and hypertension.

[0035] In embodiments, treating HFpEF comprises:(a) stabilizing the subject’s Left Ventricular Diastolic Diameter (LVEDD) relative to the LVEDD of a subject not treated with the aldosterone synthase inhibitor;(b) stabilizing the subject’s Left Ventricular End Systolic Diameter (LVESD) relative to the LVESD of a subject not treated with the aldosterone synthase inhibitor; (c) stabilizing the subj ect’ s Left Ventricular End Diastolic Volume (LVEDV) relative to the LVEDV of a subject not treated with the aldosterone synthase inhibitor;(d) stabilizing the subject’s Left Ventricular End Systolic Volume (LVESV) relative to the LVESV of a subject not treated with the aldosterone synthase inhibitor; (e) preventing an increase in the subject’s Mitral Valve (MV) E / E’ ratio or reducing the subject’s Mitral Valve (MV) E / E’ ratio relative to the MV E / E’ ratio of a subject not treated with the aldosterone synthase inhibitor;(f) preventing an increase in the subject’s MV E / A’ ratio or reducing the subject’s MV E / A’ ratio relative to the MV E / A’ ratio of a subject not treated with the aldosterone synthase inhibitor;(g) reducing levels of endothelial dysfunction related biomarkers Endothelin-1 and Intercellular adhesion molecule- 1 relative to the levels of Endothelin-1 and Intercellular adhesion molecule- 1 of a subject not treated with the aldosterone synthase inhibitor; and / or(h) reducing levels of one or more biomarkers associated with HFpEF, preferably wherein the one or more biomarkers are selected from the group consisting of: MF AIM, SVEPl: EGF-like domains 4-6; N-terminal pro-BNP, SVEP: Sushi 15-18; NRP1, Spondin-1, ITIH3, BNP, and IGFBP-7 relative to the levels of said one or more biomarkers of a subject not treated with the aldosterone synthase inhibitor.

[0036] In embodiments, treating HFpEF comprises reduction in the frequency and / or likelihood of hospitalization for heart failure (HHF), preferably wherein the reduction is a reduction in the subject’s odds ratio (OR) of HHF of at least 10%, at least 20%, at least 50%, 10- 20%, 20-50%, or 50-75%.

[0037] In embodiments, treating HFpEF comprises reduction in the likelihood of cardiovascular (CV) death, preferably wherein the reduction is a reduction in the subject’s odds ratio (OR) of CV death of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0038] In embodiments, treating HFpEF comprises reduction in the frequency or likelihood of heart failure (HF) events, preferably wherein the reduction is a reduction in the subject’s odds ratio (OR) of HF events of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0039] In embodiments, treating HFpEF comprises improvement in the subject’s exercise capacity as measured by an increase in the subject’s six-minute walk distance, preferably wherein the improvement is by at least 5 meters, more preferably by at least 10 meters.

[0040] In embodiments, treating HFpEF comprises an increase in the subject’s peak oxygen consumption, preferably wherein said increase is by at least 2.5 mL / kg / min, by at least 5 mL / kg / min, by at least 10 mL / kg / min, by 2.5-5 mL / kg / min, 5-10 mL / kg / min, or 10-20 mL / kg / min.

[0041] In embodiments, treating HFpEF comprises improvement in the subject’s Kansas City Cardiomyopathy Questionnaire (KCCQ) Clinical Summary Score (CSS), preferably wherein the improvement is by at least 5 points, by at least 10 points, by at least 20 points, by 5-10 points, by 10-20 points, or by 20-50 points.

[0042] This invention relates to a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject afflicted with HFpEF, the method including administering to the subject an aldosterone synthase inhibitor once per day in an amount effective to treat HFpEF in the subject.

[0043] In some embodiments the subject is afflicted with both hypertension and HFpEF.

[0044] In some embodiments treating HFpEF includes reduction in the frequency and / or likelihood of hospitalization for heart failure (HHF), preferably wherein the reduction is a reduction in the subject's odds ratio (OR) of HHF of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0045] In some embodiments treating HFpEF includes reduction in the likelihood of cardiovascular (CV) death, preferably wherein the reduction is a reduction in the subject's odds ratio (OR) of CV death of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0046] In some embodiments treating HFpEF includes reduction in the frequency or likelihood of heart failure (HF) events, preferably wherein the reduction is a reduction in the subject's odds ratio (OR) of HF events of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%..

[0047] In some embodiments treating HFpEF includes improvement in the subject's exercise capacity as measured by an increase in the subject's six-minute walk distance, preferably wherein the improvement is by at least 5 meters, more preferably by at least 10 meters.

[0048] In some embodiments treating HFpEF includes an increase in the subject's peak oxygen consumption, preferably wherein said increase is by at least 2.5 mL / kg / min, by at least 5 mL / kg / min, by at least 10 mL / kg / min, by 2.5-5 mL / kg / min, 5-10 mL / kg / min, or 10-20 mL / kg / min.

[0049] In some embodiments treating HFpEF includes improvement in the subject's Kansas City Cardiomyopathy Questionnaire (KCCQ) Clinical Summary Score (CSS), preferably wherein the improvement is by at least 5 points, by at least 10 points, by at least 20 points, by 5-10 points, by 10-20 points, or by 20-50 points.

[0050] In some embodiments, the aldosterone synthase inhibitor is administered in an amount effective to treat left ventricular diastolic dysfunction (LVDD) in the subject.

[0051] In some embodiments the aldosterone synthase inhibitor is administered to the subject in an amount sufficient to inhibit 50% or more of aldosterone synthase's activity for between 8 and 18 hours of a 24-hour period, preferably wherein the amount is sufficient to inhibit 50% or more of aldosterone synthase's activity for between 10 and 14 hours of a 24-hour period.

[0052] In some embodiments the aldosterone synthase inhibitor is administered to the subject once per day in the morning.

[0053] In some embodiments the aldosterone synthase inhibitor is administered to the subject once per day in the evening.

[0054] In some embodiments the aldosterone synthase inhibitor is administered to the subject daily: (a) for at least one week; (b) for at least two weeks; (c) for at least four weeks; (d) for at least eight weeks; (e) for at least twelve weeks; (f) for at least twenty-four weeks; (g) for at least fifty weeks; (h) for at least one hundred weeks; or (i) for at least two hundred weeks, to thereby treat HFpEF in the subject.

[0055] In some embodiments the aldosterone synthase inhibitor is lorundrostat or a pharmaceutically acceptable salt thereof.

[0056] In some embodiments the amount of the lorundrostat or pharmaceutically acceptable salt thereof is 25-100 mg per day, preferably 25-75 mg per day, more preferably 40-60 mg per day, even more preferably 50 mg per day.

[0057] In some embodiments the lorundrostat or pharmaceutically acceptable salt thereof is the monohydrobromide salt of lorundrostat.

[0058] In some embodiments the method further comprising administering to the subject a GLP-1 treatment concurrently or concomitantly.

[0059] In some embodiments the GLP-1 treatment is selected from Dulaglutide (Trulicity®), Exenatide (Byetta®), Exenatide extended release (Bydureon®), Liraglutide (Victoza®), Lixisenatide (Adlyxin®), Semaglutide injection (e.g. Ozempic® or Wegovy®), and Semaglutide tablets (Rybelsus®).

[0060] In some embodiments, said treating comprises increasing the subject’s E / A ratio, preferably wherein the increase in the subject’s E / A ratio is greater than the increase in E / A ratio caused by administration of the aldosterone synthase inhibitor or GLP-1 treatment alone, more preferably wherein the increase in the subject’s E / A ratio caused by administration of the aldosterone synthase inhibitor and the GLP-1 treatment is additive, even more preferably wherein the subject’s E / A ratio is restored to a normal level.Aspects of the Invention

[0061] Aspect 1. A method of treating left ventricular diastolic dysfunction (LVDD) in a subject afflicted with LVDD, the method comprising administering to the subject an aldosterone synthase inhibitor once per day in an amount effective to treat LVDD in the subject.

[0062] Aspect 2. The method of aspect 1, wherein treating LVDD comprises: (a) increasing the subject's E / A ratio relative to the subject's E / A ratio prior to treatment with the aldosterone synthase inhibitor and / or relative to the subject's E / A ratio in the absence of treatment with the aldosterone synthase inhibitor; (b) increasing the subject's E' / A' ratio relative to the subject's E' / A' ratio prior to treatment with the aldosterone synthase inhibitor and / or relative to the subject's E' / A' ratio in the absence of treatment with the aldosterone synthase inhibitor; and / or (c) increasing the subject's isovolumic relaxation time (IVRT) relative to the subject's IVRT prior to treatment with the aldosterone synthase inhibitor and / or relative to the subject's IVRT in the absence of treatment with the aldosterone synthase inhibitor.

[0063] Aspect 3. The method of aspect 1 or 2, wherein the subject is afflicted with both LVDD and heart failure with preserved ejection fraction (HFpEF), preferably wherein the aldosterone synthase inhibitor is administered to the subject in an amount effective to treat both LVDD and HFpEF in the subject.

[0064] Aspect 4. The method of any one of aspects 1 to 3, wherein the subject is afflicted with LVDD, HFpEF, and hypertension.

