Methods and materials for treating ectopic tissue mineralization

Abstract

This document provides methods and materials for reducing formation and / or slowing progression of ectopic tissue mineralization. For example, methods and materials for using ataciguat to reduce ectopic tissue mineralization formation within a mammal and / or to slow ectopic tissue mineralization progression within a mammal are provided. Ataciguat and compositions containing ataciguat also are provided.

Description

[0001] Attorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0002] METHODS AND MATERIALS FOR TREATING ECTOPIC TISSUE MINERALIZATION

[0003] TECHNICAL FIELD

[0004] This document relates to methods and materials involved in treating ectopic tissue mineralization. For example, this document provides methods and materials for using ataciguat to reduce formation and / or to slow progression of ectopic tissue mineralization.

[0005] BACKGROUND

[0006] Ectopic tissue mineralization refers to the deposition of mineralized complexes in tissues other than those in which it normally occurs. Ectopic tissue mineralization can occur in various tissues such as organs, muscles, tendons, and other soft tissues and may impair normal tissue function.

[0007] SUMMARY

[0008] This document provides methods and materials for reducing formation and / or slowing progression of ectopic tissue mineralization. For example, this document provides ataciguat, compositions containing ataciguat, and methods for using ataciguat and / or compositions containing ataciguat to treat mammals having, or likely to develop, ectopic tissue mineralization.

[0009] As demonstrated herein, loss of sGC signaling (e.g., through genetic inactivation of enzymes in pathways that regulate nitric oxide signaling) resulted in significant increases in ectopic tissue calcification. These results demonstrate that ataciguat can be used to treat ectopic tissue mineralization and / or to slow progression of ectopic tissue mineralization formation. Having the ability to reduce formation and / or slow progression of ectopic tissue mineralization as described herein can allow clinicians to treat patients who have ectopic tissue mineralization, allowing patients to live longer and happier lives.

[0010] In general, one aspect of this document features a method for reducing ectopic tissue mineralization formation or slowing ectopic tissue mineralization progression within a tissue of a mammal. The method can include (or consist essentially of, or consist of) administering a composition containing ataciguat to the mammal having ectopicAttorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0011] tissue mineralization or likely to develop ectopic tissue mineralization within the tissue, wherein formation of the ectopic tissue mineralization within the tissue is reduced or progression of the ectopic tissue mineralization within the tissue is slowed, wherein the tissue is tendonous tissue, ligamentous tissue, muscle tissue, skeletal muscle tissue, nervous system tissue, or skin tissue. The mammal can be a human. Fomiation of ectopic tissue mineralization within the mammal can be delayed, or progression of ectopic tissue mineralization within the mammal can be slowed. Formation of ectopic tissue mineralization within the mammal can be delayed for at least 6 months, or progression of ectopic tissue mineralization within the mammal can be slowed by at least 50 percent. The mammal can be a mammal having the ectopic tissue mineralization. The mammal can be a mammal likely to develop the ectopic tissue mineralization. Ataciguat or a pharmaceutically acceptable salt thereof can be used as described herein:

[0012]

[0013] (ataciguat).

[0014] In another aspect, this document features a pharmaceutical composition containing (or consisting essentially of, or consisting of) ataciguat or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0015] In another aspect, this document features a method for reducing ectopic tissue mineralization formation. The method can include (or consist essentially of, or consist of) administering a composition containing ataciguat or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided herein, to a mammal having ectopic tissue mineralization formation or likely to develop ectopic tissue mineralization, wherein formation of ectopic tissue mineralization within the mammal is reduced. The mammal can be a human. Formation of ectopic tissue mineralization within the mammal can beAttorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0016] delayed. Formation of ectopic tissue mineralization within the mammal can be delayed for at least 6 months. The mammal can be a mammal having ectopic tissue mineralization. The mammal can be a mammal likely to develop ectopic tissue mineralization. The mammal can be a mammal having ectopic tissue mineralization or likely to develop ectopic tissue mineralization within a tissue or organ of the mammal. The mammal can be a mammal having ectopic tissue mineralization of the tissue. The tissue can be vascular tissue, cardiovascular tissue, heart valve tissue, tendonous tissue, ligamentous tissue, muscle tissue, skeletal muscle tissue, nervous system tissue, or skin tissue. The mammal can be a mammal having ectopic tissue mineralization of the organ. The organ can be a heart or a kidney.

[0017] In another aspect, this document features a method for slowing ectopic tissue mineralization progression. The method can include (or consist essentially of, or consist of) administering a composition containing ataciguat or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided herein, to a mammal having ectopic tissue mineralization, wherein progression of ectopic tissue mineralization within the mammal is slowed. The mammal can be a human. Progression of ectopic tissue mineralization within the mammal can be slowed by at least 10 percent. Progression of ectopic tissue mineralization within the mammal can be slowed by at least 25 percent. Progression of ectopic tissue mineralization within the mammal can be slowed by at least 50 percent. The mammal can be a mammal having ectopic tissue mineralization within a tissue of the mammal. The tissue can be vascular tissue, cardiovascular tissue, heart valve tissue, tendonous tissue, ligamentous tissue, muscle tissue, skeletal muscle tissue, nervous system tissue, or skin tissue. The mammal can be a mammal having ectopic tissue mineralization within an organ of the mammal. The organ can be a heart or a kidney.

[0018] In another aspect, this document features a method for reducing ectopic tissue mineralization formation in vascular or cardiovascular tissue. The method can include (or consist essentially of, or consist of) administering a composition containing ataciguat or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided herein to a mammal having ectopic tissue mineralization in vascular or cardiovascular tissue or likely to develop ectopic tissue mineralization in vascular or cardiovascularAttorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0019] tissue, wherein formation of the ectopic tissue mineralization in vascular or cardiovascular tissue within the mammal is reduced. The mammal can be a human.

