Methods and materials for gender-dependent treatment of cardiovascular dysfunction

Abstract

To provide methods and materials involved in treating cardiovascular conditions such as calcific aortic valve stenosis.SOLUTION: For example, methods and materials for using sGC agonists or a combination of sGC agonists and PDE5A inhibitors to reduce calcification of heart valves and / or vessels or to slow progression of aortic sclerosis to calcific aortic valve stenosis are provided.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 832,139, filed April 10, 2019.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under TR000954 and HL092235 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] 1.Technical Field This document relates to methods and materials involved in the treatment of cardiovascular conditions, such as calcific aortic stenosis. For example, this document provides methods and materials using a soluble guanylate cyclase (sGC) agonist of the oxidized or non-oxidized form of sGC, or a combination of an sGC agonist and a cGMP-specific phosphodiesterase 5A (PDE5A) inhibitor, to reduce calcification of heart valves and / or blood vessels or to slow the progression of aortic sclerosis to calcific aortic stenosis in male patients. [Background technology]

[0004] 2. Background information Calcific aortic stenosis is a disease in which the aortic valve opening narrows. Symptoms of calcific aortic stenosis can vary depending on the severity of the valve stenosis. Patients with mild to moderate calcific aortic stenosis may be asymptomatic, while symptoms commonly occur in patients with severe calcific aortic stenosis. Symptoms can include progressive shortness of breath with exercise, fainting, chest pain, and sudden death. Summary of the Invention

[0005] This document provides methods and materials related to the treatment of gender-based cardiovascular conditions in mammals, such as calcific aortic stenosis. For example, this document provides methods and materials for using an sGC agonist or a combination of an sGC agonist and a PDE5A inhibitor in male patients to reduce calcification of heart valves and / or blood vessels or to slow the progression of aortic sclerosis to calcific aortic stenosis. As described herein, sGC agonists such as ataciguat can be used to slow the progression of aortic sclerosis to calcific aortic stenosis, but such treatment is more effective in males than in females. The ability to effectively slow the progression of aortic sclerosis to calcific aortic stenosis allows male patients to live longer and happier lives. In some cases, selectively slowing the progression of aortic sclerosis to calcific aortic stenosis in male patients allows female patients to avoid unnecessary treatment with sGC agonists. This document also provides methods and materials for using antifibrotic agents to treat females identified as having aortic sclerosis. In some cases, for example, if calcification is observed after treatment with antifibrotic agents, females can be treated with one or more sGC agonists. The methods disclosed herein can also be used to reduce blood pressure in mammals with or at risk of hypertension, particularly in the supine position. Furthermore, the methods disclosed herein can include treating diastolic dysfunction, alleviating elevated pulse pressure, and reducing blood pressure, particularly in the supine position. In some cases, these methods can be performed in a gender-dependent manner, where males are treated and females are excluded from treatment.

[0006] In one aspect, this document features a method of treating a mammal based on the mammal's sex, the method comprising: (a) identifying the mammal as having or at risk of developing heart valve or vascular calcification, (b) identifying the mammal as a male mammal or a female mammal, and (c) if the mammal is a male mammal, administering an sGC agonist to the male mammal, thereby slowing the progression of heart valve or vascular calcification in the male mammal, and if the mammal is a female mammal, not administering the sGC agonist to the female mammal, and optionally administering a non-sGC agonist treatment to the female mammal, thereby slowing the progression of heart valve or vascular calcification in the female mammal. The mammal may be a human. sGC agonists include ataciguat (5-chloro-2-[[(5-chloro-2-thienyl)sulfonyl]amino]-N-[4-(4-morpholinylsulfonyl)phenyl]-benzamide; HMR1766), YC-I (5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol), BAY 58-2667 (4-[((4-carboxybutyl)(2-[(4-phenethylbenzyl)oxy]phenethyl)amino)methyl[benzo]hydrochloride), and BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), BAY-41-8543 (2-[1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-(4-morpholinyl)-4,6-pyrimidinediamine), BAY 63-2521 (methyl(4,6-diamino-2-(1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)(methyl)carbamate), CFM-1571 (3-[3-(dimethylamino)propoxy]-N-(4-methoxyphenyl)-1-(phenylmethyl)1H-pyrazole-5-carboxamide hydrochloride), A-350619 (3-[2-(4-chlorophenylthio)phenyl]-N-(4-dimethylaminobutyl)acrylamide), vericiguat (methyl(4,6-diamino-2-(5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)carbamate), praliciguat (1,1,1,3,3,3-hexafluoro-2-[({5-fluoro-2-[1-(2-fluorobenzyl)-5-(1,2-oxazol-3-yl)-1H-pyrazol-3-yl]pyrimidin-4-yl}amino)methyl]propan-2-ol), olinciguat ((2R)-3,3,3-trifluoro-2-{[(5-fluoro-2-{1-[(2-fluorophenyl)methyl]-5-(1,2-oxazol-3-yl)-1H-pyrazole -3-yl}pyrimidin-4-yl)amino]methyl}-2-hydroxypropanamide), bis-heteroarylpyrazole IWP-051 (5-fluoro 2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl)pyrimidin-4(3H)-one), IW-6463, GSK2181236A (1-(6-{2-[({3-methyl-4'-[(trifluoromethyl)oxy]-4-biphenyl}methyl)oxy]phenyl}-2-pyridinyl)-5-(trifluoromethyl)-1H-pyrazole -4-carboxylic acid), IWP-550, IWP-854 (4-(5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl)pyrimidin-4-yl)-1-(3-methyl-3H-diazirin-3-yl)-N-(37-oxo-41-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azahentetracontyl)-7,10, 13,16-tetraoxa-4-azanonadecane-19-amide), IWP-953, nerociguat (methyl (4,6-diamino-2-(1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)carbamate), MGV354 ((S)-1-(6-(3-((4-(1-(cyclopropanecarbonyl)piperidin-4-yl)-2-methylphenyl)amino)-2,3-dihydro-1H-inden-4-yl)pyridin-2-yl)-5-methyl-1H-pyrazole-4-carboxylic acid), BI 703704 ((1R,5S,The PDE5A inhibitor may be 8s)-3-(4-(5-methyl-2-((2-methyl-4-(piperidine-1-carbonyl)benzyl)oxy)phenyl)thiazol-2-yl)-3-azabicyclo[3.2.1]octane-8-carboxylic acid), S3448 (2-[[(4-chlorophenyl)sulfonyl]amino]-4,5-dimethoxy-N-[4-(4-thiomorpholinylsulfonyl)phenyl]benzamide), or BAY 60-2770 (4-[[(4-carboxybutyl)[2-[5-fluoro-2-[[4'-(trifluoromethyl)[1,1'-biphenyl]-4-yl]methoxy]phenyl]ethyl]amino]methyl]benzoic acid). The method may further comprise administering a PDE5A inhibitor to the male mammal. The method may further include identifying the male mammal as having an elevated plasma level of lysophosphatidic acid (LPA). The method may include administering an anti-fibrotic agent to the female mammal to slow the progression of cardiac valve or vascular calcification in the female mammal.

[0007] In another aspect, this document features a method of treating a population of mammals identified as having or at risk of developing cardiac valve or vascular calcification, the population including at least one male mammal and at least one female mammal, the method comprising administering an sGC agonist to at least one male mammal, thereby slowing the progression of cardiac valve or vascular calcification in the at least one male mammal, and not administering an sGC agonist to the at least one female mammal. The mammal may be a human. The sGC agonist can be ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.The method can further comprise administering a PDE5A inhibitor to at least one male mammal.At least one male mammal can also be identified as having elevated plasma levels of LPA.

[0008] In another aspect, this document features a method of treating a population of mammals based on sex, the method comprising: (a) identifying the sex of mammals in the population determined to have or be at risk for developing cardiac valve or vascular calcification; and (b) administering an sGC agonist to the mammals identified as male mammals, not administering an sGC agonist to the mammals identified as female mammals, and optionally administering a non-sGC agonist treatment to the female mammals. The mammal may be human. The sGC agonist may be ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770. The method may further comprise administering a PDE5A inhibitor to the mammal identified as a male mammal. The mammal identified as a male mammal may also be determined to have an elevated plasma level of LPA. The method can include administering an anti-fibrotic agent to the mammal, identified as a female mammal.

[0009] In yet another aspect, this document features a method for avoiding the unnecessary use of an sGC agonist to treat a mammal having or at risk of developing cardiac valve or vascular calcification, the method comprising identifying the mammal as a female mammal having or at risk of developing cardiac valve or vascular calcification, and excluding the female mammal from treatment with an sGC agonist based at least in part on the female sex of the female mammal. The mammal may be a human. The sGC agonist may be ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

[0010] This document also features a method of treating a mammal based on the mammal's sex, comprising: (a) identifying the mammal as having or at risk of developing aortic sclerosis, (b) identifying the mammal as a male mammal or a female mammal, and (c) if the mammal is a male mammal, administering an sGC agonist to the male mammal, thereby slowing the progression of aortic sclerosis to calcific aortic stenosis in the male mammal, and if the mammal is a female mammal, not administering the sGC agonist to the female mammal, and optionally administering a non-sGC agonist treatment to the female mammal, thereby slowing the progression of aortic sclerosis to calcific aortic stenosis in the female mammal. The mammal may be a human. The sGC agonist may be ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.The method may further comprise administering a PDE5A inhibitor to the male mammal.The method may further comprise identifying the male mammal as having an elevated plasma level of LPA. The method can include administering to the female mammal an anti-fibrotic agent.

[0011] In another aspect, this document features a method of treating a population of mammals identified as having or at risk of developing aortic sclerosis, the population including at least one male mammal and at least one female mammal, the method comprising administering an sGC agonist to at least one male mammal, thereby slowing the progression of aortic sclerosis to calcific aortic stenosis in the at least one male mammal, and not administering the sGC agonist to the at least one female mammal. The mammal may be a human. The sGC agonist may be vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448, or BAY 60-2770. The method may further include administering a PDE5A inhibitor to at least one male mammal.The method may further include identifying at least one male mammal as having an elevated plasma level of LPA.