[0065] Aspect 5. The method of aspect 3 or 4, wherein treating HFpEF comprises: (a) stabilizing the subject's Left Ventricular Diastolic Diameter (LVEDD) relative to the LVEDD of a subject not treated with the aldosterone synthase inhibitor; (b) stabilizing the subject's Left Ventricular End Systolic Diameter (LVESD) relative to the LVESD of a subject not treated with the aldosterone synthase inhibitor; (c) stabilizing the subject's Left Ventricular End Diastolic Volume (LVEDV) relative to the LVEDV of a subject not treated with the aldosterone synthase inhibitor; (d) stabilizing the subject's Left Ventricular End Systolic Volume (LVESV) relative to the LVESV of a subject not treated with the aldosterone synthase inhibitor; (e) preventing an increase in the subject's Mitral Valve (MV) E / E' ratio or reducing the subject's Mitral Valve (MV) E / E' ratio relative to the MV E / E' ratio of a subject not treated with the aldosterone synthase inhibitor; (f) preventing an increase in the subject's MV E / A' ratio or reducing the subject's MV E / A' ratio relative to the MV E / A' ratio of a subject not treated with the aldosterone synthase inhibitor; and / or (g) reducing levels of endothelial dysfunction related biomarkers Endothelin-1 and Intercellular adhesion molecule-1 relative to the levels of Endothelin-1 and Intercellular adhesion molecule-1 of a subject not treated with the aldosterone synthase inhibitor; reducing levels of one or more biomarkers associated with HFpEF, preferably wherein the one or more biomarkers are selected from the group consisting of: MF AIM, SVEP1: EGF-like domains 4-6; N-terminal pro-BNP, SVEP: Sushi 15-18; NRP1, Spondin-1, ITIH3, BNP, and IGFBP-7 relative to the levels of said one or more biomarkers of a subject not treated with the aldosterone synthase inhibitor.

[0066] Aspect 6. The method of any one of aspects 3 to 5, wherein treating HFpEF comprises reduction in the frequency and / or likelihood of hospitalization for heart failure (HHF), preferably wherein the reduction is a reduction in the subject's odds ratio (OR) of HHF of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0067] Aspect 7. The method of any one of aspects 3 to 32, wherein treating HFpEF comprises reduction in the likelihood of cardiovascular (CV) death, preferably wherein the reduction is a reduction in the subject's odds ratio (OR) of CV death of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0068] Aspect 8. The method of any one of aspects 3 to 7, wherein treating HFpEF comprises reduction in the frequency or likelihood of heart failure (HF) events, preferably wherein the reduction is a reduction in the subject's odds ratio (OR) of HF events of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0069] Aspect 9. The method of any one of aspects 3 to 7, wherein treating HFpEF comprises improvement in the subject's exercise capacity as measured by an increase in the subject's six-minute walk distance, preferably wherein the improvement is by at least 5 meters, more preferably by at least 10 meters.

[0070] Aspect 10. The method of any one of aspects 3 to 8, wherein treating HFpEF comprises an increase in the subject's peak oxygen consumption, preferably wherein said increase is by at least 2.5 mL / kg / min, by at least 5 mL / kg / min, by at least 10 mL / kg / min, by 2.5-5 mL / kg / min, 5-10 mL / kg / min, or 10-20 mL / kg / min.

[0071] Aspect 11. The method of any one of aspects 3 to 9, wherein treating HFpEF comprises improvement in the subject's Kansas City Cardiomyopathy Questionnaire (KCCQ) Clinical Summary Score (CSS), preferably wherein the improvement is by at least 5 points, by at least 10 points, by at least 20 points, by 5-10 points, by 10-20 points, or by 20-50 points.

[0072] Aspect 12. A method of treating heart failure with preserved ejection fraction (HFpEF) in a subject afflicted with HFpEF, the method comprising administering to the subject an aldosterone synthase inhibitor once per day in an amount effective to treat HFpEF in the subject.

[0073] Aspect 13. The method of aspect 11, wherein the subject is afflicted with both hypertension and HFpEF.

[0074] Aspect 14. The method of aspect 11 or 12, wherein treating HFpEF comprises reduction in the frequency and / or likelihood of hospitalization for heart failure (HHF), preferably wherein the reduction is a reduction in the subject's odds ratio (OR) of HHF of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0075] Aspect 15. The method of any one of aspects 11 to 13, wherein treating HFpEF comprises reduction in the likelihood of cardiovascular (CV) death, preferably wherein the reduction is a reduction in the subject's odds ratio (OR) of CV death of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0076] Aspect 16. The method of any one of aspects 11 to 14, wherein treating HFpEF comprises reduction in the frequency or likelihood of heart failure (HF) events, preferably wherein the reduction is a reduction in the subject's odds ratio (OR) of HF events of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

[0077] Aspect 17. The method of any one of aspects 11 to 15, wherein treating HFpEF comprises improvement in the subject's exercise capacity as measured by an increase in the subject's six-minute walk distance, preferably wherein the improvement is by at least 5 meters, more preferably by at least 10 meters.

[0078] Aspect 18. The method of any one of aspects 11 to 16, wherein treating HFpEF comprises an increase in the subject's peak oxygen consumption, preferably wherein said increase is by at least 2.5 mL / kg / min, by at least 5 mL / kg / min, by at least 10 mL / kg / min, by 2.5-5 mL / kg / min, 5-10 mL / kg / min, or 10-20 mL / kg / min.

[0079] Aspect 19. The method of any one of aspects 11 to 17, wherein treating HFpEF comprises improvement in the subject's Kansas City Cardiomyopathy Questionnaire (KCCQ) Clinical Summary Score (CSS), preferably wherein the improvement is by at least 5 points, by at least 10 points, by at least 20 points, by 5-10 points, by 10-20 points, or by 20-50 points.

[0080] Aspect 20. The method of any one of aspects 11 to 18, wherein the aldosterone synthase inhibitor is administered in an amount effective to treat left ventricular diastolic dysfunction (LVDD) in the subject.

[0081] Aspect 21. The method of any one of aspects 1 to 19, wherein the aldosterone synthase inhibitor is administered to the subject in an amount sufficient to inhibit 50% or more of aldosterone synthase's activity for between 8 and 18 hours of a 24-hour period, preferably wherein the amount is sufficient to inhibit 50% or more of aldosterone synthase's activity for between 10 and 14 hours of a 24-hour period.

[0082] Aspect 22. The method of any one of aspects 1 to 20, wherein the aldosterone synthase inhibitor is administered to the subject once per day in the morning.

[0083] Aspect 23. The method of any one of aspects 1 to 20, wherein the aldosterone synthase inhibitor is administered to the subject once per day in the evening.

[0084] Aspect 24. The method of any one of aspects 1 to 22, wherein the aldosterone synthase inhibitor is administered to the subject daily: (a) for at least one week; (b) for at least two weeks; (c) for at least four weeks; (d) for at least eight weeks; (e) for at least twelve weeks; (f) for at least twenty -four weeks; (g) for at least fifty weeks; (h) for at least one hundred weeks; or (i) for at least two hundred weeks, to thereby treat LVDD and / or HFpEF in the subject.

[0085] Aspect 25. The method of any one of aspects 1 to 23, wherein the aldosterone synthase inhibitor is lorundrostat or a pharmaceutically acceptable salt thereof.

[0086] Aspect 26. The method of aspect 24, wherein the amount of the lorundrostat or pharmaceutically acceptable salt thereof is 25-100 mg per day, preferably 25-75 mg per day, more preferably 40-60 mg per day, even more preferably 50 mg per day.

[0087] Aspect 27. The method of any one of aspects 24 to 25, wherein the lorundrostat or pharmaceutically acceptable salt thereof is the monohydrobromide salt of lorundrostat.

[0088] Aspect 28. The method of any one of aspects 1 to 25, the method further comprising administering to the subject a GLP-1 treatment concurrently or concomitantly.

[0089] Aspect 29. The method of aspect 27, wherein the GLP-1 treatment is selected from Dulaglutide (Trulicity®), Exenatide (Byetta®), Exenatide extended release (Bydureon®), Liraglutide (Victoza®), Lixisenatide (Adlyxin®), Semaglutide injection (e.g. Ozempic® or Wegovy®), and Semaglutide tablets (Rybelsus®).

[0090] Aspect 30. The method of any one of aspects 27 to 28, wherein said treating comprises increasing the subject's E / A ratio, preferably wherein the increase in the subject's E / A ratio is greater than the increase in E / A ratio caused by administration of the aldosterone synthase inhibitor or GLP-1 treatment alone, more preferably wherein the increase in the subject's E / A ratio caused by administration of the aldosterone synthase inhibitor and the GLP-1 treatment is additive, even more preferably wherein the subject's E / A ratio is restored to a normal level.Definitions

[0091] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / ormaterials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0092] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[0093] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0094] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0095] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0096] As used herein “lorundrostat” refers to the disubstituted 1, 2, 4-Triazine compound which is represented by Formula (A):3-[4-[[trans-4-(acetoamino)cyclohexyl]carbamoylmethyl]piperazin-l-yl]-5-(p-tolyl)-l,2,4-triazine (A).

[0097] “Lorundrostat HBr” refers to the hydrobromide (HBr) salt of lorundrostat. Weights and / or strengths of “lorundrostat HBr,” and “the compound” of the invention refer to the weight of the free base (i.e. lorundrostat) in the HBr salt and not the weight of the HBr salt.

[0098] Lorundrostat and pharmaceutically acceptable salts thereof can be made by processes described, for example, in US Patent No. 10,029,993 and European Publication No. 3549935, the disclosures of which are incorporated by reference herein in their entirety.

[0099] As used herein, “a subject afflicted with heart failure with preserved ejection fraction (HFpEF)” means a subject who has been identified as being afflicted with HFpEF. In some embodiments of the invention, the method of treating a subject afflicted with HFpEF includes an affirmative step of receiving an identification of the subject as being afflicted with HFpEF. In some embodiments, where the subject is also afflicted with hypertension, the method includes an affirmative step of receiving an identification of the subject as being afflicted with hypertension.

[0100] As used herein “receiving an identification of the subject as being afflicted with HFpEF” includes receiving information regarding a prior diagnosis of HFpEF as well as diagnosing the subject with HFpEF. Thus, some embodiments of the invention include a step of receiving information regarding a prior diagnosis of HFpEF in the subject, while other embodiments include a step of diagnosing the subject with HFpEF. Similarly, as used herein “receiving an identification of the subject as being afflicted with hypertension” includes receiving information regarding a prior diagnosis of hypertension as well as diagnosing the subject with hypertension. Thus, some embodiments of the invention include a step of receiving informationregarding a prior diagnosis of hypertension in the subject, while other embodiments include a step of diagnosing the subject with hypertension.

[0101] In embodiments, cardiovascular (CV) death is as described in Hicks et al. 2015, the contents of which are specifically incorporated by reference herein for its definition of CV death.