[0020] Formation of ectopic tissue mineralization in vascular or cardiovascular tissue within the mammal can be delayed. Formation of ectopic tissue mineralization in vascular or cardiovascular tissue within the mammal can be delayed for at least 6 months. The mammal can be a mammal having ectopic tissue mineralization in vascular or cardiovascular tissue. The mammal can be a mammal likely to develop ectopic tissue mineralization in vascular or cardiovascular tissue.

[0021] In another aspect, this document features a method for slowing ectopic tissue mineralization progression in vascular or cardiovascular tissue. The method can include (or consist essentially of, or consist of) administering a composition containing ataciguat or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided herein to a mammal having ectopic tissue mineralization in vascular or cardiovascular tissue, wherein progression of the ectopic tissue mineralization in vascular or cardiovascular tissue is slowed. The mammal can be a human. Progression of ectopic tissue mineralization in vascular or cardiovascular tissue within the mammal can be slowed by at least 10 percent. Progression of ectopic tissue mineralization in vascular or cardiovascular tissue within the mammal can be slowed by at least 25 percent.

[0022] Progression of ectopic tissue mineralization in vascular or cardiovascular tissue within the mammal can be slowed by at least 50 percent.

[0023] In another aspect, this document features a method for reducing ectopic tissue mineralization formation in skeletal muscle tissue. The method can include (or consist essentially of, or consist of) administering a composition comprising ataciguat or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided herein to a mammal having ectopic tissue mineralization in skeletal muscle tissue or likely to develop ectopic tissue mineralization in skeletal muscle tissue, wherein formation of the ectopic tissue mineralization in skeletal muscle tissue within the mammal is reduced. The mammal can be a human. Formation of ectopic tissue mineralization in skeletal muscle tissue within the mammal can be delayed. Formation of ectopic tissue mineralization in skeletal muscle tissue within the mammal can be delayed for at least 6 months. The mammal can be a mammal having ectopic tissue mineralizationAttorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0024] in skeletal muscle tissue. The mammal can be a mammal likely to develop ectopic tissue mineralization in skeletal muscle tissue.

[0025] In another aspect, this document features a method for slowing ectopic tissue mineralization progression in skeletal muscle tissue. The method can include (or consist essentially of, or consist of) administering a composition containing ataciguat or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided herein to a mammal having ectopic tissue mineralization in skeletal muscle tissue, wherein progression of the ectopic tissue mineralization in skeletal muscle tissue is slowed. The mammal can be a human. Progression of ectopic tissue mineralization in skeletal muscle tissue within the mammal can be slowed by at least 10 percent.

[0026] Progression of ectopic tissue mineralization in skeletal muscle tissue within the mammal can be slowed by at least 25 percent. Progression of ectopic tissue mineralization in skeletal muscle tissue within the mammal can be slowed by at least 50 percent.

[0027] 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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0028] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0029] DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a graph plotting expression of DDAH1, DDAH2, PRMT1, and SLC7A2 in aortic valve tissue from humans with end-stage aortic stenosis undergoing valve replacement surgery. Expression of DDAH1 and SLC7A2 was significantly increased whereas expression of PRMT1 was reduced in calcified valve tissue such that:Attorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0031] 1) DDAH1 and DDAH2, the enzymes that degrade the endogenous inhibitor of nitric oxide synthase ADMA, are both upregulated in diseased tissue.

[0032] Figures 2A-2B contain results demonstrating changes in aortic valve calcification (Figure 2 A) following 6 months of Western diet feeding, and changes in aortic valve function (Figure 2B) in LA-D+ / +and LA-D+ / ' mice. Reductions in cusp separation distance are indicative of poorer valve function. Valvular calcification and valvular dysfunction were significantly increased in LA-D+ / ‘ mice compared to LA-D+ / +littemiates.

[0033] Figures 3A-3C contain results demonstrating changes in canonical BMP signaling (pSMADl / 5 / 8, Figure 3A), Osterix protein levels (Figure 3B), and SMAD target genes (Idl, Id2, and Hey, Figure 3C; note that Osterix (Osx) transcript levels were not detectable) in aortic valve from LA-D+ / +and LA-D+ / ’mice. Increased valvular calcification and valvular dysfunction observed in LA-D+ / ' mice (Figures 2A-2B) was associated with increases in pSMAD 1 / 5 / 8 protein levels, osteogenic transcription factor proteins, and target genes of canonical BMP signaling.

[0034] Figures 4A-4B contain results demonstrating changes USF-1 protein levels (Figure 4A) and the expression levels of USF-1 target genes osteopontin and alkaline phosphatase (Figure 4B) in aortic valve tissue from LA-D+ / +and LA-D+ / ' mice. USF-1 protein levels and SPP1 expression were increased in LA-D+ / ' mice, suggesting that there was increased USF-1 signaling in LA-D " mice.

[0035] Figures 5A-5C contain results demonstrating changes in protein levels of osteopontin (Figure 5A) and protein (Figure 5B) and mRNA (Figure 5C) levels of tartrate -resistant alkaline phosphatase (TRAP) in aortic valve tissue from LA-D+ / +and LA-D+Amice, suggesting that post- translational regulation of SPP1 (e.g., protein phosphorylation) may limit the anti-calcific effects of SPP1 in valve tissue.

[0036] Figures 6A-6B contain results demonstrating changes in expression of genes related to ADMA degradation, synthesis, and transport (Figure 6A), USF-1 protein levels (Figure 6B), and USF-1 target gene expression (Figure 6B) in aortic valve interstitial cells following 24 hours of treatment with vehicle, ADMA, or PD404182. Treatment with exogenous ADMA did not significantly alter gene expression or protein levels; howeverAttorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0037] inhibition ofDDAHl with PD404182 significantly increased both SPP1 expression and USF-1 protein levels.