[0012] In another aspect, this document features a method of treating a population of mammals based on sex, the method comprising: (a) identifying the sex of mammals in the population determined to have or be at risk of developing aortic sclerosis; and (b) administering an sGC agonist to the mammals identified as male mammals, not administering an sGC agonist to the mammals identified as female mammals, and optionally administering a non-sGC agonist treatment to the female mammals. The mammal may be human. The sGC agonist may be ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770. The method may further comprise administering a PDE5A inhibitor to the mammal identified as a male mammal. The mammal identified as a male mammal may further be determined to have an elevated plasma level of LPA. The method can include administering an anti-fibrotic agent to the mammal, identified as a female mammal.

[0013] In yet another aspect, this document features a method for avoiding the unnecessary use of an sGC agonist to treat a mammal having or at risk of developing aortic sclerosis, the method comprising identifying the mammal as a female mammal and identifying the mammal as having or at risk of developing aortic sclerosis, and excluding the female mammal from treatment with an sGC agonist based at least in part on the female sex of the female mammal. The mammal of this method may be a human. The sGC agonist may be ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

[0014] In another aspect, this document features a method of treating a mammal, the method including (a) identifying the mammal as having or at risk for hypertension when in the supine position, and (b) administering an sGC agonist to the mammal, thereby reducing blood pressure in the supine position. The mammal may be a human. The sGC agonist may be ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448, or BAY 60-2770. The method may further comprise administering a PDE5A inhibitor to the mammal. The method may further comprise identifying the mammal as a male mammal prior to administration. Administration may not reduce blood pressure when the mammal is in an upright position.

[0015] In yet another aspect, this document features a method of treating a population of mammals identified as having or at risk of developing supine hypertension, the population including at least one male mammal and at least one female mammal, the method comprising administering an sGC agonist to at least one male mammal, thereby reducing supine blood pressure in the at least one male mammal and preventing the harmful side effects of hypertension, and not administering the sGC agonist to the at least one female mammal. The mammal may be a human. The sGC agonist may be ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770. The method may further comprise administering a PDE5A inhibitor to at least one male mammal. The method may comprise administering an anti-fibrotic agent to at least one female mammal. Administration may not reduce blood pressure when the mammal is in an upright position.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not limiting.