[0102] In embodiments, a “heart failure (HF) event” is as described in Appendix 7 of Hicks et al. 2015, the contents of which are specifically incorporated by reference herein for its definition of HF event. Thus, a “HF event” includes: presentation of the patient for an urgent, unscheduled clinic / office / ED visit or hospital admission, with a primary diagnosis of HF, where the patient exhibits new or worsening symptoms of HF on presentation, has objective evidence of new or worsening HF, and receives initiation or intensification of treatment specifically for HF. Objective evidence consists of at least 2 physical examination findings OR at least 1 physical examination finding and at least 1 laboratory criterion of new or worsening HF on presentation.

[0103] “Treating” or “treatment” as used herein (and as well-understood in the art) also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, "treatment" as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.

[0104] “Treating” and “treatment” as used herein include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may be a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of aparticular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In embodiments, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient. In embodiments, the treating or treatment is not prophylactic treatment.General

[0105] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments.

[0106] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.

[0107] Additional obj ects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0108] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0109] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.EXAMPLESExample 1Background

[0110] The ZSF1 (Zucker fatty and spontaneously hypertensive) rat is an animal model for heart failure with preserved ejection fraction (HFpEF) (Schauer 2020). ZSF1 rats are produced by crossing rat strains with two separate leptin receptor mutations (fa and facp): the lean female ZDF rat (+ / fa) and the lean male SHHF rat (+ / facp). The offspring of this cross may comprise both mutations (fa:facp), may comprise neither mutation (+ / +), or may comprise only one mutation (either + / fa or + / facp). The rats comprising both mutations (fa:facp) are obese and develop insulin resistance, hyperglycaemia, and mild hypertension (“ZSF1 -obese”), while the offspring comprising neither mutation or only one mutation (“ZSFl-lean”) are lean and exhibit no signs of obesity or diabetes. Studies have shown that ZSFl-obese animals develop signs of HFpEF, in particular elevated E / e' (the ratio of early transmitral flow velocity (E) to early diastolic mitral annular tissue velocity (e')), elevated left ventricular end-diastolic pressure (LVEDP), and preserved Left Ventricular Ejection Fraction (LVEF) (Bowen 2017; Davila 2019; Leite 2019; Miranda-Silva 2020).Study Design SummarySpecies

[0111] Male, ZSF1 Lean (strain code: 379) and Obese (strain code: 378) (Charles River Laboratories) rats; approximately 11 weeks of age at surgery.Model

[0112] Uni-nephrectomy (right) (UNx) of ZSF1 obese rats at study week -1.

[0113] 0.9% NaCl via drinking water.Study GroupsDoseSurgery Drinking Route / Study Group Model Test Article (mg / kg / (Week -1) Water Frequency Enrollment dose)ZSFl- 1 Sham Tap Vehicle N / A PO, BID 10leanZSFl- 0.9%2 UNx Vehicle N / A PO, BID 16obese NaClZSF1- 0.9% Lorundrostat3 UNx 50 PO, BID 16 obese NaCl HBrZSF1- 0.9% Lorundrostat4 UNx 75 PO, BID 16obese NaCl HBr

[0114] The timeline of study procedures and measurements is outlined in Figure 1.Dosing and Test Article Formulation

[0115] Vehicle or Lorundrostat HBr: Twice daily (BID) (8 / 16-hour interval; 8:00AM and 4:00PM) dose via oral gavage (PO) for 12 weeks.Mortality and Morbidity

[0116] Twice weekly body weight.

[0117] Daily monitoring of clinical signs.Fasting Blood Glucose

[0118] 5-hour fasting.

[0119] Timepoints: Week 0 (pre-treatment), week 6, and week 12.

[0120] Measured by Glucometer.In-Life Blood Collections

[0121] Timepoints: Week 0, Week 6 and Week 12.(a) Route: Tail vein(b) Collect blood for serum and plasma for clinical chemistry analysis, hsCRP biomarker, and potential aldosterone analysis.

[0122] Timepoints: Week 7(a) Route: Tail vein(b) Collect blood at 1 hour of post AM dose and immediately prior to PM dose for plasma compound exposureUrine Collections

[0123] Timepoints: Week 6 and week 12.

[0124] 24-hour collections at room temperature for clinical chemistry.Echocardiograph

[0125] Timepoints: Week 0 (pre-treatment), week 6 and week 12.

[0126] Cardiac structure and function (systolic and diastolic) via echocardiography.Necropsy

[0127] Collect blood for plasma.

[0128] Harvest heart and weigh whole heart, left ventricle (including IV septum), right ventricle free wall and right and left atria.(a) Freeze and fix heart tissue for potential analyses.

[0129] Harvest and weigh left kidney.(a) Snap freeze and fix kidney samples for potential analyses.

[0130] Harvest, weigh, and discard lungs.

[0131] Collect left tibia and measure tibia length.Endpoints

[0132] Body weight

[0133] Whole heart, left ventricle, right ventricle, left atria, right atria, kidney, and lung weights (normalized to tibia length).

[0134] Cardiac structure and function (systolic and diastolic) via echocardiography.

[0135] Fasting blood glucose.

[0136] Clinical chemistry

[0137] Urine: protein, albumin, creatinine, and urea.

[0138] Serum: creatinine, BUN, and glucose.Study Timeline

[0139] The study timeline is set forth in Figure 1.Dosing and FormulationDosing GroupsDose DoseDose Route / Study Group Model Test Article Concentration(mg / kg / dose) Volume Frequency Enrollment (mg / mL) (kg / mL)ZSF1- N / A 51 Vehicle N / A 10leanZSF1- N / A 5 Route: PO2 Vehicle N / Aobese Frequency: 16BID at3 ZSF1- Lorundrostat 50 10 5 8:00AM and 16 obese HBr 4:00PMZSF1- Lorundrostat 15 54 75 16obese HBrTest Article FormulationVehicle Formulation (Ultrapure Water or DI Water, pH 2):

[0140] The vehicle will be prepared within 2 weeks of use. An appropriate volume of ultrapure water or DI water will be added to a glass container and continuously stirred while the initial pH is recorded. The pH will be adjusted using IN, 6N, or ION HCL until a pH of 2.00 ± 0.10 is achieved. The formulated vehicle will be stored at 4°C until use.Lorundrostat HBr Formulation (10 and 15 mg / mL):

[0141] Test article formulations will be prepared and used within one week. The vehicle formulation will be removed from refrigerated storage and stirred at room temperature for at least 15 minutes. Dosing formulations will be prepared by adding a weighed amount of test article to at least 70% volume of vehicle (ultrapure water or DI water, pH 2). The TA will be protected from light while formulating and homogenized as needed. If lumps are found in the TA, they can be lightly broken up with a mortar and pestle by adding 2 to 5 drops of vehicle. The pH will be measured and adjusted to 2.00 ± 0.10 using 6N HC1, then will be stirred for at least 30 minutes at room temperature to ensure a homogenous solution. The formulation will be stored at 4°C and protected from light until use and used within 8 days of preparation.Test Article Adverse or Physiological Effects

[0142] There are no known adverse effects for Lorundrostat HBr.Test Article Administration

[0143] Animals will receive vehicle, Lorundrostat HBr at 50 mg / kg, 75 mg / kg, or Empagliflozin at 30 mg / kg via oral gavage. Dosing volumes will be calculated based on the most current individual body weight obtained. Dosing will start at 8:00AM and 4:00PM daily for vehicle or Lorundrostat HBr. The dosing of Empagliflozin at 30 mg / kg will be once daily starting at 8:00AM.0.9% NaCl Supplemented Drinking Water Formulation

[0144] Group 2-4 animals will be switched from standard drinking water to 0.9% NaCl water shortly after baseline Echocardiography. The 0.9% NaCl supplemented drinking water will be prepared by at least weekly. A weighed amount of NaCl will be added to a measured volume of standard drinking water to obtain a 0.9% NaCl drinking water solution. The solution will be mixed until completely dissolved then added to water bottles.Subject DetailsAnimalsSpecies / Strain Source Sex Number of Age at Age at Animals Delivery Surgery Rat Charles Male 10 10 weeks 11 weeks ZSF1 Lean River(Strain: 379) LaboratoriesRat Charles Male 64 10 weeks 11 weeks ZSF1 Obese River(Strain: 378) LaboratoriesAcclimation

[0145] After arrival, the ZSF1 lean and ZSF1 obese rats will be acclimated for a period of at least 6 days prior to study initiation.Husbandry InformationHousing Feed Water(FrequencyPer Cage Cage Description Environmental and Type)ConditionGroup housed (up Rat standard • Photoperiod: 12- Purina 5008 Group 1: Water bottles to 2 rats / cage) polycarbonate with hour light, 12- Rodent Chow (meets human drinking except when com cob / wood hour dark (may be Diet standards) undergoing urine pulp bedding interrupted forcollection, they willbe single housed in study-related Group 2-5: 0.9% NaCl on metabolic cages. activities) study day 0 after baseline • Temperature: 72 Echocardiography.+ / -8 °F• Relativehumidity: 30% -70%In-Life Study ConductGeneral Procedures

[0146] Health Monitoring: Daily, standard cage-side health monitoring; Abnormal findings recorded.

[0147] Body Weight: Twice weeklyRight Kidney Nephrectomy Surgical Procedure

[0148] All surgeries are performed under sterile conditions. Rats are anesthetized under 2-5% isoflurane. The incision site is shaved and scrubbed with betadine. The rat is placed on a 37°C heating pad and draped for surgery. Care is taken to maintain body temperature at 36.5±1°C. A 1-2cm incision is made in the midline abdomen to allow access to the right kidney. The renal vessels and ureter are isolated and ligated with a single suture (5-0 silk). The distal portion will be cut, and the kidney removed. The abdominal incision is then closed with an uninterrupted 5-0 Vicryl absorbable suture, 0.25% Marcaine is applied directly to the incision, which is then closed with wound clips. Animals in group 1 undergo the same procedure except for removal of the right kidney.