[0038] Figure 7 is a graph plotting expression ofDDAHl, DDAH2, PRMT1, and SLC7A2 in aortic punch biopsies from humans with advanced coronary artery disease undergoing coronary artery bypass surgery. Expression ofDDAHl, DDAH2, and SLC7A2 did not differ between normal and diseased aortic vessel punch tissue; however, PRMT1 expression was significantly reduced in the aorta of diseased samples.

[0039] Figures 8A-8B contain results demonstrating changes in aortic calcification following 6 months of Western diet feeding (Figure 8 A) and changes in endotheliumdependent relaxation to acetylcholine in aortic rings (Figure 8B) from LA-D+ / +and LA-D+ / _mice. While mice of both genotypes developed advanced atherosclerotic plaques, LA-D+ / ’ mice demonstrated increased intimal plaque calcification compared to LA-D+ / +mice.

[0040] Figure 9 contains results demonstrating changes in vasomotor function in thoracic aorta from LA-D+ / +and LA-D+ / _mice following 6 months of Western diet feeding. There were no significant changes in vascular relaxation to sodium nitroprusside or vascular constriction to prostaglandin F2a or serotonin (5-HT).

[0041] Figures 10A-10C contain results demonstrating changes in canonical BMP signaling (pSMADl / 5 / , Figure 10A), osterix protein levels (Figure 10B), and SMAD target genes (Idl, Id2, Heyl, Osx, Figure 10C) in aorta from LA-D+ / +and LA-D+ / ' mice. Vascular calcification was associated with increases in canonical BMP signaling, but not increased expression of markers of mature osteoblast like cells or pSMAD target genes, suggesting a functional disconnect between upstream BMP signaling and induction of downstream ectopic osteogenic gene programs.

[0042] Figures 11 A-l IB contain results demonstrating changes in USF-1 protein levels (Figure 11 A) and the USF-1 target genes osteopontin and alkaline phosphatase (Figure 1 IB) in aorta from LA-D+ / +and LA-D+ / _mice. No increases in USF-1 or its target genes were observed in aorta from LA-D+ / ' mice.

[0043] Figures 12A-12C contain results demonstrating changes in protein levels of osteopontin (Figure 12A) and protein (Figure 12B) and mRNA (Figure 12C) levels of tartrate -resistant alkaline phosphatase (TRAP) in aorta from LA-D+'+and LA-D " mice.Attorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0044] There was no significant difference in SPP1 protein levels and TRAP protein and mRNA levels were variable, suggesting a possible dysregulation of mechanisms that protect against calcification in aorta.

[0045] Figures 13A-13B contain results demonstrating changes in expression of genes related to ADMA degradation, synthesis, and transport (Figure 13A), USF-1 protein levels, and USF-1 target gene expression (Figure 13B) in aortic smooth muscle cells following 24 hours of treatment with vehicle, ADMA, or PD404182. Treatment of cells with exogenous ADMA did not alter expression of DDAH1, DDAH2, ADMA transporters, USF-1 protein levels, or USF-1 molecular targets. Inhibition of DDAH1 with PD404182 failed to drive changes in expression or protein levels of DDAH isoforms or USF-1 protein or targets.

[0046] Figures 14A-14D are graphs plotting expression levels of sGC subunits GUCY1A2 (Figure 14A), GUCY1A3 (Figure 14B), GUCY1B2 (Figure 14C), and GUCY1B3 (Figure 14D) in mouse quadricep muscle tissue after a 3-month in vivo treatment with ataciguat (WD+ATA) as compared to control (WD).

[0047] Figure 15 is a graph plotting relative expression levels of GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3 in mouse quadricep muscle tissue.

[0048] Figures 16A-16D are graphs plotting expression levels of sGC subunits GUCY1A2 (Figure 16A), GUCY1A3 (Figure 16B), GUCY1B2 (Figure 16C), and GUCY1B3 (Figure 16D) in male and female mouse quadricep muscle after treatment with ataciguat (WD+ATA) compared to control (WD).

[0049] Figures 17A-17B are graphs plotting expression levels of RUNX2 (Figure 17A) and Sp7 (Figure 17B) in mouse quadricep muscle tissue after 3 months of in vivo treatment with ataciguat (WD+ATA) compared to control (WD).

[0050] Figure 18 is a graph plotting RUNX2 expression in male and female mice treated with ataciguat (WD+ATA) compared to control (WD).

[0051] Figure 19 is a graph plotting expression levels of ID 1, a classical target gene of canonical BMP signaling, in aortic valve interstitial cells in vitro. The graph shows responses to increasing doses of ataciguat (1 pM, 2 pM, or 5 pM) in the absence or presence of 100 pg / mL BMP2. Ataciguat effectively attenuated ID1 expression (i.e.,Attorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0052] canonical BMP signaling) in a dose-dependent manner in unstimulated and BMP2-stimulated conditions.

[0053] Figure 20 is a graph plotting expression levels of SPP1 in mouse quadriceps muscle tissue after treatment with ataciguat (WD+ATA) compared to control (WD).

[0054] Figure 21 is a graph plotting expression levels of SPP1 in male and female mouse quadricep muscle after treatment with ataciguat (WD+ATA).

[0055] Figures 22A-22B are graphs plotting expression of CDKN2A in mouse quadricep muscle tissue after treatment with ataciguat (WD+ATA). Figure 22 A includes data for all mice, and Figure 22B separates the male mouse data from the female mouse data.

[0056] DETAILED DESCRIPTION

[0057] This document provides methods and materials for reducing formation and / or slowing progression of ectopic tissue mineralization. For example, this document provides ataciguat, compositions containing ataciguat, and methods for using ataciguat and / or compositions containing ataciguat to treat mammals having, or likely to develop, ectopic tissue mineralization.