[0017] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0018] [Figure 1A] Figures 1A and 1B: Graphs plotting phospho-VASP239 levels in aortic valve tissue from male (Figure 1A) and female (Figure 1B) mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) versus control (WD). These studies demonstrated that ataciguat effectively activated soluble guanylyl cyclase in treated mice. Phospho-VASP239 levels were assessed using immunohistochemistry and confocal microscopy. [Figure 1B] Continuation of Figure 1A. [Figure 2A-2B] Figure 2A shows graphs plotting the levels of phospho-SMAD1 / 5 / 8, key mediators of bone morphogenetic protein (BMP) signaling, in male (Figure 2A) and female (Figure 2B) mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) or control (WD). These studies demonstrated that ataciguat effectively reduced canonical bone morphogenetic signaling in both treated mice. Phospho-SMAD1 / 5 / 8 levels were assessed using immunohistochemistry and confocal microscopy. [Figure 3A-3B] Figures 3A-3D are a series of graphs plotting the levels of molecular markers of tissue calcification in male and female mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) or control (WD). Ataciguat effectively reduced both osteopontin (SPP1; Figures 3A and 3B) and Runx2 (a master regulator of bone formation; Figures 3C and 3D) in male (Figures 3A and 3C) and female (Figures 3B and 3D) mice, consistent with the reduction in tissue calcification observed in vivo (see Figures 4A and 4B). mRNA levels were assessed using quantitative real-time RT-PCR. [Figure 3C-3D] Continuation of Figures 3A-3B. [Figure 4A-4B]4A and 4B are graphs plotting the level of calcification in aortic valve tissue in male (FIG. 4A) and female (FIG. 4B) mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) or control (WD). Calcification was assessed histologically using alizarin red staining and subsequent brightfield microscopic imaging. [Figure 5A-5B] 5A and 5B are graphs plotting the levels of canonical transforming growth factor beta (TGFβ) signaling in aortic valve tissue from male (FIG. 5A) and female (FIG. 5B) mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) or control (WD). TGFβ signaling was assessed based on the levels of SMAD2 / 3 phosphorylation measured using immunohistochemistry and confocal microscopy. [Figures 6A-6B] Figures 6A-6D: Graphs plotting the levels of collagen isoform expression (a major determinant of tissue fibrosis) in male (Figures 6A and 6C) and female (Figures 6B and 6D) mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) or control (WD). HMR1766 did not significantly affect the expression of collagen 1a1 (the major structural collagen isoform) in male mice (Figure 6A), but significantly reduced Col1a1 expression in female mice (Figure 6B). Furthermore, the expression of Col15a1 (an important basement membrane structural isoform) increased in male mice (Figure 6C) but tended to decrease in female mice (Figure 6D). Taken together, these studies demonstrated that ataciguat did not alter or increase collagen isoform expression in males, but reduced collagen isoform expression in females. mRNA levels were assessed using quantitative real-time RT-PCR. [Figure 6C-6D] Continuation of Figures 6A-6B. [Figures 7A-7B]Figure 7A shows graphs plotting levels of matrix metalloproteinase 2 (MMP2, a major determinant of collagen fibril degradation / remodeling) expression in male (Figure 7A) and female (Figure 7B) mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) or control (WD). HMR1766 did not significantly affect MMP2 mRNA levels in either male or female mice. It was noted that female mice tended to have higher MMP2 mRNA levels than males in the same litter. Taken together, these data suggest that HMR1766 did not appear to affect matrix remodeling enzyme expression, but that female mice may be more prone to excessive matrix remodeling over time. mRNA levels were assessed using quantitative real-time RT-PCR. [Figures 8A-8D] Figures 8A and 8B show graphs plotting collagen fiber structure in male (Figures 8A and 8B) and female (Figures 8C and 8D) mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) or control (WD). While ataciguat did not affect relative collagen fiber width in male mice, ataciguat increased the proportion of thinner fibers (and reduced the proportion of thicker fibers) in female mice. Importantly, these changes are consistent with molecular changes in collagen isoforms and MMP2 expression in each sex. Collagen fiber width was assessed histologically using picrosirius red staining followed by microscopic imaging under circularly polarized light (leveraging the birefringent properties of collagen). [Figure 9A-9B] Figure 9A shows graphs plotting aortic valve function in male (Figure 9A) and female (Figure 9B) mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) or control (WD). Data were obtained using a previously validated approach measuring apex separation distance by high-resolution echocardiography, where a larger separation distance indicates better function. The data suggested that only male mice received therapeutic benefit from long-term treatment with ataciguat. [Figures 10A-10B]Figures 10A-10D: Graphs plotting measurements of ventricular function in male (Figures 10A and 10C) and female (Figures 10B and 10D) mice with established aortic valve disease after treatment with ataciguat / HMR1766 (WD+ATA) or control (WD). Consistent with previous reports showing resistance to systolic dysfunction despite moderate pressure overload in mice, ejection fraction was well preserved in WD-fed mice and was not further improved by ataciguat (Figures 10A and 10B). Diastolic function (measured using E / e', a clinically known predictor of poor outcome in many patient populations) was impaired in WD-fed mice of both sexes (normal values ​​in mice are approximately 20-25) and significantly improved in male mice (Figure 10C) but not in female mice (Figure 10D) after ataciguat treatment. Ventricular function was assessed using high-resolution echocardiography. [Figures 10C-10D] This is a continuation of Figures 10A-10B. [Figures 11A-11B] 11A-11D: Graphs plotting sitting systolic (FIGS. 11A and 11B) and diastolic (FIGS. 11C and 11D) blood pressure in humans with mild to moderate aortic stenosis before and after 14 days of treatment with placebo (FIGS. 11A and 11C) or ataciguat / HMR1766 (FIGS. 11B and 11D) (randomized, double-blind study design, indicated doses). As shown in FIGS. 11B and 11D, 100 mg / day of ataciguat significantly reduced blood pressure in the sitting position, while 200 mg / day had no significant effect on blood pressure. This suggested that higher doses of ataciguat would not put patients at risk for blood pressure-lowering side effects (such as dizziness). [Figures 11C-11D] Continuation of Figures 11A-11B. [Figures 12A-12B]12A-12D: Graphs plotting the change in systolic (FIGS. 12A and 12B) and diastolic (FIGS. 12C and 12D) blood pressure during standing in humans with mild to moderate aortic stenosis before and after 14 days of treatment with placebo (FIGS. 12A and 12C) or ataciguat (FIGS. 12B and 12D) (randomized, double-blind study design, indicated doses). Treatment with ataciguat did not significantly or consistently alter the blood pressure response to the orthostatic stress of transitioning from a sitting to standing position. This again suggested that ataciguat would not put patients at risk for blood pressure-lowering side effects. [Figures 12C-12D] Continuation of Figures 12A-12B. [Figures 13A-13B] 13A and 13B are graphs plotting the change in heart rate during standing in humans with mild to moderate aortic stenosis before and after 14 days of treatment with placebo (FIG. 13A) or ataciguat / HMR1766 (FIG. 13B) (randomized, double-blind study design, indicated doses). Consistent with the observed changes in blood pressure, treatment with ataciguat did not significantly or consistently alter the heart rate response to the orthostatic stress of transitioning from a sitting to standing position. This again suggests that ataciguat will not put patients at risk for blood pressure-lowering side effects and that excessive tachycardia is not a compensatory mechanism masking the undesirable effects of ataciguat on vascular tone / vasomotor regulation. [Figures 14A-14B] 14A-14D: Graphs plotting supine systolic (FIGS. 14A and 14B) and diastolic (FIGS. 14C and 14D) blood pressure in humans with mild to moderate aortic stenosis before and after 14 days of treatment with placebo (FIGS. 14A and 14C) or ataciguat / HMR1766 (FIGS. 14B and 14D) (randomized, double-blind study design, indicated doses). Treatment with ataciguat significantly reduced both systolic and diastolic blood pressure, suggesting that ataciguat may be a viable strategy for reducing vascular stiffness and nocturnal hypertension without exposing patients to symptoms related to a drop in blood pressure while standing. [Figure 14C-14D] Continuation of Figures 14A-14B. [Figures 15A-15B]Figures 15A-15D: Graphs plotting the change in systolic (Figures 15A and 15B) and diastolic (Figures 15C and 15D) blood pressure with progressive head-up tilt in humans with mild to moderate aortic stenosis before and after 14 days of treatment with placebo (Figures 15A and 15C) or ataciguat / HMR1766 (Figures 15B and 15D) (randomized, double-blind study design, indicated doses). Treatment with ataciguat did not consistently or significantly alter the blood pressure response to head-up tilt compared to pre-treatment or placebo-treated subjects. These observations are consistent with the more functional "standing test" data (Figures 12A-12D and 13A-13B), which was implemented as a more sensitive and controlled measure of orthostatic tolerance. [Figures 15C-15D] Continuation of Figures 15A-15B. [Figures 16A-16B] 16A and 16B are graphs plotting the change in heart rate with progressive head-up tilt in humans with mild to moderate aortic stenosis before and after 14 days of treatment with placebo (FIG. 16A) or ataciguat / HMR1766 (FIG. 16B) (randomized, double-blind study design, indicated doses). Treatment with ataciguat did not consistently or significantly alter the heart rate response to head-up tilt compared to pre-treatment or placebo-treated subjects, again suggesting that ataciguat will not put patients at risk for blood pressure-lowering side effects and that excessive tachycardia is not a compensatory mechanism masking the undesirable effects of ataciguat on vascular tone / vasomotor regulation. [Figures 17A-17C] 17A-17C are graphs plotting the change in aortic valve calcification in humans with mild to moderate aortic stenosis before and after 6 months of treatment with placebo or ataciguat / HMR1766 (200 mg / day, once daily) (randomized, double-blind study design). When data from subjects of both genders were combined, treatment with ataciguat significantly attenuated the progression of valve calcification (FIG. 17A). However, when the data were separated by gender, it became clear that male patients (FIG. 17B) received significantly greater therapeutic benefit from treatment with ataciguat than females (FIG. 17C). [Figures 18A-18C]Figures 18A-18F: Graphs plotting the change in aortic valve function in humans with mild to moderate aortic stenosis before and after 6 months of treatment with placebo or ataciguat / HMR1766 (200 mg / day, once daily) (randomized, double-blind study design). Using changes in aortic valve area (Figures 18A-18C) as a measure of valve function, it was concluded that treatment with ataciguat can attenuate the progression of aortic valve dysfunction in subjects of both genders combined (Figure 18A). Similar to changes in valve calcium, the therapeutic benefit was primarily conferred to male patients (Figure 18B) compared with female patients (Figure 18C). Although changes in peak transvalvular velocity (Figures 18D-18F) as a measure of valve dysfunction may be considered to indicate negligible effects of ataciguat on the progression of valve dysfunction, this measure is critically influenced by changes in left ventricular systolic function. As shown in Figures 19A-19F, the improvement in left ventricular systolic function accounted for the artifactual "masking" of therapeutic benefit when using velocity measurements. [Figures 18D-18F] Continuation of Figures 18A-18C. [Figures 19A-19C] 19A-19F: Graphs plotting the change in left ventricular systolic function in humans with mild to moderate aortic stenosis before and after 6 months of treatment with placebo or ataciguat / HMR1766 (200 mg / day, once daily) (randomized, double-blind study design). Using left ventricular ejection fraction (FIGS. 19A-19C) or left ventricular stroke volume (FIGS. 19D-19F) as a measure of valve function, it was concluded that treatment with ataciguat may prevent the decline in left ventricular function observed in the placebo group when all subjects were combined (FIGS. 19A and 19D). However, when only male subjects were evaluated, the data suggest that ataciguat not only prevented the decline in left ventricular function but also improved left ventricular systolic function (Figures 19B and 19E), which may serve to mask the preserved valvular function, promoting the "artifactual" increase in peak transvalvular velocity observed in Figure 18. Female subjects did not appear to derive a consistent benefit from ataciguat on left ventricular systolic function (Figure 19C) and even demonstrated a potential deterioration in cardiac output (Figure 19F). [Figures 19D-19F]Continuation of Figures 19A-19C. [Figures 20A-20C] Figures 20A-20F: Graphs plotting the change in left ventricular diastolic function in humans with mild to moderate aortic stenosis before and after 6 months of treatment with placebo or ataciguat / HMR1766 (200 mg / day, once daily) (randomized, double-blind study design). Using changes in E / e' (Figures 20A-20C) or E / A ratios (Figures 20D-20F), the data suggested that ataciguat could attenuate the further progression of diastolic dysfunction associated with aortic stenosis when all subjects were combined (Figures 20A and 20D). However, when only male subjects were examined, it was clear that males were the primary recipients of any therapeutic benefit conferred by long-term ataciguat treatment (Figures 20B and 20E). In contrast, females received negligible therapeutic benefit in terms of left ventricular diastolic function from chronic treatment with ataciguat compared to placebo-treated subjects (Figures 20C and 20F). [Figures 20D-20F] This is a continuation of Figures 20A-20C. [Figures 21A-21C] 21A-21C are graphs plotting the change in aortic pulse pressure in seated humans with mild to moderate aortic stenosis before and after 6 months of treatment with placebo or ataciguat / HMR1766 (200 mg / day, once daily) (randomized, double-blind study design). When all subjects were combined, the data suggested that ataciguat attenuated pulse pressure (an index of arterial stiffness) (FIG. 21A). However, when examining data for male subjects only, it was clear that males were the primary recipients of any therapeutic benefit with respect to pulse pressure or arterial stiffness conferred by long-term ataciguat treatment (FIG. 21B). In contrast, females did not derive any clear therapeutic benefit from long-term treatment with ataciguat in terms of changes in pulse pressure function, compared with placebo-treated female subjects (FIG. 21C). [Figure 22]Figure 1 shows graphs resulting from the use of an unbiased machine learning / neural network algorithm to determine whether baseline phenotypic variables can predict progression of aortic valve dysfunction and response to treatment in patients. Patient gender, age, treatment group (ataciguat or placebo), and measurements from blood tests and echocardiograms were used as feature values ​​to train a neural network regression model to predict change in aortic valve area. Model performance was assessed through 10-fold cross-validation. The inset in the lower right shows the top eight variables that provided the most useful information for the model's decision-making process. Crucially, treatment group (placebo vs. ataciguat) and gender (male vs. female) were the two strongest predictive components identified from this unbiased approach. [Figure 23] 1 is a graph plotting the expression of the bone morphogenetic protein (BMP) target gene, Osterix (also known as the transcription factor Sp7), in aortic valve interstitial cells treated with nerociguat. Nerociguat concentrations between 1 nM and 1 μM reduced BMP signaling (arrows represent the range of effect). [Figure 24] 1 is a graph plotting the expression of Osterix in aortic valve interstitial cells treated with vericiguat. Only relatively high doses of vericiguat were effective in reducing BMP target gene expression (represented by arrows). [Figure 25] 1 is a graph plotting Osterix expression in aortic valve interstitial cells treated with rificiguat. Similar to nerociguat, rificiguat appeared to lose its effectiveness at higher concentrations in vitro. [Figure 26] 1 is a graph plotting Osterix expression in aortic valve interstitial cells treated with BAY41-8543. BAY41-8543 tended to promote a dose-dependent decrease in BMP target gene expression, and was highly effective at 10 μM. [Figure 27]This graph plots the expression of Osterix in aortic valve interstitial cells treated with lysophosphatidic acid (LPA; specifically, 18:0 lyso-PA) with or without ataciguat (ATA) to assess LPA-induced BMP signaling and BMP target gene expression. Treatment with 18:0 LPA increased BMP target gene expression (center bar), whereas ataciguat dramatically suppressed this effect (right bar). [Figure 28] 1 is a graph plotting the relationship between plasma LPA levels and baseline levels of valvular calcium (measured using CT scans) in patients with mild to moderate aortic stenosis. Higher plasma LPA levels were associated with greater calcium levels in this patient cohort. [Figure 29] 1 is a graph plotting the relationship between baseline plasma LPA levels and change in valve calcium (measured using CT scans) in patients with mild to moderate aortic stenosis treated with ataciguat for 6 months. Patients with higher levels of LPA tended to show greater attenuation of excessive BMP signaling when treated with ataciguat. DETAILED DESCRIPTION OF THE INVENTION

[0019] This document provides methods and materials for the treatment of cardiovascular conditions that are at least partially based on gender, such as calcific aortic stenosis.For example, this document provides methods and materials for using one or more sGC agonists or the combination of one or more sGC agonists and one or more PDE5A inhibitors to reduce the calcification of heart valves and / or blood vessels in male patients, or to slow the progression of aortic sclerosis to calcific aortic stenosis.As described herein, sGC agonists appear to be less effective in females than in males.Methods and materials for excluding females from such treatment are also provided herein, as are alternative methods for treating females.

[0020] In some cases, this document provides a method of treating a mammal based on sex, the method comprising identifying the mammal as having or at risk of developing a cardiovascular condition (e.g., heart valve calcification, vascular calcification, aortic sclerosis, arterial stiffness, or left ventricular systolic or diastolic dysfunction) and identifying the sex of the mammal. If the mammal is male, the method may comprise administering an sGC agonist to slow the progression of the cardiovascular condition (e.g., heart valve or vascular calcification or progression of aortic sclerosis to calcific aortic stenosis) in the male mammal. If the mammal is female, the method may exclude administering an sGC agonist to the mammal. In some cases, if the mammal is female, the method may comprise administering a non-sGC agonist treatment to slow the progression of the cardiovascular condition. In some cases, if the mammal is female, the method may comprise administering an anti-fibrotic agent to slow the progression of the cardiovascular condition. In some cases, if calcification occurs in a female mammal following administration of an anti-fibrotic agent, an sGC agonist can then be administered to the female mammal.