[0149] Long-acting Buprenorphine will be given pre-operatively at a dose of 1 mg / kg (SC) for post-operative pain relief. Immediately following surgery, animals are allowed to recover in a cage placed on a 37°C heating pad. They are then group housed and allowed free access to food and water. Hydrogel is placed into cages for up to 3 days following surgery to facilitate hydration. Fasting Blood Glucose Measurement

[0150] Animals will be fasted for 5 hours prior to blood glucose measurement. Blood glucose will be measured at study week 0 (pre-treatment), week 6, and week 12 via glucometer ( Alphatrac).In-I.ife Blood CollectionFor Clinical Chemistry and Potential Aldosterone and hsCRP AssayAnimals: All groupsCollection Week 0 (Baseline) prior to fasting.Time: Week 6 and Week 12, immediately following the completion of urine collectionCollection Tail VeinRoute:Blood Volume: Week 0: 300 pL of whole blood for plasmaWeek 6 and Week 12: 700 pL of whole blood for serum and plasma Collection Tube Serum separator tube(anticoagulant): K.2 EDTA plasma separator tubeWeek 6 and 12: 300 pL tubesSample Week 0:Processing: • -300 pL of whole blood will be collected and placed in a prechilled K2 EDTA plasma separator tubeso Samples will be mixed thoroughly and kept on wet ice prior to processing.o Plasma is separated by centrifugation at 2,000 g (4,400 rpm, Eppendorf 5417R) for 10 min at 4°C.o Plasma will be transferred into two Eppendorf tubes and stored at -80°C■ -60 pL for potential aldosterone assay■ -60 pL for potential hsCRP biomarker analysis Week 6 and Week 12:• -400 pL of whole blood will be collected and placed in a serum separator tube.o Maintain samples at room temperature for at least 30 minutes to allow for clot formation.o Centrifuge at 2,000 g (4,400 rpm, Eppendorf 5417R) for 10 min at 20°C.o Transfer serum (-150 pL) samples into Eppendorf tube and store at -80°C until clinical chemistry analysis.• -300 pL of whole blood will be collected and placed in a prechilled K2 EDTA plasma separator tubes (300 pL tubes)o Samples will be mixed thoroughly and kept on wet ice prior to processing.o Plasma is separated by centrifugation at 2,000 g (4,400 rpm, Eppendorf 5417R) for 10 min at 4°C.o Plasma will be transferred into two Eppendorf tubes and stored at -80°C■ -60 pL for potential aldosterone assay■ -60 pL for potential hsCRP analysisSample Serum: -80°CStorage: Plasma: -80°CFor Test Compound ExposureAnimals: All groupsCollection Time: Week 7, 1-hour post AM does and immediately prior to PM dose Collection Tail VeinRoute:Blood Volume: 250 pL of whole bloodCollection Tube K2 EDTA plasma separator tube (300 pL tube)(anticoagulant):Sample -250 pL of whole blood will be collected and placed in a pre-chilled 300 Processing: pL K2 EDTA plasma separator tube.• Samples will be mixed thoroughly and kept on wet ice prior to processing.• Plasma is separated by centrifugation at 2,000 g (4,400 rpm, Eppendorf 5417R) for 10 min at 4°C.• Plasma (>100 pL) will be transferred into Eppendorf tube Sample Storage: Plasma: -80°C until shipment to Sponsor’s designated laboratory forBioanalysisUrine CollectionAnimals: All groupsCollection Time: • Week 6 and Week 12• All urine collections start between 8:00-10:00AMCollection 24-hour collection at room temperature using metabolic cages Method:Sample • Record urine volumeProcessing: • Urine samples are centrifuged at 850 g (2,000 rpm, Sorvall ST40) for 10 minutes at 4°C to remove particulate matter.• Urine samples will be transferred into tubes and stored at -80°C until clinical chemistry analysis.Sample Storage: -80°C until clinical chemistry analysisEchocardiographyTime Points: Study week 0 (pre-treatment), week 6 and week 12Procedure: Images will be obtained from rats under light anesthesia with 1-2%isoflurane using a VisualSonics Vevo 3100® Ultrasound Echocardiography System and MX201 18 MHz transducer. M-Mode (short-axis) images of the left ventricle, Pulse Wave and Tissue Doppler images will be collected and analyzed for LV geometry, systolic anddiastolic function.Terminal PhaseHemodynamic AssessmentCollection Time:Week 12Anesthesia: Inactin via IVPrior to Anesthesia: Obtain body weightHemodynamic Assessment: • Systemic arterial pressure and heart rate via catheter placed into the carotid artery• Systemic blood pressure will be recorded with fluid-filled catheters and pressure transducers connected to a Ponemah™ data acquisition system (Data SciencesInternational, St. Paul, MN).Blood and Tissue Collection and ProcessingCollection Route: Vena cava or cardiac puncture if unable to obtain blood via vena cavaBlood Volume: >5 mb of whole bloodCollection Tube • Serum separator tube(anticoagulant): • K.2 EDTA plasma separator tubeSample Processing: • ~2 mb of whole blood will be collected and placed in a serum separator tube.o Maintain samples at room temperature for at least 30 minutes to allow for clot formation.o Centrifuge at 2,000 g (4,400 rpm, Eppendorf 5417R) for 10 min at 20°C.o Transfer serum samples into Eppendorf tube• ~3 mb of whole blood will be collected and placed in prechilled K2 EDTA plasma separator tubeo Samples will be kept on wet ice until processing. o Centrifuge at 2,000 g (4,400 rpm, Eppendorf 5417R) for 10 minutes at 4°C.o Transfer plasma samples into 3 Eppendorf tubes:■ 100 pL for shipment to Sponsor’s designated laboratory for Bioanalysis■ Remaining for biomarker analysisTissue Collection and • Heart:Processing: o Harvest whole heart, rinse in cold lx PBS, blot dry, and weigh.o Weigh left ventricle (including IV septum), right ventricle, left atria, and right atria separately o A 3-4 mm transversely cut basal section of the left ventricle (including IV septum) is placed into a histocassette and fixing in 10% neutral buffered formalin (NBF) at room temperature for 24-48 hours prior to transferring into 70% ethanol.o Bisect apical section of LV free wall (after removing IV septum), place into two separate tubes (one for potential mRNA and one for potential protein analysis) and snap freeze in liquid nitrogen.• Lungs:o Harvest lungs, rinse in cold lx PBS, and blot dry.o Weigh and discard.• Left Kidneyo Harvest left kidney, remove capsule, rinse in cold lx PBS, blot dry, and weigh.o Fixed Samples:■ Cut a 3 mm mid-transverse section of left kidney. ■ Place into a histocassette and fix in 10% NBF at room temperature for 24-48 hours prior to transferring into 70% ethanol.• Frozen Samples:o Place cranial (top) and caudal (bottom) kidney poles in two separate tubes and snap freeze the tissues in liquid nitrogeno Collect left tibia and measure tibia length.Sample Storage: • Serum: -80°C• Plasma: -80°C• Fixed Samples: Room temperature in 70% ethanol• Frozen Kidney Poles: -80°CSample Analysis

[0151] Clinical Chemistry AnalysisClinical Chemistry Analyzer: RX DaytonaAnalytes: Urine: protein, albumin, creatinine, and urea.Serum: creatinine, BUN, and glucose.ResultsBaseline Body Weight and Blood Glucose

[0152] At baseline (week 0), the study groups had the following average body weight and blood glucose levels. Numbers in parenthesis indicate mean standard error (SE).Study Group Body Weight (g) Blood Glucose (mg / dL) ZSF1 Lean Sham 312.8 (4.8) 131 (4)ZSF1 Obese Unx Vehicle 423.3 (4.5) 146 (3)ZSF1 Obese Unx Lor - 50 mg / kg 423.8 (5.4) 145 (3)ZSF1 Obese Unx Lor - 75 mg / kg 422.7 (5.2) 145 (3)Body weight over time

[0153] Body weights of the study groups were measured over time. As shown in Figure 2, lorundrostat HBr had no effect on body weight. ZSF1 -obese rate body weight was in line with the literature.Aldosterone measurements

[0154] Aldosterone levels were measured in the study groups at baseline and Week 6 (Figure 3), providing evidence of suppression of aldosterone by lorundrostat HBr.Echocardiography measurements

[0155] Left Ventricular Diastolic Diameter (LVEDD) is a measure of the size of the heart’s left ventricle at the end of diastole. As shown in Figure 4, the ZSFl-obese control rats demonstrated increased LVEDD, while ZSFl-obese rats treated with lorundrostat HBr (75mg / kg) exhibited stabilized LVEDD.

[0156] Left Ventricular End Systolic Diameter (LVESD) is a measure of the size of the heart’s left ventricle at the end of systole. As shown in Figure 5, the ZSFl-obese control rats demonstrated increased LVESD, while ZSFl-obese rats treated with lorundrostat HBr (75mg / kg) exhibited stabilized LVESD.

[0157] Left Ventricular End Diastolic Volume (LVEDV) is a measure of the volume in the left ventricle at the end of diastole. As shown in Figure 6, the ZSFl-obese control rats demonstrated increased LVEDV, while ZSFl-obese rats treated with lorundrostat HBr (75 mg / kg) exhibited stabilized LVEDV.

[0158] Left Ventricular End Systolic Volume (LVESV) is a measure of the volume in the left ventricle at the end of systole. As shown in Figure 7, the ZSFl-obese control rats demonstrated increase LVESV, while ZSFl-obese rats treated with lorundrostat HBr (75 mg / kg) exhibited stabilized LVESV.

[0159] Mitral Valve E / E’ ratio (MV E / E’ ratio) is a measure of left ventricle filling pressure, derived from the following two measurements:(a) E wave: The early diastolic mitral inflow velocity; and(b) E’ wave: The early diastolic mitral annular velocity.

[0160] As shown in Figure 8, the ZSFl-obese control rats demonstrated increased MV E / E’ ratio. By contrast, lorundrostat HBr (50 mg / kg and 75 mg / kg) prevented an increase in MV E / E’ ratio ZSFl-obese rats.