[0058] As described herein, ataciguat can be administered to a mammal (e.g., a human) to reduce ectopic tissue mineralization formation and / or to slow ectopic tissue mineralization progression within the mammal. For example, ataciguat can be administered to a mammal (e.g., a human) having ectopic tissue mineralization (or likely to develop ectopic tissue mineralization) to reduce ectopic tissue mineralization and / or to slow ectopic tissue mineralization progression.

[0059] Any type of mammal having or likely to develop ectopic tissue mineralization can be treated as described herein. Examples of mammals that can have or be likely to develop ectopic tissue mineralization and can be treated as described herein include, without limitation, humans, non-human primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats. In some cases, a human having ectopic tissue mineralization can be treated as described herein. In some cases, a human likely to develop ectopic tissue mineralization can be treated as described herein.

[0060] When treating a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization as described herein, the ectopic tissue mineralization can beAttorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0061] associated with a disease or condition. Examples of diseases and conditions that can be associated with ectopic tissue mineralization and treated as described herein to reduce ectopic tissue mineralization and / or to slow ectopic tissue mineralization progression include, without limitation, age-associated tendinous degeneration and calcification, chronic kidney disease, atherosclerosis, aortic valve stenosis, osteoarthritis, calcific tendinitis, diabetes-associated calcific tendinitis, hypothyroidism-associated calcific tendinitis, ligamentous calcification, myositis ossificans, pseudoxanthoma elasticum, Keutel syndrome, heterotopic ossification, heterotopic calcification, x-linked hypophosphatemia, fibrodysplasia ossificans progressiva, ankylosing spondylitis, diffuse idiopathic skeletal hyperostosis, hypertrophic osteoarthritis, Paget’s disease, diabetes, post-traumatic arthritis, osteonecrosis, rheumatoid arthritis, vitamin and mineral imbalances, accelerated aging conditions, Hutchinson-Gilford syndrome, Werner syndrome, and radiation-induced soft tissue calcification of tendons and other connective tissues. Ectopic tissue mineralization, particularly ectopic ossification, may be associated with previous tissue trauma or injury, for example, following surgery or traumatic blast injuries commonly seen in warfare.

[0062] When treating a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization as described herein, the ectopic tissue mineralization can be present in any appropriate tissue within the mammal. Examples of tissues that can undergo ectopic tissue mineralization and that can be treated as described herein include, without limitation, skin, cartilage, muscle (e.g., skeletal muscle such as quadricep, gastrocnemius, bicep, or hamstring), tendons, joints, valves, vessels, arteries, brain and nervous system tissue, ligaments, calcific bursitis, and other soft tissues. In some cases, when treating a mammal having or likely to develop ectopic tissue mineralization as described herein, the ectopic tissue mineralization can be present in an organ within the mammal. Examples of organs that can undergo ectopic tissue mineralization can include, without limitation, heart, kidney, lung, breast, liver, and brain.

[0063] Any appropriate method can be used to identify a mammal (e.g., a human) as being a mammal having ectopic tissue mineralization. For example, urinalysis, blood tests, x-ray, computed tomography scanning, fluoroscopic x-ray imaging, ultrasound imaging, physical palpation, self-reported symptoms consistent with mineralization (e.g.,Atorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0064] pain with movement), and / or other clinical assessments can be used to identify a mammal (e.g., a human) as being a mammal having ectopic tissue mineralization. Any appropriate method can be used to identify a mammal (e.g., a human) as being a mammal likely to develop ectopic tissue mineralization. For example, patient questioning or review of a patient’s medical history, recent tissue injury, chronic joint inflammation, systemic inflammatory conditions, and / or other clinical risk factors associated with soft tissue mineralization can be used to identify a mammal (e.g., a human) as being a mammal likely to develop ectopic tissue mineralization.

[0065] In some cases, ataciguat can be administered to a mammal having ectopic tissue mineralization (or likely to develop ectopic tissue mineralization) to reduce ectopic tissue mineralization formation by delaying or preventing the formation of ectopic tissue mineralization within the mammal. For example, ataciguat can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having or likely to develop ectopic tissue mineralization) to reduce the formation of ectopic tissue mineralization within the mammal. In some cases, the methods and materials described herein can be effective to delay the formation of ectopic tissue mineralization within a mammal by, for example, at least 6 months (e.g., at least about 7 months, at least about 8 months, at least about 10 months, at least about 1 year, at least about 1.5 years, at least about 2 years, at least about 2.5 years, at least about 3 years, at least about 4 years, at least about 5 years, 10 to 20 years, or for the remaining lifetime of the mammal being treated). In some cases, the formation of ectopic tissue mineralization within a mammal being treated as described herein (e.g., by administration of ataciguat) can be delayed for as long as the mammal is administered ataciguat.

[0066] In some cases, ataciguat can be administered to a mammal having ectopic tissue mineralization to slow progression of the ectopic tissue mineralization. For example, ataciguat can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having ectopic tissue mineralization) to slow progression (e.g., to slow further growth) of that ectopic tissue mineralization within the mammal. In some cases, the methods and materials described herein can be effective to slow progression of ectopic tissue mineralization within a mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the progression of existing ectopic tissue mineralizationAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0067] within a mammal being treated as described herein (e.g., by administration of ataciguat) can be reduced to no observable growth for as long as the mammal is administered ataciguat.

[0068] Any appropriate form of ataciguat (5-chloro-2-[[(5-chloro-2-thienyl)sulfonyl]amino]-N-[4-(4-morpholinylsulfonyl)phenyl]-benzamide; HMR1766) can be administered to a mammal (e.g., a human) having (or likely to develop) ectopic tissue mineralization to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein.