[0021] In some cases, the methods provided herein may involve treating a population of mammals, including at least one male and at least one female, identified as having or at risk of developing a cardiovascular condition (e.g., cardiac valve calcification, vascular calcification, aortic sclerosis, or left ventricular systolic or diastolic dysfunction). Such methods may involve administering an sGC agonist to at least one male mammal, thereby slowing the progression of the cardiovascular condition in the at least one male mammal, and not administering the sGC agonist to at least one female mammal. Thus, the methods provided herein may avoid the unnecessary use of sGC agonists to treat female mammals identified as having or at risk of developing a cardiovascular condition, such as cardiac valve calcification, vascular calcification, aortic sclerosis, or left ventricular systolic or diastolic dysfunction. In some cases, the methods may involve administering an anti-fibrotic agent to at least one female mammal. If calcification occurs in at least one female mammal following treatment with an anti-fibrotic agent, the mammal can be treated with an sGC agonist.

[0022] The methods disclosed herein can also be used to reduce blood pressure in mammals with or at risk of hypertension (e.g., supine hypertension).The methods can also be used to treat diastolic dysfunction and reduce elevated pulse pressure.In some cases, these methods can be performed gender-dependently, such that male mammals are treated with sGC agonists (with or without PDE5A inhibitors) and females are excluded from treatment.In some cases, these methods can be performed gender-dependently, such that female mammals are treated with antifibrotic agents.

[0023] Any type of mammal with cardiovascular conditions, such as calcific aortic stenosis, can be treated as described herein.For example, male humans and other primates, such as monkeys, with cardiovascular conditions, such as calcific aortic stenosis, can be treated with one or more sGC agonists, or one or more sGC agonists and one or more PDE5A inhibitors.In some cases, male dogs, cats, horses, cows, pigs, sheep, mice and rats can be treated with one or more sGC agonists or one or more sGC agonists and one or more PDE5A inhibitors as described herein.In some cases, female humans or other female mammals (such as non-human primates, dogs, cats, horses, cows, pigs, sheep, mice and rats) with cardiovascular conditions, such as calcific aortic stenosis, can be treated with one or more anti-fibrotic agents as described herein.

[0024] Any suitable method can be used to identify mammals with a cardiovascular condition, such as calcific aortic stenosis or aortic sclerosis. For example, echocardiography or computed tomography scans can be used to identify humans with aortic sclerosis who are at risk of progressing to calcific aortic stenosis.

[0025] In some cases, mammals can be identified for treatment based on elevated levels of LPA, a phospholipid derivative that can act as a signaling molecule and mitogen. Increased LPA levels can indicate an increased risk of aortic valve calcification and stenosis. Furthermore, as described in the Examples herein, elevated LPA levels can be a primary indicator of response to sGC agonists. As used herein with respect to blood levels of LPA, the term "elevated level" refers to any level above a threshold level (e.g., a threshold plasma level) for a control population of healthy mammals without the cardiovascular conditions described herein (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 age-matched healthy mammals). In some cases, the threshold LPA level can be, but is not limited to, 5 mg / dL or higher (e.g., 10 mg / dL, 20 mg / dL, 25 mg / dL, 30 mg / dL, 40 mg / dL, or 50 mg / dL). Any suitable method can be used to determine blood levels of LPA. For example, polypeptide detection methods, such as immunoassays (e.g., ELISA or radioimmunoassays) and mass spectrometry (e.g., LC-MS / MS) can be used to determine the level of LPA or a specific LPA species (e.g., 18:0 LPA) in a plasma or whole blood sample. In some cases, radioimmunoassays can be used to determine blood or plasma levels of LPA.

[0026] Once identified as having a cardiovascular condition, e.g., calcific aortic stenosis or aortic sclerosis that may progress to calcific aortic stenosis, and identified as a male mammal, the male mammal can be administered, or instructed to self-administer, one or more sGC agonists or a combination of one or more sGC agonists and one or more PDE5A inhibitors.

[0027] Examples of sGC agonists include, but are not limited to, ataciguat (5-chloro-2-[[(5-chloro-2-thienyl)sulfonyl]amino]-N-[4-(4-morpholinylsulfonyl)phenyl]-benzamide; HMR1766), YC-I (5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol), BAY 58-2667 (4-[((4-carboxybutyl)(2-[(4-phenethylbenzyl)oxy]phenethyl)amino)methyl[benzo]hydrochloride; cinaciguat), BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), BAY-41-8543 (2-[1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-(4-morpholinyl)-4,6-pyrimidinediamine), BAY 63-2521 (methyl (4,6-diamino-2-(1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl) (methyl)carbamate; riociguat), CFM-1571 (3-[3-(dimethylamino)propoxy]-N-(4-methoxyphenyl)-1-(phenylmethyl)-1H-pyrazole-5-carboxamide hydrochloride), A-350619 (3-[2-(4-chlorophenylthio)phenyl]-N-(4-dimethylaminobutyl)acrylamide), vericiguat (BAY 1021189, also known as MK-1242; methyl (4,6-diamino-2-(5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)carbamate), praliciguat (IW-1973, IWP-121; 1,1,1,3,3,3-hexafluoro-2-[({5-fluoro-2-[1-(2-fluorobenzyl)-5-(1,2-oxazol-3-yl)-1H-pyrazol-3-yl]pyrimidin-4-yl}amino)methyl]propan-2-ol), olinciguat (IW-1701;(2R)-3,3,3-trifluoro-2-{[(5-fluoro-2-{1-[(2-fluorophenyl)methyl]-5-(1,2-oxazol-3-yl)-1H-pyrazol-3-yl}pyrimidin-4-yl)amino]methyl}-2-hydroxypropanamide), bis-heteroarylpyrazole IWP-051 (5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl)pyrimidin-4(3H)one), IW-6463, GSK2181236A (1-(6-{2-[({3-methyl-4'-[(trifluoromethyl)oxy]-4-biphenyl}methyl)oxy]phenyl}-2-pyridinyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid), IWP-550, IWP-854 (4-(5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl)pyrimidin-4-yl)-1-(3-methyl-3H-diazirin-3-yl)-N-(37-oxo-41-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9 ,12,15,18,21,24,27,30,33-undecaoxa-36-azahentetracontyl)-7,10,13,16-tetraoxa-4-azanonadecane-19-amide), IWP-953, nerociguat (methyl(4,6-diamino-2-(1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)carbamate;BAY 60-4552), MGV354 ((S)-1-(6-(3-((4-(1-(cyclopropanecarbonyl)piperidin-4-yl)-2-methylphenyl)amino)-2,3-dihydro-1H-inden-4-yl)pyridin-2-yl)-5-methyl-1H-pyrazole-4-carboxylic acid), BI 703704 ((1R,5S,8s)-3-(4-(5-methyl-2-((2-methyl-4-(piperidine-1-carbonyl)benzyl)oxy)phenyl)thiazol-2-yl)-3-azabicyclo[3.2.1]octane-8-carboxylic acid), S3448 (2-[[(4-chlorophenyl)sulfonyl]amino]-4,5-dimethoxy-N-[4-(4-thiomorpholinylsulfonyl)phenyl]benzamide), and BAY 60-2770 (4-[[(4-carboxybutyl)[2-[5-fluoro-2-[[4'-(trifluoromethyl)[1,1'-biphenyl]-4-yl]methoxy]phenyl]ethyl]amino]methyl]benzoic acid). See Buys et al., Nitric Oxide 78:72-78, 2018; Sandner et al., "Soluble Guanylate Cyclase Stimulators and Activators," in Handbook of Experimental Pharmacology, Springer, Berlin, Heidelberg, pp. 1-40, 2018; and Friebe et al., Naunyn-Schmiedeberg's Arch Pharmacol 390, 1177-1188, 2017.

[0028] An sGC agonist may be an sGC "stimulator" or an sGC "activator." An sGC stimulator binds to sGC and enhances nitric oxide (NO)-mediated cGMP signaling. While sGC stimulators can increase sGC activity in the absence of NO, in the presence of NO, sGC stimulators can act synergistically with NO to amplify NO signaling (Buys et al., supra). In contrast, sGC activators bind to the heme pocket of the sGC enzyme and therefore can only activate heme-free sGC independently of NO (Buys et al., supra).

[0029] Therefore, in some cases, the sGC agonist may be a stimulator that synergistically increases sGC enzyme activity with NO. Examples of sGC stimulators that can be used as described herein include, but are not limited to, riociguat (BAY 63-2521), vericiguat (BAY 1021189), praliciguat (IW-1973, IWP-121), olinciguat (IW-1701), nerociguat (BAY 60-4552), IW-6463, A-350619, BAY 41-2272, BAY 41-8543, CFM-1571, GSK2181236 A, IWP-051, IWP-550, IWP-854, IWP-953, and YC-1. In some cases, the sGC agonist may be an sGC activator. Examples of sGC activators that can be used as described herein include, but are not limited to, cinaciguat (BAY 58-2667), ataciguat (HMR1766), MGV354, BI 703704, S3448, BAY 60-2770.

[0030] Examples of PDE5A inhibitors include, but are not limited to, sildenafil, vardenafil, tadalafil, EMD 360527, DA 8159, UK-343-664 (Walker et al., Xenobiotica, 31:651-664 (2001)), UK-427-387, UK-357903 ([1-ethyl-4-{3-[3-ethyl-6,7-dihydro-7-oxo-2-(2-pyridylmethyl)-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-2-(2-methoxyethoxy)-5-pyridylsulfonyl}piperazine]) (Gardiner et al., J. Pharmacol. Exp. Ther., 312:265-271 (2005)), UK-371800 (Pfizer), UK-313794 (Pfizer), UK-343664 (Abel et al., Xenobiotica, 31:665-76 (2001)), TA-1790 (Tanabe Seiyaku), CP-248 (Osi Pharmaceuticals), CP-461 (Osi Pharmaceuticals), exisulind (Deguchi et al., Molecular Cancer Therapeutics, 803-809 (2002); (Osi Pharmaceuticals)), pyrazolinone, EMD82639 (Merck KgaA, Darmstadt, DE; (4-(4-[2-ethyl-phenylamino)-methylene]-3-methyl-5-oxo-4,5-dihydro-pyrazole-1-yl)-benzoic acid; Senzaki et al., FASEB J., 15:1718-1726(2001) and Scutt et al., BMC Pharmacol., 4:10 (2004)), EMD360527 (Merck KgaA, Darmstadt, DE; [7-(3-chloro-4-methoxy-benzylamino)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-ylmethoxy]-acetic acid; Scutt et al., BMC Pharmacol., 4:10 (2004)), EMD221829 (Merck KgaA, Darmstadt, DE; 4-[4-(3-chloro-4-methoxy-benzylamino)-benzo[4,5]thieno[2,3-d]-pyrimidin-2-yl]-cyclohexanecarboxylic acid, ethanolamine salt; Scutt et al., BMC Pharmacol., 4:10 (2004)), EMDl 71827 (Merck KgaA, Darmstadt, DE; 5-[4-(3-chloro-4-methoxy-benzylamino)-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-2-yl]-pentanoic acid; Scutt et al., BMC Pharmacol., 4:10 (2004)), DA-8259 (3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo-[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide), E-4021 (Dukarm et al., Am. J. Respir. Crit. Care Med., 160:858-865 (1999)), pentoxifylline, and FR22934 (Fujisawa). Additional examples of PDE5A inhibitors can be found in U.S. Patent Nos. 6,916,927, 6,911,542, 6,903,099, 6,878,711, 6,872,721, 6,858,620, 6,825,197, 6,774,128, 6,723,719, 6,699,870, 6,670,366, 5,859,006 and 5,250,534, and International Patent Application Publication Nos. WO 03 / 063875, WO 03 / 1012761, WO 2004 / 037183 and WO 98 / 38168. In some cases, the sGC agonist used as described herein can be an activator of oxidized or non-oxidized sGC. .