[0161] Mitral Valve E / A ratio (MV E / A ratio) is the ratio of the early (E) to late (A) ventricular filling velocities. As shown in Figure 9, ZSF1 obese control rats demonstrated increased MV E / A Ratio. By contrast, lorundrostat HBr (50 and 75mg / kg) prevented an increase in MV E / A Ratio.

[0162] Fractional shortening (FS) is an echocardiographic parameter used to assess left ventricular systolic function. It measures the percentage of size reduction of the left ventricle during systole, providing insight into how well the heart contracts. FS is calculated using the following formula: FS (%) = (LVEDD - LVESD) / LVEDD x 100.

[0163] As shown in Figure 10, lorundrostat HBr exhibited no effect on Fractional Shortening, with all study groups exhibiting FS within the normal range.

[0164] Ejection fraction (EF) is a key measurement of heart function that represents the percentage of blood pumped out of the left ventricle with each heartbeat. It is calculated using the following formula: EF(%) = (SV / EDV)x100, where SV is Stroke Volume (amount of blood pumped out) and EDV is End Diastolic Volume (total amount of blood in the ventricle).

[0165] As shown in Figure 11 lorundrostat HBr exhibited no effect on EF, with all study groups exhibiting EF within the normal range.

[0166] As shown in Figure 12 lorundrostat HBr exhibited no effect on Left Ventricular Anterior Wall Thickness.

[0167] As shown in Figure 13 lorundrostat HBr exhibited no effect on Left Ventricular Posterior Wall Thickness.Blood Glucose measurements

[0168] As shown in Figure 14, lorundrostat HBr stabilized blood glucose levels relative to the control group.Biomarker Analysis

[0169] Endothelial dysfunction related biomarkers (Endothelin-1 and Intercellular adhesion molecule-1) were measured in the study groups. As shown in Figure 15, endothelial dysfunction related biomarkers were increased in obese ZSF1 rats and dose dependent endothelial function improvement was observed in the study groups treated with lorundrostat HBr.Example 2Introduction and purpose of the study

[0170] The aim of the study described in the present Example was to evaluate the effects of lorundrostat, a highly selective aldosterone synthase inhibitor, in a model of obesity and associated metabolic syndrome and related co-morbidities including hypertension and other cardiovascular risk, in particular Left Ventricular Diastolic Dysfunction (LVDD). The study was conducted in a free choice Diet-Induced Obese (DIO) at model either alone or in combination with semaglutide.Materials and MethodsGuidelines

[0171] All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (revised 1996 and 2011, 2010 / 63 / EU) and French laws.Test Item 1Name and dose administered Lorundrostat HBr p.o. BID Lorundrostat HBr p.o. BID 50 mg / kg 75 mg / kgConcentration in the Concentration: 5 mg / mL Concentration: 7.5 mg / mL formulation (e g. mg / mL) Dose: 50mg / kg Dose: 75mg / kgDose volume: 10 mL / kg Dose volume: 10 mL / kgVehicle Osmosed water, ph2Test Item 2Name and dose administered Semaglutide 0.04mg / kg s.c. QDConcentration in the Dose: 0.04mg / kgformulation (e.g. mg / mL) Dose volume: 5mL / kgConcentration: 0.008 mg / mLVehicleName Osmosed water, pH 2Dosing volume (mL / kg) Dose volume: lOmL / kgTest System and ManagementSpecification and justificationSpecie RatBreed Sprague DawleySex MaleAge 5 -weekNumber 72 in order to obtain 60 evaluable animals at the end of the experimental phaseWeight 200-225g at deliveryJustification Male is chosen to avoid hormonal cycleIdentification

[0172] Animals were identified with an electronic chip.Housing

[0173] Animals were housed in enriched and ventilated housing cages - rat cage GR900 (905 cm2) throughout the experimental phase. Animals’ cages litters were changed at least once a week.

[0174] Animals were housed in groups of 2 animals on a normal 12-hour light cycle (at 7:30pm lights off), 22 ± 2 °C and 50 ± 10 % relative humidity.Acclimation

[0175] At least 5 days of acclimation.Food and water

[0176] During the acclimation phase, standard chow diet (Ref# 105 from Safe Diets, France) and tap water was provided ad libitum.

[0177] After the acclimation phase and until the end of the study, rats were fed either the same chow diet (group #1), or a free choice diet (free choice between the chow diet with normal tap water or a high fat / cholesterol diet (40.8% fat, 14.8% protein, 44.4% carbohydrates and 0.5% cholesterol, reference# E8400A01R V32 from Safe Diets, France) with 10% fructose enriched tap water.TreatmentsDosage regimen and test groupsGroup n= Treatment Route Dose Treatment period 1 12 Chow diet + vehicle po, BID N / A2 12 Free choice diet + vehicle po, BID N / A3 12 Free choice diet + lorundrostat po, BID 50 mg / kg For 4 weeks HBr from week 14 4 12 Free choice diet + lorundrostat po, BID 75 mg / kgHBr5 12 Free choice diet + semaglutide s.c. QD 0.04 mg / kg6 12 Free choice diet + semaglutide s.c. QD 0.04 mg / kg ++ lorundrostat HBr 30 mg / kg / po, BID 75 mg / kgMethods

[0178] The design of the study of the present example is summarized in Figure 17.

[0179] Body weight, food intake and water intake were measured once per week during the entire experimental period.

[0180] After the 12-week chow or free choice diet induction period, baseline echocardiography was performed in all rats (n=72).

[0181] All rats (n=72) were weighed, blood collected (100µl of blood / EDTA for 50µl of plasma aliquot) for aldosterone plasma measurements and then rats were 6-hour fasted at ~08:00am prior to being bled (150µL-200µl / EDTA for 4 plasma aliquots) at ~2:00pm to measure baseline parameters i.e., blood glucose, plasma insulin, total cholesterol, adiponectin, and leptin levels. HOMA-IR was calculated from fasting blood glucose and plasma insulin levels.

[0182] Rats were then randomized into homogenous treatment groups (n=10 per treatment group), according to their (1) echocardiography parameters (E / A ratio, E7A’ ratio and ejection fraction confirming HFpEF phenotype), (2) HOMA-IR, and (3) body weight.

[0183] After the first dosing, blood was collected at 1, 3 and 6 hours post first dose in all rats from groups 3, 4 and 6.

[0184] At the end of the treatment period, echocardiography was repeated. As described above for baseline, blood was collected for aldosterone plasma level and then rats were 6-hour fasted for fasting blood glucose, plasma insulin measurement and calculation of HOMA-IR as well as plasma total cholesterol, adiponectin and leptin levels.

[0185] Then were anesthetized to perform invasive intraventricular pressure. At the end of the procedure, heart and kidneys were collected, weighed and stored as described in the table below.Echocardiography

[0186] Before and at the end of the treatment period (1 hour after the dosing), echocardiography was performed using two-dimensional echocardiograph (VF16-5 probe, Siemens, Acuson NX3 Elite).

[0187] Animals were anesthetized by inhalation of 4% isoflurane in 0.4 l / min O2, and then maintained with 1.5-2% isoflurane.

[0188] Numeric images of the heart were obtained in both parasternal long-axis and short-axis views to examine cardiac dimensions and systolic function. Those views were used to guide calculations of percentage fractional shortening (FS) and percentage ejection fraction (EF, Simpson method) and to determine heart rate, left ventricle dimensions and wall thickness.

[0189] Diastolic function was assessed using pulse-wave doppler imaging from apical four chambers view of the mitral flow and the mitral annulus. Early (E) and late (A) peak transmitral flow velocity, isovolumic relaxation time (IVRT) and annular tissue velocity of the mitral valve (E’ and A’ peaks) were measured. Thereafter, E / A E7A’ and E / E’ ratio were calculated.Assessment of intraventricular pressure

[0190] On the next days after final echocardiography and 1 hour after dosing, rats were anesthetized by inhalation of 4% isoflurane in 0.6 l / min O2, mechanical ventilation, and maintenance with 2% isoflurane. A 2-cm incision was performed to the right of the trachea to visualize the right carotid artery. The artery was dissected free, and an ultraminiature conductance catheter was inserted into carotid artery and advanced into the ascending aorta to measure blood pressure for a few minutes. Thereafter, the catheter was further advanced into the left ventricle to record internal pressure.Humane Endpoints

[0191] In case of bad health statute scoring:(a) Severe scoring: body weight loss up to 20%, convulsing, lying on its side without reaction, or(b) Moderate scoring: several days with the following criteria without improvement or cumulative following criteria: hypothermia, ached back, prostrate, dehydrated the concerned rat will be euthanized by IP injection of pentobarbital (150mg / kg, 5mL / kg).

[0192] In case of hypothermia and / or dehydration, the concerned rat will be housed temporarily in warmed cabinet and / or injected with Ringer Lactate solution (up to 10mL / kg) for maximum 4 days. If it does not improve the health statute, rat will be euthanized. Sponsor will be informed each time this kind of event happens.Anesthesia & Analgesia methods if applicable

[0193] Blood collection (depending on route used for bleeding), echocardiography and LV invasive pressure measurement examination will be conducted under anesthesia with isoflurane.Methods of euthanasia if applicable

[0194] According to the defined humane endpoints, euthanasia will be performed by IP injection of pentobarbital (150mg / kg, 5mL / kg).Tissue and blood samples