[0069] In some cases, a mammal (e.g., a human) having (or likely to develop) ectopic tissue mineralization can be administered or instructed to self-administer ataciguat. For example, a mammal (e.g., a human) having (or likely to develop) ectopic tissue mineralization can be administered a composition containing ataciguat to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein. In some cases, a mammal (e.g., a human) having (or likely to develop) ectopic tissue mineralization can be instructed to self-administer a composition containing ataciguat to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein.

[0070] In some cases, when a composition containing ataciguat is administered to a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization, the composition can be designed for oral or parenteral (including, without limitation, subcutaneous, intramuscular, intravenous, intradermal, intra-cerebral, intrathecal, intraabdominal, and intraperitoneal injections) administration to the mammal.

[0071] Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. In some cases, a pharmaceutically acceptable composition including ataciguat can be administered locally or systemically. For example, a composition containing ataciguat can be administered systemically by injection to a mammal (e.g., a human).Atorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0072] In some cases, a composition containing ataciguat can be in the form of a sterile injectable suspension (e.g., a sterile injectable aqueous or oleaginous suspension). This suspension may be formulated using, for example, suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Examples of acceptable vehicles and solvents that can be used include, without limitation, saline, mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In some cases, sterile, fixed oils can be used as a solvent or suspending medium. In some cases, a bland fixed oil can be used such as synthetic mono- or di-glycerides.

[0073] When ataciguat is formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein, the composition can deliver ataciguat in any manner. In some cases, a composition including ataciguat can be an immediate release composition (e.g., an immediate release oral dosage form). In some cases, a composition including ataciguat can be a controlled (e.g., delayed and / or sustained) release compositions (e.g., a controlled release oral dosage form). For example, a controlled release composition including ataciguat can be designed to release ataciguat over 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0074] When designing a composition (e.g., a pharmaceutically acceptable composition) containing ataciguat for administration to a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein, the composition (e.g., pharmaceutically acceptable composition) can include any appropriate effective amount of ataciguat. For example, a therapeutically effective amount of ataciguat can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, cyclodextrins (e g., beta-cyclodextrins such as KLEPTOSE®), dimethyl sulfoxide (DMSO), sucrose, lactose, starch (e.g., starch glycolate), cellulose,Atorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0075] cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline such as phosphate buffered saline, protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and com oil. If required, the solubility and bioavailability of ataciguat in a pharmaceutically acceptable composition can be enhanced using lipid excipients and / or block copolymers of ethylene oxide and propylene oxide. See, e.g., U.S. Patent No. 7,014,866 and U.S. Patent Application Publication Nos.

[0076] 2006 / 0094744 and 2006 / 0079502.

[0077] In some cases, a composition containing ataciguat can be designed to be in unitdose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.

[0078] Ataciguat can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to reduce formation and / or to slow progression of ectopic tissue mineralization).

[0079] An effective amount of ataciguat that can be administered to a mammal as described herein can be any amount that reduces formation of ectopic tissue mineralization within the mammal and / or slows progression of ectopic tissue mineralization within the mammal without producing significant toxicity to the mammal. For example, an effective amount of ataciguat can be from about 0.0001 mg ofAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0080] ataciguat / kg of body weight to about 8000 mg of ataciguat / kg of body weight (e.g., from about 0.0001 mg / kg to about 5000 mg / kg, from about 0.0001 mg / kg to about 1000 mg / kg, from about 0.0001 mg / kg to about 500 mg / kg, from about 0.0001 mg / kg to about 100 mg / kg, from about 0.0001 mg / kg to about 50 mg / kg, from about 0.0001 mg / kg to about 20 mg / kg, from about 0.0001 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.1 mg / kg, from about 0.0001 mg / kg to about 0.01 mg / kg, from about 0.0001 mg / kg to about 0.001 mg / kg, from about 0.001 mg / kg to about 8000 mg / kg, from about 0.01 mg / kg to about 8000 mg / kg, from about 0.1 mg / kg to about 8000 mg / kg, from about 1 mg / kg to about 8000 mg / kg, from about 10 mg / kg to about 8000 mg / kg, from about 20 mg / kg to about 8000 mg / kg, from about 50 mg / kg to about 8000 mg / kg, from about 100 mg / kg to about 8000 mg / kg, from about 500 mg / kg to about 8000 mg / kg, from about 1000 mg / kg to about 8000 mg / kg, from about 5000 mg / kg to about 8000 mg / kg, from about 0.001 mg / kg to about 5000 mg / kg, from about 0.01 mg / kg to about 1000 mg / kg, from about 0.1 mg / kg to about 500 mg / kg, from about 1 mg / kg to about 100 mg / kg, from about 10 mg / kg to about 50 mg / kg, from about 0.0001 mg / kg to about 0.01 mg / kg, from about 0.001 mg / kg to about 0.1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 20 mg / kg, from about 10 mg / kg to about 50 mg / kg, from about 20 mg / kg to about 100 mg / kg, from about 50 mg / kg to about 500 mg / kg, from about 100 mg / kg to about 1000 mg / kg, from about 500 mg / kg to about 5000 mg / kg, or from about 1000 mg / kg to about 8000 mg / kg). In some cases, between about 0.1 mg and 200 mg (e.g., between about 0.1 mg and 150 mg, between about 0.1 mg and 100 mg, between about 0.1 mg and 50 mg, between about 0.1 mg and 20 mg, between about 0.1 mg and 10 mg, between about 0.1 mg and 5 mg, between about 0.1 mg and 1 mg, between about 1 mg and 200 mg, between about 5 mg and 200 mg, between about 10 mg and 200 mg, between about 20 mg and 200 mg, between about 50 mg and 200 mg, between about 100 mg and 200 mg, between about 150 mg and 200 mg, between about 1 mg and 150 mg, between about 5 mg and 100 mg, between about 10 mg and 50 mg, between about 0.1 mg and 5 mg, between about 1 mg and 10 mg, between about 5 mg and 20 mg, between about 10 mg and 50 mg, between about 20 mg and 100 mg, between about 50 mg and 150 mg, or between about 100 mg and 200 mg) of ataciguat can beAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0081] administered to an average sized human (e.g., about 70 kg human) daily for about 20 weeks. If a particular mammal fails to respond to a particular amount, then the amount of ataciguat can be increased by, for example, two-fold. After receiving this higher amount, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly. Reciprocally, if a mammal experiences side effects at an initial dose (e.g., blood pressure lowering), the amount of ataciguat can be decreased while still monitoring for efficacy with regards to ectopic tissue mineralization. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., ectopic tissue mineralization formation and / or progression) can require an increase or decrease in the actual effective amount administered.