[0031] In some cases, one or more sGC agonists (e.g., 1, 2, 3, 4, 5 or more sGC agonists) or a combination of one or more sGC agonists (e.g., 1, 2, 3, 4, 5 or more sGC agonists) and one or more PDE5A inhibitors (e.g., 1, 2, 3, 4, 5 or more PDE5A inhibitors) can be administered to male mammals to reduce calcification of heart valves and / or blood vessels, or to slow the progression of aortic sclerosis to calcific aortic valve stenosis, and / or to slow the progression of established mild to moderate valve stenosis to more severe stenosis.The administration of such drugs in the early or moderate disease stage can also be used to prevent or reduce organ dysfunction (e.g., left ventricular adaptation failure or renal dysfunction due to aortic stiffness) resulting from long-term increase in cardiovascular / valvular stiffness. One or more sGC agonists and one or more PDE5A inhibitors can be administered simultaneously (e.g., in separate compositions administered substantially simultaneously or in the same composition) or at different times (e.g., 10 to 30 minutes apart, 30 to 60 minutes apart, 1 to 2 hours apart, 2 to 4 hours apart, 4 to 6 hours apart, 6 to 12 hours apart, 12 to 24 hours apart, or more than 24 hours apart). In some cases, one or more sGC agonists or a combination of one or more sGC agonists and one or more PDE5A inhibitors can be formulated into a pharmaceutically acceptable composition. For example, a therapeutically effective amount of ataciguat can be formulated with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. The pharmaceutical composition can be formulated for administration in solid or liquid form, including, but not limited to, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

[0032] After a female mammal has been identified as having a cardiovascular condition, such as calcific aortic stenosis or aortic sclerosis that may progress to calcific aortic stenosis, the female mammal can be administered or instructed to self-administer one or more anti-fibrotic agents. In some cases, if the female mammal develops calcification after administration of the anti-fibrotic agent, the female mammal can be administered or instructed to self-administer one or more sGC agonists described above.

[0033] Examples of anti-fibrotic agents include, but are not limited to, TGFβ neutralizing antibodies, angiotensin II inhibitors, inhibitors of fibrogenic cytokine signaling (e.g., TNFα neutralizing antibodies and inhibitors or neutralizing antibodies against IL6 signaling), integrin inhibitors, and other small molecules or inhibitors or neutralizing antibodies that directly target fibrogenic signaling, indirectly target fibrogenic signaling (e.g., upstream or downstream modulators), or can suppress fibrogenic signaling. Specific examples of anti-fibrotic agents include, but are not limited to, SHP-627, hydronidone PXS-25, disiteltide, frezolimumab, LY2382770, STX-100, CWHM-12, SB-431542, THR-184, PF-06473871, RXI-109, FG-3019, imatinib, BOT-191, nilotinib, dasatinib, nintedanib, sorafenib, thalidomide, pomalidomide, etanercept, and benzamidine. Limumab, refanalin (BB-3), dextrecumab (QAX-576), tralokinumab, anakinra, rilonacept, SAR156597, carlumab, bindarit, maraviroc, RS-504393, Actimune, IFN-α, batimastat (BB-49), marimastat, macitentan, bosentan, ambrisentan, sparsentan (RE-021), atrasentan, losartan, BMS-98602 0, SAR-100842, PAR1 antagonist, curcumin, silymarin, β-caryophyllene, beraprost, iloprost, treprostinil, aviptadil, sivelestat, UK-396082, serelaxin, PRM-151, dioscin, NTU281, rapamycin, palomidin-529 (RES-529), ruxolitinib, baricitinib, omipalisib (GSK2126458), PF-562271, ta inzisertib (CC-930), MMI-0100, IMD-1041, bardoxolone methyl (CDDO-Me), antisense NF-κB, baicalein, sulfasalazine, Y-27632, bortezomib, emricasan, VX-166, Z-VAD-fmk, CTP-499, VBY-376, CA-074Me, paquinimod, HOE-077, rosiglitazone, elafibranor (GFT-505), saroglitazar,These include pioglitazone, docosahexaenoic acid, INT-767, PX-102, obeticholic acid, tulofexolate isopropyl (WAY-362450), GW4064, triamcinolone, genistein, pirfenidone, pentoxifylline, SIS-3, glycyrrhizin, anti-miR-21, ademethionine (SAM), β-aminopropionitrile (BAPN), simtuzumab (GS-6624), GM-CT-01, GR-MD-02, GCS-100, GKT137831, N-acetylcysteine, mitoquinone, salvianolic acid B, resveratrol, pyridoxamine, α-tocopherol, and IW001.

[0034] The pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene block polymer, polyethylene glycol and wool fat partial glyceride mixture.If necessary, the solubility and bioavailability of the sGC agonist and / or PDE5A inhibitor in the pharmaceutical composition can be enhanced using lipid excipients and / or ethylene oxide and propylene oxide block copolymers. See, for example, U.S. Patent No. 7,014,866 and U.S. Patent Application Publication Nos. 20060094744 and 20060079502.

[0035] The pharmaceutical compositions described herein can be designed for oral or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0036] Such injection solutions may be in the form of, for example, a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland, fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, can be used in the preparation of injections, as can natural pharmaceutically acceptable oils, such as olive oil or castor oil, including their polyoxyethylated versions. These oil solutions or suspensions can contain long-chain alcohol diluents or dispersants.

[0037] In some cases, the pharmaceutically acceptable composition comprising one or more sGC agonists and / or one or more PDE5A inhibitors can be administered locally or systemically.For example, the composition containing an sGC agonist can be administered systemically to a mammal (e.g., a human) by injection.When administering two or more sGC agonists, each sGC agonist can be administered by the same or different routes.For example, ataciguat can be administered orally, and YC-I can be administered by injection.In some cases, one or more sGC agonists can be administered by one route, and one or more PDE5A inhibitors can be administered by the same or different routes.

[0038] Compositions containing one or more sGC agonists or a combination of one or more sGC agonists and one or more PDE5A inhibitors can be administered to male mammals in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to reduce calcification of heart valves and / or blood vessels, or to slow the progression of calcific aortic sclerosis to aortic valve stenosis). Compositions containing one or more antifibrotic agents can be administered to female mammals in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to reduce calcification of heart valves and / or blood vessels, or to slow the progression of calcific aortic sclerosis to aortic valve stenosis).

[0039] As will be recognized by one of skill in the art, the effective dose may vary depending on the severity of the condition (e.g., calcific aortic stenosis), the route of administration, the age and general health of the subject, the use of excipients, the possibility of concurrent use with other therapeutic treatments, e.g., the use of other drugs, and the judgment of the treating physician.

[0040] An effective amount of a composition containing one or more sGC agonists or a combination of one or more sGC agonists and one or more PDE5A inhibitors may be any amount that reduces the severity of the symptoms of the condition being treated (e.g., calcific aortic stenosis) without causing significant toxicity to a male mammal. For example, an effective amount of an sGC agonist (e.g., YC-I) may be from about 0.5 mg / kg to about 80 mg / kg (e.g., from about 0.5 mg / kg to about 70 mg / kg, from about 0.5 mg / kg to about 60 mg / kg, from about 0.5 mg / kg to about 50 mg / kg, from about 0.5 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.5 mg / kg to about 20 mg / kg, from about 0.5 mg / kg to about 10 mg / kg, from about 0.5 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 1 mg / kg, from about 0.75 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, or from about 2 mg / kg to about 10 mg / kg). In some cases, the daily dose of an sGC agonist, such as ataciguat, is about 0.1 mg to about 500 mg (e.g., about 0.1 mg to about 0.5 mg, about 0.5 mg to about 1 mg, about 1 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 25 mg, about 25 mg to about 50 mg, about 50 mg to about 200 mg, about 50 mg to about 180 mg, about 50 mg to about 150 mg, about 50 mg to about 150 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 180 mg, about 50 mg to about 150 mg, about 50 mg to about 2 ... A dosage of about 125 mg, about 60 mg to about 200 mg, about 75 mg to about 200 mg, about 100 mg to about 200 mg, about 75 mg to about 150 mg, about 100 mg to about 150 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, or about 400 mg to about 500 mg can be administered to a mammal (e.g., a male human) for a suitable period of time. If a particular mammal does not respond to a particular amount, the amount of sGC agonist or PDE5A inhibitor can be increased, for example, by twofold. After receiving this higher amount, the mammal can be monitored for both responsiveness to treatment and symptoms of toxicity, and adjustments can be made accordingly.