[0195] Blood collection during the study:Day of collection & Site & condition of Volume & number Subsequent assays animal condition collection of aliquotsWeek 17 (1, 3 and 6 Blood on EDTA / ~400-500 µL blood at PK analysis + hrs post first dosing) Sublingual or Retro each bleeding to Aldosterone for groups 3, 4 and 6 orbital or Tail vein prepare 2 plasmafor PK & in all aliquot (min 100 µLgroups for for PK, 50µL foraldosterone aldo) per timepointWeek 12 and Week ~125-150µL blood to Aldosterone18 (non-fasting) for prepare 1 plasmaall groups aliquot (min 50 µl)Week 12 and week ~150-200µL blood 4 Plasma insulin, total 18 (fasting) for all aliquots (10µl for cholesterol, leptin groups insulin, 10 µl for adiponectin,cholesterol, 30 µl forleptin andadiponectin)Week 12 and week Tail vein One drop Blood glucose (with a 18 (fasting) for all glucometer)groups0196] Organs collected at sacrifice:Organ Weight recorded? storage condition#! Storage condition#! (fixed in formalin (flash-frozen in liquid for 48h and nitrogen then stored transferred / kept in at -80°C until ethanol 70% at RT shipment / instructions) until shipment)Heart Yes, whole organ Base of heart fixed Apical part of the heart and stored in ethanol flash frozen and stored at -80°CTibia Length for Not applicable Not applicablenormalization (discarded) (discarded)Kidneys Yes, whole organ Left kidney fixed Right kidney flash and stored in frozen and storedethanol.ParametersDescription Characteristics Condition 1 Condition 2 Animal Sprague Dawley rats, 72 ratsmaleHousing before 2 rats per cage 72 rats 13 weeks of housing treatmentHousing after 2 rats per cage 60 rats 4 weeks treatmentFormulation Lorundrostat HBr 2 times Estimated time of preparation (every 2 wks) 4 times preparation Semaglutide (weekly) 2 hours per compound Screening E / A, E7A’ and EF 72 rats 6 groups procedure HOMA-IR and BWBlood collection Sublingual / RO / tail 72 and 60 rats Week 12 & 18 (once) vein 60 rats Week 17 (3 time points)Drug treatment Oral BID / s.c. QD 60 rats For 28 days Echography Cardiac geometry / 72 rats 2 times, at baseline Systolic & diastolic 60 rats and treatment end functionMillar catheter Left ventricular 60 rats Once (terminal)PressureOrgans collection 2 organs 60 rats Weight and fixation Biochemical Glucose, Insulin, 72 rats 2 times, at baseline Analysis leptin, Cholesterol, 60 rats and treatment Adiponectin endSamples PK samples after Dry iceshipment collection on W14Data Analysis

[0197] Means ± standard deviation (S. EM, S. D) will be presented in tables, and mean ± S. EM in graphics. Statistical analysis will be performed by one-way or two-way ANOVA when appropriate to compare more than 2 groups, and with Student’s t-test to compare two groups (GraphPad Prism 9). For all tests, the significant level will be set at p < 0.05.ResultsE / A Ratio

[0198] The E / A ratio of study rats at weeks 12 and 18 (Figure 18) shows a trend of improved diastolic dysfunction in rats treated with lorundrostat alone and semaglutide alone. The combination of lorundrostat and semaglutide had the greatest effect on diastolic function, with the E / A ratio returning to the level of the lean control. E / A ratio is a measure of how well the left ventricle fills with blood during diastole. The decrease in E / A ratio of the obese rat indicates diastolic dysfunction. Treatment with lorundrostat and treatment with the combination of lorundrostat with semaglutide restored the E / A ratio to the level of the lean control.E ’ / A ’ Ratio

[0199] The E7A’ ratio is the mitral annular velocity from tissue doppler. The significant increase in E7A’ ratio of study rats treated with lorundrostat at both doses as well as in combination with semaglutide at week 18 relative to week 12 (Figure 19) confirmed improvement in heart relaxation properties.IVRT

[0200] Isolvolumic relaxation time (IVRT), is the measure of the time interval between aortic closure and mitral opening. In diastolic dysfunction the IVRT increases. Mitigated effects on IVRT were observed, with significant improvement in lorundrostat at the highest dose used either alone or with semaglutide (Figure 20, p<0.01 for both).Example 3Study 1Trial Design and Population

[0201] Study 1 was a global, randomised, double-blind, placebo-controlled trial that screened patients at 159 sites across 13 countries. Patients were recruited by investigators from clinics in primary and secondary care settings. The trial comprised of three phases: a screening period, a 2-week single-blind placebo run-in period, and a 12-week randomised, double-blind, treatment period. Upon completion, participants had the option to enroll into an open-label extension (OLE) trial with lorundrostat or to attend a 2-week safety follow-up. Participants continued taking their prescribed AHTs for the duration of the trial, with doses remaining stable for at least the first 6weeks in the double-blind period unless warranted for safety based on investigator’s discretion. All participants provided written informed consent before enrolment. The trial protocol was approved by an institutional review board or independent ethics committee at each site. The trial was conducted in accordance with the principles of the Declaration of Helsinki and the Good Clinical Practice guidelines of the International Council for Harmonization. An independent, unblinded data monitoring committee monitored safety during the trial.

[0202] Males or females aged 18 years or older with unattended automated office systolic BP (AOSBP) of 135-180 mmHg and diastolic BP of 65-110 mmHg, or isolated automated office diastolic BP of 90-110 mmHg, on stable doses of two to five AHTs, including a thiazide or thiazide-like diuretic, were eligible for inclusion. The key exclusion criteria were an estimated glomerular filtration rate (eGFR) <45 ml / min / 1.73m2, serum potassium >5.0 mmol / L at screening or >4.8 mmol / L at randomisation, and serum sodium <135 mmol / L at the screening visit. The use of epithelial sodium channel inhibitors or MRAs / potassium sparing diuretics was prohibited from 4 weeks prior to screening and throughout the trial. Full inclusion and exclusion criteria are provided in the protocol for clinical trial NCT06153693 available at clinicaltrials.gov, which is hereby incorporated by reference.Single blind run-in

[0203] Patients eligible at screening proceeded to a 2-week single-blind placebo run-in period. At the end of this period, eligible patients had a baseline visit to confirm eligibility for randomisation into the double-blind treatment period.Randomisation and masking

[0204] Participants were randomly assigned in a ratio of 1:2:1 to receive placebo QD for 12 weeks, lorundrostat 50mg QD for 12 weeks, or lorundrostat 50mg QD for 6 weeks followed by lorundrostat 100mg QD for 6 weeks for participants who met the following criteria at Week 6: AOSBP >130 mmHg, serum potassium <4.8 mmol / L, serum sodium >135 mmol / L, and eGFR >45 mL / min / 1.73m2and <25% reduction in eGFR from baseline. Participants in this group who did not meet all the above criteria at Week 6 remained on lorundrostat 50mg QD for the remaining 6 weeks of double-blind treatment. Trial medication dose could be reduced, temporarily held, or permanently discontinued for safety concerns at investigator discretion. Randomisation used random permuted blocks, and was stratified by geography (USA, Canada, Europe, Australia). Therandomization sequence was implemented by a Randomisation and Trial Supply Management system. Placebo tablets were identical in colour, shape, and packaging to the tablets containing lorundrostat. Participants were taking two tablets at each dosing to assure blinding of dose escalation at Week 6. Placebo administration was masked to participants during the single-blind run-in. The randomized trial treatment assignment was masked to participants, investigators, those assessing the outcomes, and those analyzing the data until the final database lock. Patients were enrolled into the trial by Investigators.Procedures

[0205] Lorundrostat tablets or matching placebo tablets were administered orally once daily at approximately the same time each morning. Participants were reminded about taking their trial medication using a smart phone application (AiCure).

[0206] Unattended AOBP was assessed at screening, randomisation (baseline), and Weeks 2, 4, 6, 8, and 12 during the double-blind treatment phase and at an optional safety follow-up visit. Each site was provided with an identical automated oscillometric BP device (MicroLife WatchBP Office AFIB, Microlife USA, Inc., Clearwater, FL), training, and an instruction manual for collecting BP measurements. BP was measured with an appropriately sized cuff on the same arm of the participant throughout the trial at approximately the same time, preferably morning. After an initial 5-minute rest period, five unattended seated measurements were performed with a 1-minute period between each reading; the last four measures were used to calculate the average. A standing measurement followed the seated measurements to monitor for orthostatic hypotension. Patient Characteristics

[0207] Between November 24, 2023 and September 16, 2024, 3161 patients were screened, 1575 entered the placebo-run in, 1083 were randomised to the double-blind treatment period, and 1078 received at least one dose of either placebo for 12 weeks (n=270), lorundrostat 50mg for 12 weeks (n=538), or lorundrostat 50mg for 6 weeks with the potential to increase to 100mg (n=270).

[0208] The randomised groups were comparable in baseline demographic and clinical characteristics (Table 1). Mean age was 61.6 years, 508 (46.9%) were female, 733 (67.7%) were White, 311 (28.7%) were Black or African American, and 685 (63.3%) had BMI >30 kg / m2. At randomization, 432 (39.9%) participants were on 2 AHTs and 651 (60.1%) were on >3 AHTs.These included 929 (85.6%) who were on an angiotensin-converting enzyme inhibitor (ACEi) or angiotensin receptor blocker (ARB), 553 (51.1%) on a CCB, and 1038 (96.3%) on a diuretic. Ninety-two (34.1%) participants were escalated to 100mg at Week 6. The trial was completed by 991 (91.5%) participants. The most common reasons for early discontinuation were withdrawal of consent (60, 5.5%), lost to follow-up (13, 1.2%), and other (13, 1.2%).Table 1 - Participant demographics and characteristics at baseline in the intention-to-treat populationPlacebo Lorundrostat Lorundrostat 50 mg 50 mg to 100 mg (n=272)(n=541) (n=270) Age (years) 61.8 (10.4) 61.7 (10.6) 61.4 (10.3) SexMale 139 (51.1) 294 (54.3) 142 (52.6) Female 133 (48.9) 247 (45.7) 128 (47.4) RaceWhite 177 (65.1) 366 (67.7) 190 (70.4) Black or African American 87 (32.0) 152 (28.1) 72 (26.7) Asian 6 (2.2) 14 (2.6) 4 (1.5) Native Hawaiian or Other Pacific Islander 1 (0.4) 1 (0.2) 0 (0) American Indian or Alaska Native 0 (0) 0 (0) 1 (0.4) Multiracial 1 (0.4) 2 (0.4) 0 (0)Not reported 0 (0) 6 (1.1) 3 (1.1) EthnicityHispanic or Latino 101 (37.1) 191 (35.3) 102 (37.8) Body mass indexkg / m232.60 (7.45) 33.00 (6.87) 32.81 (6.65) <30 kg / m2102 (37.5) 190 (35.1) 104 (38.5) >30 kg / m2168 (61.8) 351 (64.9) 166 (61.5) Estimated glomerular filtration rate at randomizationmL / min / 1.73m291.2 (16.37) 90.1 (17.77) 92.8 (17.55) <60 ml / min / 1.73 m210 (3.7) 38 (7.1) 15 (5.6) >60 ml / min / 1.73 m2260 (96.3) 500 (92.9) 255 (94.4) Diabetes 89 (33.0) 173 (32.2) 76 (28.2) Number of antihypertensive drugs2 113 (41.5) 213 (39.4) 106 (39.3) >3 159 (58.5) 328 (60.6) 164 (60.7) Antihypertensive drug classThiazide or thiazide-like diuretic 259 (95.2) 520 (96.1) 259 (95.9) ACE inhibitor or ARB 225 (82.7) 467 (86.3) 237 (87.8) Calcium channel blocker 135 (49.6) 278 (51.4) 140 (51.9) GLP-1 RA drug 9 (3.31) 30 (5.55) 13 (4.81) SGLT2i drug 13 (4.8) 21 (3.9) 14 (5.2) AOBP at randomisation (mm Hg)Systolic 148.8 (12.2) 149.0 (12.2) 146.6 (11.1) Diastolic 87.1 (9.1) 87.8 (9.0) 86.3 (9.2) Data are n (%) or mean (SD). ACE=angiotensin-converting enzyme; AOBP, automated office blood pressure; ARB=angiotensin receptor blocker; GLP-1 RA, glucagon-like peptide-1 receptor agonists; SGLT2i, sodium-glucose co-transporter 2 inhibitors.Study 2Trial Design and Population