[0082] A composition containing ataciguat can be administered to a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization in any appropriate frequency. The frequency of administration can be any frequency that reduces formation of ectopic tissue mineralization within the mammal and / or slows progression of ectopic tissue mineralization within the mammal without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a week to about three times a day, or from about twice a month to about six times a day, or from about twice a week to about once a day. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing ataciguat can include rest periods. For example, a composition containing ataciguat can be administered daily over a two week period followed by a two week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., ectopic tissue mineralization formation and / or progression) can require an increase or decrease in administration frequency.Atorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0083] A composition containing ataciguat can be administered to a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization for any appropriate duration. An effective duration for administering a composition containing ataciguat can be any duration that reduces formation of ectopic tissue mineralization within the mammal and / or slows progression of ectopic tissue mineralization within the mammal without producing significant toxicity to the mammal. In some cases, the effective duration can vary from several days to several weeks, months, or years. In general, the effective duration for the treatment of ectopic tissue mineralization as described herein can range in duration from several months to several years. In some cases, an effective duration can be for as long as an individual mammal is alive. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition (e g., ectopic tissue mineralization formation and / or progression) being treated.

[0084] In some cases, methods for treating a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization as described herein (e.g., by administering ataciguat) can include administering to the mammal ataciguat as the sole active ingredient to treat ectopic tissue mineralization formation and / or progression within the mammal. For example, a composition containing ataciguat can include ataciguat as the sole active ingredient in the composition that is effective to reduce formation of ectopic tissue mineralization within the mammal and / or to slow progression of ectopic tissue mineralization within the mammal.

[0085] In some cases, methods for treating a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization as described herein (e.g., by administering ataciguat) can include administering to the mammal ataciguat in combination with one or more cGMP-specific phosphodiesterase 5A (PDE5A) inhibitors. In some cases, ataciguat can be administered together as a single composition with the one or more PDE5A inhibitors. In some cases, ataciguat and the one more PDE5A inhibitors can be administered to the mammal being treated at the same time as separate compositions. In some cases, ataciguat can be administered to the mammal first followed by the one more PDE5A inhibitors. In some cases, the one more PDE5A inhibitors can be administered toAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0086] the mammal first followed by ataciguat. Examples of PDE5A inhibitors include, without limitation, sildenafil, vardenafil, tadalafil, EMD 360527, DA 8159, UK-343-664 (Walker et al., Xenobiotica, 31:651-664 (2001)), UK-427-387, UK-357903 ([l-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-pyridylsulphonyl}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., Mol. Cancer Ther., 803-809 (2002); (Osi Pharmaceuticals)), pyrazolinone, EMD82639 (Merck KgaA, Darmstadt, DE; (4-(4-[2-ethyl-phenylamino)-methylene]-3-methyl-5-oxo-4,5-di-hydro-pyrazol-l-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)-l-methyl-3-propyl-lH-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, ethanolamin salt; Scutt et al., BMC Pharmacol., 4:10 (2004)), EMD171827 (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-(l-Methyl-7-oxo-3-propyl-6,7-dihydro-lH-pyrazolo-[4,3-d]pyrimidin-5-yl)-N-[2-(l-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide), E-4021 (Dukarm et al., Am. J. Respir. Crit. Care Med., 160:858-865 (1999)), pentoxifylline, andFR22934 (Fujisawa). Additional examples of PDE5A inhibitors can be set forth 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 No. WO 03 / 063875, WO 03 / 1012761, WO 2004 / 037183, and WO 98 / 38168.

[0087] In some cases, a course of treatment and the severity of ectopic tissue mineralization formation and / or progression can be monitored. Any appropriate method can be used to determine whether or not treatment with ataciguat as described herein is effective to reduce ectopic tissue mineralization formation and / or to slow ectopic tissue mineralization progression within the mammal being treated. For example, ectopic tissueAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0088] mineralization progression during a course of treatment described herein can be assessed using imaging techniques at different time points.

[0089] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0090] EXAMPLES

[0091] Example 1 - Increases in DDAH1 are adaptive in aortic valve disease This example identifies increases in DDAH1, a modulator of nitric oxide signaling, per se as adaptive in aortic disease and protective against fundamental molecular processes that drive osteogenesis, valvular calcification, and stenosis.

[0092] Human tissue samples

[0093] Non-calcified, non-stenotic human aortic valve tissue was acquired from explanted hearts at the time of transplant or from hearts not suitable for transplant, as described previously (Hagler et al., Cardiovasc. Res., 99:175-184 (2013)). Calcified valve tissue was acquired from patients with severe aortic valve stenosis undergoing aortic valve replacement surgery.

[0094] Non-atherosclerotic aortic tissue samples were obtained from explanted hearts that were not suitable for transplant. Aortic tissue samples from patients with severe atherosclerotic heart disease were obtained from patients undergoing coronary artery bypass surgery (i.e., tissue punches removed for initiation of anastomosis on ascending aorta).