[0041] An effective amount of a composition containing one or more anti-fibrotic agents can be any amount that reduces the severity of the symptoms of the condition being treated (e.g., calcific aortic stenosis) without causing significant toxicity to the female mammal. For example, an effective amount of an antifibrotic agent can be from about 0.01 mg / kg to about 80 mg / kg (e.g., from about 0.01 mg / kg to about 0.1 mg / kg, from about 0.1 mg / kg to about 0.5 mg / kg, from about 0.5 mg / kg to about 70 mg / kg, from about 0.5 mg / kg to about 60 mg / kg, from about 0.5 mg / kg to about 50 mg / kg, from about 0.5 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.5 mg / kg to about 20 mg / kg, from about 0.5 mg / kg to about 10 mg / kg, from about 0.5 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 1 mg / kg, from about 0.75 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, or from about 2 mg / kg to about 10 mg / kg). In some cases, the daily dose of the antifibrotic agent is about 0.1 mg to about 500 mg (e.g., about 0.1 mg to about 0.5 mg, about 0.5 mg to about 1 mg, about 1 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 25 mg, about 25 mg to about 50 mg, about 50 mg to about 200 mg, about 50 mg to about 180 mg, about 50 mg to about 150 mg, about 50 mg to about 125 mg) A dose of about 60 mg to about 200 mg, about 75 mg to about 200 mg, about 100 mg to about 200 mg, about 75 mg to about 150 mg, about 100 mg to about 150 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, or about 400 mg to about 500 mg) can be administered to a mammal (e.g., a female human) for a suitable period of time. If a particular mammal does not respond to a particular amount, the amount of antifibrotic agent can be increased, for example, by twofold. After receiving this higher dose, the mammal can be monitored for both responsiveness to treatment and symptoms of toxicity, and adjustments can be made accordingly.

[0042] The effective amount may be constant or may be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors may affect the actual effective amount used for a particular application. For example, the frequency of administration, the duration of treatment, the use of multiple therapeutic agents, the route of administration, and the severity of the condition (e.g., calcific aortic stenosis) may require an increase or decrease in the actual effective amount administered.

[0043] The frequency of administration can be any frequency that reduces the severity of the symptoms of the condition (for example, calcific aortic stenosis) being treated without causing significant toxicity to male mammals.For example, the frequency of administration can be about once a week to about three times a day, or about twice a month to about six times a day, or about twice a week to about once a day.The frequency of administration can be constant or can vary during treatment.In some cases, the course of treatment of male mammals with the composition containing one or more sGC agonists or the combination of one or more sGC agonists and one or more PDE5A inhibitors, or the course of treatment of female mammals with the composition containing one or more antifibrotic agents, can include a rest period.For example, the composition containing one or more sGC agonists or the combination of one or more sGC agonists and one or more PDE5A inhibitors can be administered daily for two weeks, followed by a rest period of two weeks, and this regimen can be repeated multiple times. Like the effective amount, various factors can affect the actual administration frequency used for a specific application.For example, the effective amount, the duration of treatment, the use of multiple treatment agents, the administration route, and the severity of the condition (e.g., calcific aortic stenosis) may require an increase or decrease in the administration frequency.The effective period for administering a composition containing one or more sGC agonists or a combination of one or more sGC agonists and one or more PDE5A inhibitors can be any period that reduces the severity of the symptoms of the condition being treated (e.g., calcific aortic stenosis) without causing significant toxicity to male mammals.Therefore, the effective period can vary from several days to several weeks, several months, or several years.In general, the effective period for treating calcific aortic stenosis can vary from several months to several years.In some cases, the effective period can be the survival time of an individual mammal.Multiple factors can affect the actual effective period used for a specific treatment. For example, the effective duration can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the condition being treated.

[0044] In certain cases, the course of treatment and the severity of one or more symptoms related to the condition being treated can be monitored. Any suitable method can be used to determine whether the severity of the symptoms is reduced. For example, the severity of the symptoms of calcific aortic stenosis can be evaluated at different time points using imaging technology.

[0045] This document also provides methods and materials to assist medical or research professionals in determining whether a mammal identified as having or likely to have a cardiovascular condition (e.g., cardiac valve calcification, vascular calcification, aortic sclerosis, arterial stiffness, or left ventricular systolic or diastolic dysfunction) is likely to benefit from treatment with an sGC agonist or an sGC agonist and a PDE5A inhibitor. Medical professionals can be, for example, physicians, nurses, medical laboratory technicians, and pharmacists. Research professionals can be, for example, principal investigators, research technicians, postdoctoral trainees, and graduate students. (1) Determining blood LPA levels and (2) communicating information about the levels to the professional can assist the professional in determining whether a mammal has or likely has a cardiovascular condition and / or whether a mammal having a cardiovascular condition is likely to respond to an sGC agonist (e.g., ataciguat).

[0046] Any method can be used to communicate information to another person (e.g., a professional). For example, the information can be given directly or indirectly to the professional. Furthermore, any type of communication can be used to communicate the information. For example, mail, email, telephone, and face-to-face interaction can be used. Information can also be communicated to a professional by making the information available electronically to the professional. For example, information can be communicated to a professional by placing the information in a computer database so that the professional can access the information. Furthermore, information can be communicated to a hospital, clinic, or research facility acting as an agent for the professional.

[0047] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0048] [Example 1] Materials and Methods Preclinical animal research: ldlr - / - / apoB 100 / 100 These mice are apolipoprotein B100-only mice lacking the low-density lipoprotein receptor, which consistently develop severe, hemodynamically significant calcific aortic stenosis, making them well suited for studying the effects of pharmacological interventions on the initiation and progression of calcific aortic stenosis.

[0049] ldlr - / - / apoB 100 / 100 The two groups of mice were placed on a Western Diet (WD) for six months, a time point at which the mice were allowed to develop mild to moderate valvular stenosis. At six months, the mice were randomized into two groups: (1) sequential western diet (WD) / sequential disease progression groups, or (2) Continuous western diet + ataciguat in food (WD+ATA) / treatment group.

[0050] At 9 months, mice underwent echocardiography (high-resolution ultrasound) and blood pressure (tail cuff) measurements and were sacrificed for histological and molecular analysis of valve tissue (histology, immunohistochemistry, quantitative real-time RT-PCR).

[0051] Phase I Clinical Trial Design: Patients with mild to moderate calcific aortic stenosis (1.0 cm) with well-preserved left ventricular function (ejection fraction >50%) were enrolled in a randomized, double-blind, placebo-controlled trial. 2 ~2.0cm 2 Patients with a ≥100% vasopressin (VPS) and a ≥100% vasopressin (VPS) were recruited. After screening, patients were randomized into two groups: (1) 14 days of placebo treatment, or (2) 14 days of ataciguat treatment.

[0052] Patients underwent a series of tests to assess baseline blood pressure (sitting and supine body positions), orthostatic tolerance (transition to standing, tilt test), and blood tests to assess liver enzyme function.

[0053] Phase II Clinical Trial Design: Patients with mild to moderate calcific aortic stenosis (1.0 cm or greater) with well-preserved left ventricular function (ejection fraction >50%) and significant valvular calcification (calcium level >300 units by computed tomography scan) were selected for participation in this randomized, double-blind, placebo-controlled trial. 2 ~2.0cm 2 Patients with a ≥100% vasopressin (VPS) and a ≥100% vasopressin (VPS) were recruited. After screening, patients were randomized into two groups: (1) Placebo treatment for at least 6 months (2) At least 6 months of ataciguat treatment

[0054] Patients underwent computed tomography measurements of aortic valve calcification, echocardiographic assessment of ventricular and aortic valve function, and DEXA scans for assessment of whole-body bone mineral density at baseline and at 6-month intervals from the start of treatment.

[0055] In vitro cell culture studies: Aortic valve interstitial cells were isolated using the expansion method, expanded in DMEM / F-10 medium containing 10% FBS, and studied at passages 2–10. Cells were treated under each experimental condition for 24 hours, at which point mRNA was harvested using lysis buffer. After mRNA isolation and reverse transcription, gene expression was measured using quantitative real-time RT-PCR and TaqMan primers. Changes in gene expression were assessed by the ddCt method and expressed as fold changes normalized to the control condition.

[0056] LPA study in humans with aortic valve calcification: After diluting samples with diluent buffer, human plasma LPA levels were measured using a colorimetric, sandwich / quantitative ELISA assay (AB212165). Computed tomography scans were performed using a clinical-grade, high-resolution scanning method established for the assessment of aortic valve calcification. Calcium load was measured from three-dimensional multiplanar images and expressed in arbitrary units (AU).

[0057] [Example 2] Ataciguat increases sGC signaling and attenuates BMP signaling in vitro To confirm that ataciguat effectively activates sGC in cells, murine aortic valve interstitial cells (mVICs) were isolated from C57BL6 / J mice treated with ataciguat or other sGC agonists as internal controls. In otherwise naive cells, treatment with ataciguat significantly upregulated sGC-dependent signaling (phospho-VASP). 239 The levels of phospho-VASP (as indicated by the ATP-dependent ATP synthase activity) were significantly increased compared to vehicle-treated cells. Importantly, both sodium nitroprusside and DEA-NONOATE significantly increased phospho-VASP. 239 levels increased to a similar extent, suggesting that sGC exists in a reduced and heme-free state in mVIC.

[0058] To determine whether ataciguat can attenuate the signaling cascade central to the initiation and progression of valvular calcification in vitro, cells were treated with BMP2 with or without ataciguat. In these studies, ataciguat reduced phospho-SMAD1 / 5 / 8 signaling (e.g., canonical BMP signaling) as well as the expression of osteogenic genes induced by BMP2 (e.g., osterix).

[0059] [Example 3] Ataciguat slows the progression of established FCAVS in vivo A therapeutically relevant treatment strategy was used in which ldlr-deficient apoB100-only mice were fed a western diet for 6 months (to induce mild / moderate FCAVS) followed by a western diet plus ataciguat (150 ppm in the diet). To confirm that ataciguat effectively binds to oxidized sGC in aortic valve tissue in vivo, pVASP was administered. 239 Immunohistochemistry was used to demonstrate that α-amyloid β was significantly increased in mice of both sexes (Figures 1A and 1B). Consistent with other reports in mouse models of hyperlipidemia, treatment with ataciguat did not alter blood pressure in mice of either sex.