[0209] Study 2 was a multicenter, prospective, randomized, double-blind, placebo-controlled, phase 2b clinical trial was performed at 103 sites within the United States. The trial enrolled adults ages 18 years of age or older who were receiving stable doses of 2 to 5 antihypertensive medications and had an automated office systolic blood pressure between 140 and 180 mmHg and diastolic blood pressure between 65 and 110 mmHg, or a diastolic blood pressure of 90 to 110 mmHg. Exclusion criteria included an estimated glomerular filtration rate of less than 45 mL per minute per 1.73 m2of body surface area, serum potassium greater than 5.0 mmol / L, or serum sodium less than 135 mmol / L. Full inclusion and exclusion criteria are provided in the protocol for clinical trial NCT05769608 available at clinicaltrials.gov, which is hereby incorporated by reference.

[0210] For eligible participants, their individual antihypertensive medications were discontinued, and a standardized regimen initiated. Participants taking 2 antihypertensive medications upon enrollment were changed to once daily olmesartan 40 mg and indapamide 2.5 mg or hydrochlorothiazide 25 mg. For participants taking 3 to 5 antihypertensive medications, amlodipine 10 mg daily was also included in the standardized regimen. Olmesartan 20 mg or amlodipine 5 mg daily were allowed at the discretion of local investigators.

[0211] Following three weeks of standardized regimen with a concomitant single-blind placebo run-in period, 24-hour ambulatory blood pressure monitoring (ABPM) was performed. Participants in whom blood pressure remained uncontrolled (24-hour average systolic blood pressure between 130 and 180 mmHg, or a 24-hour average diastolic blood pressure of greater than 80 mmHg) were randomized 1:1:1 to receive daily doses of either placebo, 50 mg of lorundrostat for twelve weeks, or 50 mg of lorundrostat for at least 4 weeks with an increase to 100 mg for the remaining 8 weeks if office systolic blood pressure was greater than 130 mmHg at week 4, and other biomarker criteria were met. These criteria were a serum potassium less than 4.8 mmol / L, sodium greater than 135 mmol / L, estimated glomerular filtration rate greater than 45 mL / min / 1.73m2, and a less than 25% reduction in estimated glomerular filtration rate from randomization. Randomization was stratified by the number of medications in the standardized regimen. Twenty-four-hour ABPM was performed at randomization, week 4, and week 12. Following the week 12 ABPM, 151 (53.5%) participants entered a 4-week washout period.Patient Characteristics

[0212] From March 2023 until October 2024, 2617 patients were screened and 926 were provided the standardized regimen during the placebo run-in period. In all, 285 participants were randomized and 282 received at least one dose of either 50 mg of lorundrostat once daily (94 participants), 50 to 100 mg daily (94 participants), or placebo (94 participants). Baseline characteristics were broadly similar among treatment groups, although more patients with diabetes were randomized to the lorundrostat 50 to 100 mg group (Table 1). The mean participant age was 60 years, 172 (60%) were men, and 150 were Black race (53%). The trial population was representative of the United States’ population with uncontrolled and resistant hypertension (Table 2). At randomization, 177 (63%) participants were taking a 2-drug standardized regimen, while others were receiving 3. Most participants, 174 (61%) were provided indapamide, rather than hydrochlorothiazide, as the standardized regimen diuretic. Of participants randomized to the dose titration strategy of 50 to 100 mg of lorundrostat, 19 (20%) escalated to 100 mg at week 4. A total of 262 participants completed week 4 ABPM and 241 participants completed the 12-week ABPM.Table 2. Representativeness of trial populationDisease Under Investigation Uncontrolled and Resistant HypertensionSpecial Considerations related toSex and gender The age-adjusted prevalence of hypertension among US adults 20 years of age or older was estimated to be 47% in NHANES in 2017 to 2020 (50% for men and 43% for women).Data on gender identity and uncontrolled or resistant hypertension are limited. There is also no known impact on gender identity on the prevalence and outcomes associated with uncontrolled and resistant hypertension.Race and Ethnicity Uncontrolled hypertension is prevalent among all races and ethnicities. In the United States, the prevalence of uncontrolled hypertension based on NHANES 2017 to 20202 data is 73% among non-Hispanic Whites, 79% among nonHispanic Blacks, and 78% among Mexican Americans.Resistant hypertension is prevalent among all races and ethnicities. In the United States, the percentage of adults taking antihypertensive medication with apparent treatment-resistant hypertension is 19% of non-Hispanic Whites. 27% of nonHispanic Blacks, and 18% among Hispanic adults.Age As age increases the prevalence of resistant hypertension increases, as does the risk for adverse events attributed to uncontrolled hypertension, namely atherosclerotic cardiovascular disease, heart failure, stroke, and chronic kidney disease.The prevalence of resistant hypertension among age groups in the United States is 14% among those 50-59 years of age, 20% among those 60-69 years of age, and 29% among those greater than or equal to 70 years of age.Overall representativeness of The trial was designed to enroll United States patient with uncontrolled and this trial resistant hypertension based on 24-hour ambulatory blood pressure of >130 / 80 mm Hg while taking 2 or 3 standardized antihypertensive medications.The population was largely representative of the uncontrolled and resistant hypertension population in the United States, albeit with a higher a higher proportion of Black patients. This is notable because of the higher prevalence of resistant hypertension among Black adults in the United States compared with other races.Investigators recorded their participants age, sex, gender, race and ethnicity in an electronic case report form. Participant race and ethnicity were self-reported. Data cited from:Carey RM, Sakhuja S, Calhoun DA, Whelton PK. MuntnerP. Prevalence of Apparent Treatment-Resistant Hypertension in the United States. Hypertension. 2019;73(2) 424-431. Martin SS, Aday AW, Allen NB, et al. 2025 Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart Association. Circulation. 2025;151(8):e41-e660. Richardson LC, Vaughan AS. Wright JS, Coronado F.Examining the Hypertension Control Cascade in Adults With Uncontrolled Hypertension in the US. JAMA Netw Open. 2024;7(9):e2431997.Serum Analysis of Study 1 and Study 2 participants

[0213] As summarized in Figure 21, over 900 serum analytes from 1,853 samples from 986 subjects of Study 1 and Study 2 were analyzed after comprehensive QC.

[0214] 849 (86.10%) subjects had paired baseline and week-12 samples.

[0215] A custom 1,053 analyte panel (974 genes) combining SomaLogic CV panel (953 analytes) and literature- and expert-curated set (100 analytes) was used to analyze the samples.

[0216] As shown in Figure 22, lorundrostat treatment is associated with significant reductions in candidate causal risk biomarkers for heart failure. Risk biomarkers of incident disease were evaluated to determine whether treatment modulates these protein sets set as a whole. Next, individual analytes from each risk biomarker set were evaluated. It was determined that these analytes were significantly decreased under treatment.REFERENCES

[0217] Castiglione, V., et al. (2023). Left Ventricular Diastolic Dysfunction. In: Dorobantu, M., Voicu, V., Grassi, G., Agabiti-Rosei, E., Mancia, G. (eds) Hypertension and Heart Failure. Updates in Hypertension and Cardiovascular Protection. Springer, Cham.

[0218] Hicks, Karen A., et al. “2014 ACC / AHA key data elements and definitions for cardiovascular endpoint events in clinical trials: a report of the American College of Cardiology / American Heart Association Task Force on Clinical Data Standards (Writing Committee to Develop Cardiovascular Endpoints Data Standards).” Circulation 132.4 (2015): 302-361.

[0219] Schauer, Antje, et al. “ZSF1 rat as animal model for HFpEF: Development of reduced diastolic function and skeletal muscle dysfunction.” ESC Heart Failure 7.5 (2020): 2123-2134.

[0220] Bowen, T. Scott, et al. “Exercise training reveals inflexibility of the diaphragm in an animal model of patients with obesity-driven heart failure with a preserved ejection fraction.” Journal of the American Heart Association 6.10 (2017): e006416.

[0221] Davila, Alec, et al. “Adenosine kinase inhibition augments conducted vasodilation and prevents left ventricle diastolic dysfunction in heart failure with preserved ejection fraction.” Circulation: Heart Failure 12.8 (2019): e005762.

[0222] Leite, Sara, et al. “Arterial remodeling and dysfunction in the ZSF1 rat model of heart failure with preserved ejection fraction.” Circulation: Heart Failure 12.7 (2019): e005596.