[0095] Animals

[0096] To experimentally alter DDAH1 levels, we used low density lipoprotein receptordeficient, apolipoprotein BlOO-only (LA) mice that were either wild-type (D+ / +) or heterozygous (D+ / ) for DDAH1. Mice were studied following six months of Western diet feeding (TD88137, Harlan Teklad). Prior work from our group suggests that this model faithfully recaptures multiple phenotypic and molecular changes observed in tissues from aged humans with end-stage disease (Miller et al., Arterioscler. Thromb. Vase. Biol., 33:459-465 (2013); and Weiss et al., Circulation, 114:2065-2069 (2006)). Littermate-Atorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0097] matched pairs were studied at each time point whenever possible. Animals were euthanized using intraperitoneal injection of pentobarbital sodium (>100 mg / kg).

[0098] Cell culture

[0099] Aortic valves interstitial cells from C57BL / 6 mice (pooled in groups of three) were harvested. All three valve leaflets were used for cell culture. As described in previous study (Hagler et al., supra), aortic valves were pooled in keeping with standard culture techniques to ensure the growth of a large and sufficient number of cells for quantitative assays. Valvular cells were grown in complete media using standard outgrowth techniques (40% Dulbecco’s modified Eagles medium, 40% Ham’s F-10, 20% fetal bovine serum, 100 U / mL penicillin and 100 pg / mL streptomycin) between passages 6 and 8, and harvested at 90-100% confluence. Cells were treated separately for different concentration of ADMA (50 pmol / L) and PD404182 (50 pmol / L) or normal saline as a control. Twenty -four hour treatments were used to represent a standard duration at which transcriptional changes can be observed.

[0100] Quantitative Real-Time RT-PCR

[0101] Quantitative real-time PCR was performed on a StepOne Plus RT-PCR machine (Applied Biosystems). Gene expression for DDAH isoforms, ADMA transporters (SLC7A2), osteogenic pathways, and USF-1 related genes were measured using TaqMan Gene Expression Assay primers (Life Technologies). Gene expression levels were normalized by GADPH and hypoxanthine phosphoribosyltransferase 1 (HPRT1), both commonly used housekeeper genes and expressed using the AACt method.

[0102] Table 1. Primer sets used for gene expression assays. All primer sets were purchased from Life Technologies.

[0103]

[0104] Atorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0105]

[0106] Circulating glucose and ADMA levels

[0107] Mice were fasted for 12 hours prior to euthanasia. Whole blood glucose was measured at the time of sacrifice using a point-of-care device (AccuChek). Plasma samples were collected at the time of sacrifice and stored at -80°C. Plasma ADMA concentration was measured using ELISA / EIA kits (Immundiagnostik AG).

[0108] Cardiac and aortic valve function (in vivo)

[0109] Left ventricular ejection fraction, aortic valve cusp separation distance, and peak transvalvular velocity were assessed under light isoflurane anesthesia using echocardiography (GE Vivid 7 ultrasound system, 13 MHz linear probe) as described previously (Casaclang-Verzosa et al., J Vis Exp 120:54110 (2017)).

[0110] In brief, echocardiography was performed on all animals using 2-D, M-mode, pulse- wave and tissue Doppler imaging techniques. Each mouse was sedated using 1.5% isoflurane delivered via a nose cone, while keeping heart rate above 450 bpm. Left ventricular ejection fraction, fractional shortening and LV mass were calculated from the maximal diastolic and systolic dimensions obtained using M-mode imaging.

[0111] Aortic valve cusp separation distance was measured using M-mode imaging. Peak aortic velocity was obtained from Doppler flow velocity spectral display. The mitral, tricuspid and pulmonic valves were assessed using color flow imaging.Atorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0112] Blood pressure (in vivo)

[0113] Systolic blood pressure was measured non-invasively at six month time point using a tail-cuff method (CODA system, Kent Scientific). More specifically, blood pressure was measured for 5 consecutive days, with the first 3 days considered to be “acclimatization” to the stress of gentle restraint, and the mean of systolic blood pressure measurements during the last two days being used for data analysis.

[0114] RNA Extraction and cDNA Synthesis

[0115] Thoracic aortic arch tissue was carefully taken from test animal with perivascular adipose tissue removed, homogenized and stored in TRIzol regent (Invitrogen). Aortic valve tissue were carefully dissected and stored in TRIzol reagent. All aortic and aortic valve tissue was stored at -80°C prior to RNA extraction. RNA samples were isolated using spin columns and chloroform extractions (Ambion), as described previously (Hagler et al., supra).

[0116] Vasomotor function

[0117] Rings from descending thoracic aorta were used for studies of vasomotor function, as described previously (Roos et al., J. Physiol. Heart Circ. Physiol., 305: H1428-1439 (2013)). Responses to serotonin, acetylcholine, sodium nitroprusside, and prostaglandin F2alpha (PGF-2u) were evaluated in all mice.

[0118] Immunohistochemistry

[0119] Murine descending thoracic aorta (1-2 mm length), aortic valve, and kidney were embedded in tissue freezing medium (Triangle Biomedical Sciences). Cross-sections of the frozen embedded tissue were subsequently cryosectioned (10 pm thickness), and used for histological and immunohistochemical analyses. Osterix (1:500 dilution), USF-1 (1:200 dilution), and pSMADl / 5 / 8 (1:100 dilution, Cell Signaling Technology) primary antibodies were used on individual slides and co-stained with species matched Alexa Fluor 647 secondary antibodies. Primary antibody incubation duration was 24 hours for USF-1 and pSMADl / 5 / 8 experiments (4° C) and one hour incubation for osterix (room temperature), and secondary antibody incubation duration was 1 hour (roomAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0120] temperature). Sections were mounted in ProLong Gold DAPI solution, and then imaged on a Zeiss 780 LSM confocal microscope at 20x magnification. Subsequently, images were quantified and analyzed using the software ImageJ (NIH).

[0121] Table 2. Antibodies used for immunohistochemistry / immunofluorescence experiments. All antibody sets were purchased from Life Technologies.