[0060] To subsequently evaluate the effects of ataciguat on signaling pathways that promote the progression of FCAVS in vivo, we used immunohistochemistry and quantitative real-time RT-PCR to assess changes in canonical BMP and TGFβ signaling in aortic valve tissue. Similar to the observations in valve interstitial cells in vitro, 3-month treatment with ataciguat significantly attenuated SMAD1 / 5 / 8 phosphorylation in valve tissue from male and female mice (Figures 2A and 2B) and reduced osteogenic markers, such as Runx2 and SPP1 (Figures 3A–3D). A greater reduction in valve calcium was also observed in male mice (Figure 4). Interestingly, treatment with ataciguat increased canonical TGFβ signaling (SMAD2 / 3 phosphorylation) in valve tissue from both male and female mice (Figures 5A and 5B), but paradoxically reduced the expression of SMAD2 / 3 target genes CTGF and COL1A1 only in female mice (Figures 6A–6D). MMP2 expression was not altered by ataciguat in either male or female mice, but was significantly higher in female mice than in male mice (Figures 7A and 7B). Consistent with these molecular changes in matrix metalloproteinase and collagen isoform expression, changes in relative collagen fiber width were only evident in female mice (Figures 8A and 8B). Furthermore, consistent with the observed changes in valvular calcification, chronic treatment with ataciguat significantly attenuated the progression of valvular dysfunction and the associated decline in left ventricular diastolic function in male mice only (Figures 9A-9B and 10A-10D).

[0061] [Example 4] Ataciguat is well tolerated in vivo in patients with mild / moderate FCAVS Combining ataciguat's role as an activator of heme-free sGC with reports of increased systemic oxidative stress in patients with FCAVS (Mourino-Alvarez et al., Int. J. Cardiol., 225:99-106 (2016)), it was considered possible that administration of ataciguat might elicit an acute decrease in blood pressure or increase the propensity to experience orthostatic hypotension. To investigate this possibility, we performed a randomized, double-blind, placebo-controlled, dose-escalation study to evaluate the effect of ataciguat on orthostatic tolerance using both the orthostatic test (highly functional) and the tilt test (highly sensitive). These studies revealed that 14 days of treatment with either placebo or ataciguat at any dose did not significantly alter resting blood pressure (Figures 11A-11D) or enhance any reductions in blood pressure or heart rate in response to standing (Figures 12A-12D and 13A-13B) or head-up tilt (Figures 14A-14D, 15A-15D and 16A-16D).

[0062] [Example 5] Ataciguat slows progression of valve disease in patients with mild / moderate FCAVS To determine whether ataciguat can slow the progression of FCAVS, we conducted a randomized, double-blind, placebo-controlled trial in patients with mild / moderate valve disease. Key eligibility criteria for the study included valve calcium levels greater than 300 AU, 1.0-2.0 cm 2 Valve area of ​​0.05 mm, ejection fraction greater than 50%, and the absence of congenital valvular disease were included (full inclusion / exclusion criteria are shown in Table 1). Patients were randomized to 6 months of placebo or ataciguat treatment, with a subset of patients receiving 12 months of treatment. Baseline subject characteristics are shown in Table 2. Importantly, there were no differences in measured hemodynamics, blood biochemistry, medication, or comorbidity indexes between treatment groups at baseline. Given the relatively short duration of follow-up, the primary outcome variable was the change in valve calcium over time. Secondary and tertiary outcomes were aortic valve and left ventricular function, respectively.

[0063] After 6 months of treatment, ataciguat treatment was found to attenuate the progression of aortic valve calcification by approximately 70% compared with placebo-treated patients (Figures 17A-17C). Interestingly, and consistent with observations in mice, subgroup analysis suggested that ataciguat conferred greater benefit to men with FCAVS than to women with FCAVS. While placebo-treated patients showed progressive deterioration of aortic valve function over the 6-month follow-up period (p<0.01 vs. baseline), aortic valve function did not significantly worsen in ataciguat-treated patients (p=0.24 vs. baseline) (Figures 18A-18F). Similar to the observed changes in aortic valve calcium levels, ataciguat treatment tended to confer greater therapeutic benefit to men with FCAVS compared with women.

[0064] We next performed a study to assess whether ataciguat had any effect on cardiac function and adaptation to chronic ventricular overload presented by aortic valve dysfunction. After 6 months of treatment, we observed that ataciguat treatment prevented the decline in left ventricular systolic function (assessed by ejection fraction or stroke volume) compared with placebo-treated subjects (Figures 19A-19F). Interestingly, ataciguat tended to improve left ventricular diastolic function (assessed by either the E / e' or E / A ratio) compared with placebo-treated patients (Figures 20A-20F). A reduction in pulse pressure was also observed in ataciguat-treated males compared with placebo. Such a reduction was not evident in females (Figures 21A-21C). Surprisingly, and similar to the observed changes in aortic valve function, these data suggested that males derive greater and more consistent therapeutic benefit from ataciguat in both systolic and diastolic function compared with females.

[0065] [Table 1]

[0066] [Table 2] TIFF2025172729000004.tif88160

[0067] [Example 6] Usefulness of baseline variables in predicting progression of aortic valve dysfunction To determine whether there were readily available phenotypic characteristics that predicted change in aortic valve area over time, further analyses used a neural network algorithm trained on baseline variables. After 10-fold cross-validation, the algorithm performed reasonably well in predicting change in aortic valve function with data from all subjects. Crucially, and consistent with the data presented above, the model identified treatment group (placebo vs. ataciguat) and sex (male vs. female) as the most informative variables predicting change in valve area over time (Figure 22).

[0068] In summary, the above studies demonstrated the following: In mice, ataciguat activated sGC in valve tissue, reduced osteogenic signaling and mineralization in valve tissue from male and female animals, and did not consistently affect collagen expression in males, but did reduce collagen expression in females, which, combined with higher MMP2 expression, caused a shift toward a thinner collagen fiber phenotype in females. This study suggested that ataciguat preferentially delayed valvular and diastolic dysfunction in male mice.

[0069] In humans, ataciguat was well tolerated with respect to side effects and blood pressure control. It effectively reduced blood pressure at low doses and was more effective when subjects were supine. It also slowed the progression of valvular calcification by approximately 70%, slowed the progression of valvular dysfunction, and tended to preserve or prevent the decline of left ventricular systolic and diastolic function, although these benefits were observed primarily in men.

[0070] Finally, the unbiased neural network model revealed that treatment group (ataciguat vs. placebo) and sex (male vs. female) were the strongest predictors of treatment efficacy when predicting change in aortic valve area.

[0071] [Example 7] Effect of sGC agonists on the expression of Osterix To determine the effects of several sGC agonists on mRNA levels of osterix, a BMP target gene, in vitro studies were performed in aortic valve interstitial cells. Cells were treated with nerociguat, vericiguat, rificiguat, or BAY41-8543, and osterix mRNA levels were determined. Nerociguat and BAY41-8543 were used at concentrations ranging from 1 nM to 10 μM, while vericiguat and rificiguat were used at concentrations ranging from 1 nM to 1 μM.

[0072] Nerociguat effectively reduced BMP signaling at concentrations between 1 nM and 1 μM (Figure 23, arrow). This finding suggested that concentrations above 10 μM may be cytotoxic, as observed with some other compounds. Vericiguat was effective in reducing Osterix expression only at the highest dose tested (Figure 24, arrow). Similar to nerociguat, rificiguat appeared to lose efficacy at higher concentrations in vitro (Figure 25; effective concentrations indicated by arrows). BAY41-8543 tended to promote a dose-dependent reduction in Osterix expression and was highly effective at 10 μM (Figure 26, arrow).

[0073] [Example 8] Effects of ataciguat on BMP signaling induced by LPA To examine the effects of ataciguat on LPA-induced BMP signaling and BMP target gene expression, additional studies were performed in aortic valve interstitial cells. Cells were treated with 18:0 lyso-PA with or without ataciguat, and Osterix mRNA levels were determined. These studies showed that treatment with 18:0 lyso-PA increased Osterix expression, and that ataciguat dramatically suppressed the LPA effect (Figure 27, "LPA" vs. "LPA + ATA"), suggesting that ataciguat may be effective in treating valve disease in patients with elevated LPA levels.

[0074] The relationship between plasma LPA levels and valvular calcium levels was evaluated in patients with mild to moderate aortic stenosis. Plasma LPA was measured at baseline, and valvular calcium levels were assessed by CT scan at baseline or after sGC agonist treatment. At baseline, higher plasma LPA levels were associated with greater valvular calcium levels in this patient cohort (Figure 28). After 6 months of treatment with ataciguat, patients with higher baseline plasma LPA levels tended to receive greater therapeutic benefit (greater attenuation of excessive BMP signaling) than patients with lower plasma LPA levels (Figure 29).