[0223] Miranda-Silva, Daniela, et al. “Disturbed cardiac mitochondrial and cytosolic calcium handling in a metabolic risk-related rat model of heart failure with preserved ejection fraction.” Acta physiologica 228.3 (2020): el3378.

Claims

CLAIMSWhat is claimed is:

1. A method of treating left ventricular diastolic dysfunction (LVDD) in a subject afflicted with LVDD, the method comprising administering to the subject an aldosterone synthase inhibitor once per day in an amount effective to treat LVDD in the subject.

2. The method of claim 1, wherein treating LVDD comprises:a) increasing the subject’s E / A ratio relative to the subject’s E / A ratio prior to treatment with the aldosterone synthase inhibitor and / or relative to the subject’s E / A ratio in the absence of treatment with the aldosterone synthase inhibitor; b) increasing the subject’s E’ / A’ ratio relative to the subject’s E’ / A’ ratio prior to treatment with the aldosterone synthase inhibitor and / or relative to the subject’s E’ / A’ ratio in the absence of treatment with the aldosterone synthase inhibitor; and / orc) increasing the subject’s isovolumic relaxation time (IVRT) relative to the subject’s IVRT prior to treatment with the aldosterone synthase inhibitor and / or relative to the subject’s IVRT in the absence of treatment with the aldosterone synthase inhibitor.

3. The method of claim 1 or 2, wherein the subject is afflicted with both LVDD and heart failure with preserved ejection fraction (HFpEF), preferably wherein the aldosterone synthase inhibitor is administered to the subject in an amount effective to treat both LVDD and HFpEF in the subject.

4. The method of any one of claims 1 to 3, wherein the subject is afflicted with LVDD, HFpEF, and hypertension.

5. The method of claim 3 or 4, wherein treating HFpEF comprises:a) stabilizing the subject’s Left Ventricular Diastolic Diameter (LVEDD) relative to the LVEDD of a subject not treated with the aldosterone synthase inhibitor; b) stabilizing the subj ect’ s Left Ventricular End Systolic Diameter (LVESD) relative to the LVESD of a subject not treated with the aldosterone synthase inhibitor;c) stabilizing the subj ect’ s Left Ventricular End Diastolic Volume (LVEDV) relative to the LVEDV of a subject not treated with the aldosterone synthase inhibitor; d) stabilizing the subject’s Left Ventricular End Systolic Volume (LVESV) relative to the LVESV of a subject not treated with the aldosterone synthase inhibitor; e) preventing an increase in the subject’s Mitral Valve (MV) E / E’ ratio or reducing the subject’s Mitral Valve (MV) E / E’ ratio relative to the MV E / E’ ratio of a subject not treated with the aldosterone synthase inhibitor;f) preventing an increase in the subject’s MV E / A’ ratio or reducing the subject’s MV E / A’ ratio relative to the MV E / A’ ratio of a subject not treated with the aldosterone synthase inhibitor;g) reducing levels of endothelial dysfunction related biomarkers Endothelin-1 and Intercellular adhesion molecule- 1 relative to the levels of Endothelin-1 and Intercellular adhesion molecule-1 of a subject not treated with the aldosterone synthase inhibitor; and / orh) reducing levels of one or more biomarkers associated with HFpEF, preferably wherein the one or more biomarkers are selected from the group consisting of MF AIM, SVEPEEGF-like domains 4-6; N-terminal pro-BNP, SVEP: Sushi 15-18, NRP1, Spondin-1, ITIH3, BNP, and IGFBP-7 relative to the levels of said one or more biomarkers of a subject not treated with the aldosterone synthase inhibitor.

6. The method of any one of claims 3 to 5, wherein treating HFpEF comprises reduction in the frequency and / or likelihood of hospitalization for heart failure (HHF), preferably wherein the reduction is a reduction in the subject’s odds ratio (OR) of HHF of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

7. The method of any one of claims 3 to 6, wherein treating HFpEF comprises reduction in the likelihood of cardiovascular (CV) death, preferably wherein the reduction is a reduction in the subject’s odds ratio (OR) of CV death of at least 10%, at least 20%, at least 50%, 10- 20%, 20-50%, or 50-75%.

8. The method of any one of claims 3 to 7, wherein treating HFpEF comprises reduction in the frequency or likelihood of heart failure (HF) events, preferably wherein the reductionis a reduction in the subject’s odds ratio (OR) of HF events of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

9. The method of any one of claims 3 to 8, wherein treating HFpEF comprises improvement in the subject’s exercise capacity as measured by an increase in the subject’s six-minute walk distance, preferably wherein the improvement is by at least 5 meters, more preferably by at least 10 meters.

10. The method of any one of claims 3 to 9, wherein treating HFpEF comprises an increase in the subject’s peak oxygen consumption, preferably wherein said increase is by at least 2.5 mL / kg / min, by at least 5 mL / kg / min, by at least 10 mL / kg / min, by 2.5-5 mL / kg / min, 5-10 mL / kg / min, or 10-20 mL / kg / min.

11. The method of any one of claims 3 to 10, wherein treating HFpEF comprises improvement in the subject’s Kansas City Cardiomyopathy Questionnaire (KCCQ) Clinical Summary Score (CSS), preferably wherein the improvement is by at least 5 points, by at least 10 points, by at least 20 points, by 5-10 points, by 10-20 points, or by 20-50 points.

12. A method of treating heart failure with preserved ejection fraction (HFpEF) in a subject afflicted with HFpEF, the method comprising administering to the subject an aldosterone synthase inhibitor once per day in an amount effective to treat HFpEF in the subject.

13. The method of claim 12, wherein the subject is afflicted with both hypertension and HFpEF.

14. The method of claim 12 or 13, wherein treating HFpEF comprises reduction in the frequency and / or likelihood of hospitalization for heart failure (HHF), preferably wherein the reduction is a reduction in the subject’s odds ratio (OR) of HHF of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

15. The method of any one of claims 12 to 14, wherein treating HFpEF comprises reduction in the likelihood of cardiovascular (CV) death, preferably wherein the reduction is a reduction in the subject’s odds ratio (OR) of CV death of at least 10%, at least 20%, at least 50%, 10- 20%, 20-50%, or 50-75%.

16. The method of any one of claims 12 to 15, wherein treating HFpEF compri ses reduction in the frequency or likelihood of heart failure (HF) events, preferably wherein the reduction is a reduction in the subject’s odds ratio (OR) of HF events of at least 10%, at least 20%, at least 50%, 10-20%, 20-50%, or 50-75%.

17. The method of any one of claims 12 to 16, wherein treating HFpEF comprises improvement in the subject’s exercise capacity as measured by an increase in the subject’s six-minute walk distance, preferably wherein the improvement is by at least 5 meters, more preferably by at least 10 meters.

18. The method of any one of claims 12 to 17, wherein treating HFpEF comprises an increase in the subject’s peak oxygen consumption, preferably wherein said increase is by at least 2.5 mL / kg / min, by at least 5 mL / kg / min, by at least 10 mL / kg / min, by 2.5-5 mL / kg / min, 5-10 mL / kg / min, or 10-20 mL / kg / min.

19. The method of any one of claims 12 to 18, wherein treating HFpEF comprises improvement in the subject’s Kansas City Cardiomyopathy Questionnaire (KCCQ) Clinical Summary Score (CSS), preferably wherein the improvement is by at least 5 points, by at least 10 points, by at least 20 points, by 5-10 points, by 10-20 points, or by 20-50 points.

20. The method of any one of claims 12 to 19, wherein the aldosterone synthase inhibitor is administered in an amount effective to treat left ventricular diastolic dysfunction (LVDD) in the subject.

21. The method of any one of claims 1 to 20, wherein the aldosterone synthase inhibitor is administered to the subject in an amount sufficient to inhibit 50% or more of aldosterone synthase’s activity for between 8 and 18 hours of a 24-hour period, preferably wherein the amount is sufficient to inhibit 50% or more of aldosterone synthase’s activity for between 10 and 14 hours of a 24-hour period.

22. The method of any one of claims 1 to 21, wherein the aldosterone synthase inhibitor is administered to the subject once per day in the morning.

23. The method of any one of claims 1 to 21, wherein the aldosterone synthase inhibitor is administered to the subject once per day in the evening.

24. The method of any one of claims 1 to 23, wherein the aldosterone synthase inhibitor is administered to the subject daily:a) for at least one week;b) for at least two weeks;c) for at least four weeks;d) for at least eight weeks;e) for at least twelve weeks;f) for at least twenty -four weeks;g) for at least fifty weeks;h) for at least one hundred weeks; ori) for at least two hundred weeks,to thereby treat LVDD and / or HFpEF in the subject.

25. The method of any one of claims 1 to 24, wherein the aldosterone synthase inhibitor is lorundrostat or a pharmaceutically acceptable salt thereof.

26. The method of claim 25, wherein the amount of the lorundrostat or pharmaceutically acceptable salt thereof is 25-100 mg per day, preferably 25-75 mg per day, more preferably 40-60 mg per day, even more preferably 50 mg per day.

27. The method of any one of claims 25 to 26, wherein the lorundrostat or pharmaceutically acceptable salt thereof is the monohydrobromide salt of lorundrostat.

28. The method of any one of claims 1 to 26, the method further comprising administering to the subject a GLP-1 treatment concurrently or concomitantly.

29. The method of claim 28, wherein the GLP-1 treatment is selected from Dulaglutide (Trulicity®), Exenatide (Byetta®), Exenatide extended release (Bydureon®), Liraglutide (Victoza®), Lixisenatide (Adlyxin®), Semaglutide injection (e.g. Ozempic® or Wegovy®), and Semaglutide tablets (Rybelsus®).

30. The method of any one of claims 28 to 29, wherein said treating comprises increasing the subject’s E / A ratio, preferably wherein the increase in the subject’s E / A ratio is greater than the increase in E / A ratio caused by administration of the aldosterone synthase inhibitor or GLP-1 treatment alone, more preferably wherein the increase in the subject’s E / A ratio caused by administration of the aldosterone synthase inhibitor and the GLP-1 treatment is additive, even more preferably wherein the subject’s E / A ratio is restored to a normal level.

Images