[0075] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is illustrative of the invention and not limiting of its scope, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method of treating a mammal based on the sex of the mammal, comprising: (a) identifying a mammal as having or at risk for developing cardiac valve or vascular calcification; (b) identifying the mammal as a male mammal or a female mammal; and (c) if the mammal is a male mammal, administering an sGC agonist to the male mammal, thereby slowing the progression of calcification of the heart valves or blood vessels in the male mammal, and if the mammal is a female mammal, not administering an sGC agonist to the female mammal, and optionally administering a non-sGC agonist treatment to the female mammal, thereby slowing the progression of calcification of the heart valves or blood vessels in the female mammal. A method comprising:

2. The method of claim 1, wherein the mammal is a human.

3. The sGC agonist is Ataciguat (5-chloro-2-[[(5-chloro-2-thienyl)sulfonyl]amino]-N-[4-(4-morpholinylsulfonyl)phenyl]-benzamide; HMR1766), YC-I (5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol), BAY 58-2667 (4-[((4-carboxybutyl)(2-[(4-phenethylbenzyl)oxy]phenethyl)amino)methyl[benzoic acid]hydrochloride), BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), BAY-41-8543 (2-[1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-(4-morpholinyl)-4,6-pyrimidinediamine), BAY 63-2521 (methyl(4,6-diamino-2-(1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)(methyl)carbamate), CFM-1571 (3-[3-(dimethylamino)propoxy]-N-(4-methoxyphenyl)-1-(phenylmethyl)-1H-pyrazole-5-carboxamide hydrochloride), A-350619 (3-[2-(4-chlorophenylthio)phenyl]-N-(4-dimethylaminobutyl)acrylamide), Vericiguat (methyl(4,6-diamino-2-(5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)carbamate), Praliciguat (1,1,1,3,3,3-hexafluoro-2-[({5-fluoro-2-[1-(2-fluorobenzyl)-5-(1,2-oxazol-3-yl)-1H-pyrazol-3-yl]pyrimidin-4-yl}amino)methyl]propan-2-ol), Olinciguat ((2R)-3,3,3-trifluoro-2-{[(5-fluoro-2-{1-[(2-fluorophenyl)methyl]-5-(1,2-oxazol-3-yl)-1H-pyrazol-3-yl}pyrimidin-4-yl)amino]methyl}-2-hydroxypropanamide), Bis-heteroarylpyrazole IWP-051 (5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl)pyrimidin-4(3H)-one), IW-6463, GSK2181236A (1-(6-{2-[({3-methyl-4'-[(trifluoromethyl)oxy]-4-biphenyl}methyl)oxy]phenyl}-2-pyridinyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid), IWP-550, IWP-854 (4-(5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl)pyrimidin-4-yl)-1-(3-methyl-3H-diazirin-3-yl)-N-(37-oxo-41-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azahentetracontyl)-7,10,13,16-tetraoxa-4-azanonadecane-19-amide), IWP-953, Nerociguat (methyl(4,6-diamino-2-(1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)carbamate), MGV354 ((S)-1-(6-(3-((4-(1-(cyclopropanecarbonyl)piperidin-4-yl)-2-methylphenyl)amino)-2,3-dihydro-1H-inden-4-yl)pyridin-2-yl)-5-methyl-1H-pyrazole-4-carboxylic acid), BI 703704 ((1R,5S,8s)-3-(4-(5-methyl-2-((2-methyl-4-(piperidine-1-carbonyl)benzyl)oxy)phenyl)thiazol-2-yl)-3-azabicyclo[3.2.1]octane-8-carboxylic acid), S3448 (2-[[(4-chlorophenyl)sulfonyl]amino]-4,5-dimethoxy-N-[4-(4-thiomorpholinylsulfonyl)phenyl]benzamide), or BAY 60-2770 (4-[[(4-carboxybutyl)[2-[5-fluoro-2-[[4'-(trifluoromethyl)[1,1'-biphenyl]-4-yl]methoxy ]phenyl]ethyl]amino]methyl]benzoic acid) The method according to claim 1 or claim 2, wherein

4. The method of any one of claims 1 to 3, further comprising administering to said male mammal a PDE5A inhibitor.

5. 5. The method of any one of claims 1 to 4, further comprising identifying the male mammal as having elevated plasma levels of lysophosphatidic acid (LPA).

6. 3. The method of claim 1 or claim 2, wherein the mammal is female and the method comprises administering an anti-fibrotic agent to the female mammal to slow the progression of cardiac valve or vascular calcification in the female mammal.

7. A method of treating a population of mammals identified as having or at risk of developing cardiac valve or vascular calcification, said population including at least one male mammal and at least one female mammal, said method comprising administering an sGC agonist to at least one of said male mammals, thereby slowing the progression of cardiac valve or vascular calcification in at least one of said male mammals, and not administering said sGC agonist to at least one of said female mammals.

8. The method of claim 7, wherein the mammal is a human.

9. 9. The method of claim 7 or 8, wherein the sGC agonist is ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

10. The method of any one of claims 7 to 9, further comprising administering a PDE5A inhibitor to at least one of said male mammals.

11. 11. The method of any one of claims 7 to 10, wherein at least one of said male mammals is also identified as having elevated plasma levels of LPA.

12. 1. A method of treating a population of mammals based on sex, comprising: (a) identifying the sex of mammals in a population determined to have or be at risk of developing cardiac valve or vascular calcification; and (b) administering an sGC agonist to the mammal identified as a male mammal, and not administering an sGC agonist to the mammal identified as a female mammal, and optionally administering a non-sGC agonist treatment to said female mammal. A method comprising:

13. The method of claim 12, wherein the mammal is a human.

14. 14. The method of claim 12 or 13, wherein the sGC agonist is ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

15. 15. The method of any one of claims 12 to 14, further comprising administering a PDE5A inhibitor to said mammal, identified as a male mammal.

16. 16. The method of any one of claims 12 to 15, wherein the mammal identified as a male mammal is also determined to have an elevated plasma level of LPA.

17. 14. The method of claim 12 or claim 13, comprising administering an anti-fibrotic agent to said mammal, identified as a female mammal.

18. 1. A method for avoiding the unnecessary use of sGC agonists for treating a mammal having or at risk of developing cardiac valve or vascular calcification, comprising: identifying the mammal as a female mammal having or at risk of developing the cardiac valve or vascular calcification; and excluding said female mammal from treatment with said sGC agonist based at least in part on the female sex of said female mammal. A method comprising:

19. 19. The method of claim 18, wherein the mammal is a human.

20. 20. The method of claim 18 or 19, wherein the sGC agonist is ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

21. 1. A method of treating a mammal based on the sex of the mammal, comprising: (a) identifying a mammal as having or at risk of developing aortic sclerosis; (b) identifying the mammal as a male mammal or a female mammal; and (c) if the mammal is a male mammal, administering an sGC agonist to the male mammal, thereby delaying the progression of aortic sclerosis to calcific aortic stenosis in the male mammal, and if the mammal is a female mammal, not administering an sGC agonist to the female mammal, and optionally administering a non-sGC agonist treatment to the female mammal, thereby delaying the progression of aortic sclerosis to calcific aortic stenosis in the female mammal. A method comprising:

22. 22. The method of claim 21, wherein the mammal is a human.

23. 23. The method of claim 21 or 22, wherein the sGC agonist is ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

24. 24. The method of any one of claims 21 to 23, further comprising administering to said male mammal a PDE5A inhibitor.

25. 25. The method of any one of claims 21 to 24, further comprising identifying the male mammal as having an elevated plasma level of LPA.

26. 23. The method of claim 21 or claim 22, comprising administering to the female mammal an anti-fibrotic agent.

27. A method of treating a population of mammals identified as having or at risk of developing aortic sclerosis, said population including at least one male mammal and at least one female mammal, said method comprising administering an sGC agonist to at least one of said male mammals, thereby slowing the progression of aortic sclerosis to calcific aortic stenosis in at least one of said male mammals, and not administering said sGC agonist to at least one of said female mammals.

28. 28. The method of claim 27, wherein the mammal is a human.

29. 29. The method of claim 27 or 28, wherein the sGC agonist is ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

30. 30. The method of any one of claims 27 to 29, further comprising administering to at least one of said male mammals a PDE5A inhibitor.

31. 31. The method of any one of claims 27 to 30, further comprising identifying at least one said male mammal as having an elevated plasma level of LPA.

32. 1. A method of treating a population of mammals based on sex, comprising: (a) identifying the sex of mammals in a population determined to have or be at risk of developing aortic sclerosis; and (b) administering an sGC agonist to the mammal identified as a male mammal, and not administering an sGC agonist to the mammal identified as a female mammal, and optionally administering a non-sGC agonist treatment to said female mammal. A method comprising:

33. 33. The method of claim 32, wherein the mammal is a human.

34. 34. The method of claim 32 or 33, wherein the sGC agonist is ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

35. 35. The method of any one of claims 32 to 34, further comprising administering a PDE5A inhibitor to said mammal, identified as a male mammal.

36. 36. The method of any one of claims 32 to 35, wherein the mammal identified as a male mammal is further determined to have an elevated plasma level of LPA.

37. 34. The method of claim 32 or claim 33, comprising administering to said mammal, identified as a female mammal, an anti-fibrotic agent.

38. 1. A method for avoiding the unnecessary use of sGC agonists for treating a mammal having or at risk of developing aortic sclerosis, comprising: identifying said mammal as a female mammal and as having or at risk of developing said aortic sclerosis; and excluding said female mammal from treatment with said sGC agonist based at least in part on the female sex of said female mammal. A method comprising:

39. 39. The method of claim 38, wherein the mammal is a human.

40. 40. The method of claim 38 or 39, wherein the sGC agonist is ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

41. 1. A method of treating a mammal, comprising: (a) identifying the mammal as having or at risk for hypertension if the mammal is in a supine position; and (b) administering to said mammal an sGC agonist, thereby reducing blood pressure in the supine position. A method comprising:

42. 42. The method of claim 41, wherein the mammal is a human.

43. 43. The method of claim 41 or 42, wherein the sGC agonist is ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

44. 44. The method of any one of claims 41 to 43, further comprising administering to the mammal a PDE5A inhibitor.

45. 45. The method of any one of claims 41 to 44, further comprising identifying said mammal as a male mammal prior to said administering.

46. 46. ​​The method of any one of claims 41 to 45, wherein said administering does not reduce blood pressure when the mammal is in an upright position.

47. A method for treating a population of mammals identified as having or at risk of developing hypertension in the supine position, said population including at least one male mammal and at least one female mammal, said method comprising administering an sGC agonist to at least one of said male mammals, thereby reducing blood pressure in the supine position in at least one of said male mammals and preventing the harmful side effects of hypertension, and not administering said sGC agonist to at least one of said female mammals.

48. 48. The method of claim 47, wherein the mammal is a human.

49. 49. The method of claim 47 or 48, wherein the sGC agonist is ataciguat, YC-I, BAY 58-2667, BAY 41-2272, BAY-41-8543, BAY 63-2521, CFM-1571, A-350619, vericiguat, praliciguat, olinciguat, bis-heteroarylpyrazole IWP-051, IW-6463, GSK2181236A, IWP-550, IWP-854, IWP-953, nerociguat, MGV354, BI 703704, S3448 or BAY 60-2770.

50. 50. The method of any one of claims 47 to 49, further comprising administering to at least one of said male mammals a PDE5A inhibitor.

51. 49. The method of claim 47 or claim 48, comprising administering to at least one said female mammal an anti-fibrotic agent.

52. 51. The method of any one of claims 47 to 50, wherein said administering does not reduce blood pressure when the mammal is in an upright position.

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