[0122]

[0123] *CST, Cell Signaling Technology; LT, Life Technologies

[0124] Western blotting

[0125] Protein was obtained from lysed valvular and aortic cells. Gel electrophoresis was carried out using Bis-Tris 4-12% gels under reducing conditions, and subsequently transferred to nitrocellulose membranes. Membrane incubation with anti- USF-1, and anti-HPRTl was performed at dilutions of 1 : 1000 and for 18 h at 4°C. Thereafter, membranes were incubated with goat anti -rabbit HRP-conjugated secondary antibody for 1.5 hours at room temperature (1 :7500). Following the addition of chemiluminescent substrate, bands were detected by densitometric analysis performed using digital imaging techniques (Fluorochem M) and quantified using Cell Biosystems software. The density of pSMAD2 and pSMAD 1 / 5 / 8 protein bands was normalized to housekeeper, hypoxanthine phosphoribosyltransferase 1 (HPRT1). Quantitation was carried out using AlphaView software (ProteinSimple™).Atorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0126] Table 3. Antibodies used for western blotting experiments. All antibody sets were purchased from Life Technologies.

[0127]

[0128] Statistical analyses

[0129] Between-group differences were detected using unpaired, Bonferroni-corrected t-tests in GraphPad Prism Software. All data are expressed as mean ± SE.

[0130] Results

[0131] To determine whether ADMA synthesizing or hydrolyzing enzymes were altered in diseased tissue from aged humans with end-stage calcific aortic valve stenosis, RT-qPCR was performed on samples acquired from patients undergoing valve replacement surgery. Compared to normal valves, only expression of DDAH1 was significantly increased (Figure 1A) in valve tissue from patients with CAVS, whereas expression of the ADMA-synthesizing enzyme PRMT1 was reduced in calcified valve tissue (Figure 1A). In the context of the working hypothesis, these changes would be expected to be adaptive and protect against increases in intracellular ADMA (Morales et al., J. Biol. Chem., 290:14915-14926 (2015); and Pope et al., Pharmacol. Res., 60:461-465 (2009)). In contrast, expression of SLC7A2 - a key ADMA transporter was increased in calcified valve tissue compared to non-calcified control samples (Kawahara et al., Brain Res. Mol. Brain Res., 90:165-173 (2001); and Yeramian et al., J. Immunol., 176:5918-5924 (2006)) (Figure 1), which we propose would be maladaptive and result in deleterious increases in cytosolic ADMA (Strobel et al., Amino Acids, 45:989-1002 (2013)).

[0132] To determine the mechanistic contribution of DDAH1 in the evolution of calcific aortic valve disease, low density lipoprotein receptor-deficient / apolipoprotein B lOO-only mice (a previously validated model of valvular stenosis) were generated that were deficient in one copy of DDAH1 (LA-D+ / ). Consistent with previous reports (Hu et al.,Atorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0133] Circulation, 120:2222-2229 (2009)), these mice had significantly elevated plasma ADMA levels, increased blood pressure, and preserved left ventricular function.

[0134] In line with the working hypothesis, valvular calcification (Figure 2A) and valvular dysfunction (Figure 2B) were significantly increased in LA-D+ / ' mice compared to their LA-D+ +littermates. These increases in valvular calcification and valvular dysfunction were associated with increases in pSMADl / 5 / 8 protein levels (Figure 3A), osteogenic transcription factor proteins such as Osterix (Figure 3B), and target genes of canonical BMP signaling (Figure 3C) (Bostrom et al., J. Clin. Invest., 91:1800-1809 (1993); Miller et al., Circ. Res., 108:1392-1412 (2011); New & Aikawa, Circ. Res., 108:1381-1391 (2011); Seya et al., J. Pharmacol. Sci., 115:8-14 (2011); and Yangawa et al., J. Thorac. Cardiovasc. Surg., 144:256-262 (2012)). Based on a recent report associating increased nuclear methyl arginine levels with osteogenic differentiation (Cheng et al., Circ. Res., 117:142-156 (2015)), upstream stimulatory factor-1 (USF-1) protein levels (Figure 4A) were measured and expression of the USF-1 target genes osteopontin (SPP1) and alkaline phosphatase (ALPL, both depicted in Figure 4B) and strong evidence of increased USF-1 signaling in LA-D+ / ’ mice was found. Importantly, protein levels of SPP1 were modestly but not significantly elevated in valve tissue from LA-D+Amice (Figure 5A), suggesting that post-translational regulation of SPP1 (e.g., protein phosphorylation) may limit the anti-calcific effects of SPP1 in valve tissue (Cheng et al., supra, Jono et al., J. Biol. Chem., 275:20197-20203 (2000)). Consistent with this hypothesis, significant increases in tartrate-resistant acid phosphatase (TRAP) protein levels — an enzyme that cleaves and inactivates phosphorylated osteopontin (Hailing et al., Calcif. Tissue Int., 101:92-101 (2017); and Wolak, Atherosclerosis, 236:327-337 (2014)) — were found in aortic valves from LA-D+ / " mice compared to their LA-D+ / +littermates (Figure 5B-C).

[0135] To determine the relative contributions of ADMA and DDAH1 to valve calcification, an in vitro model system of cultured murine aortic valve interstitial cells (VICs) was used. Interestingly, treatment of VICs with exogenous ADMA did not significantly alter expression of DDAH1, DDAH2, or ADMA transporters in vitro (Figure 6A), nor did it alter USF-1 protein levels or osteopontin (SPP1) expression (Figure 6B). In contrast, inhibition of DDAH1 with PD404182 significantly increasedAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0136] both SPP1 expression and USF-1 protein levels (Figure 6B). Importantly, treatment of cells with the combination of ADMA or PD404182 did not exhibit an additive effect compared to PD404182 alone.

[0137] Collectively, these data suggest increases in DDAH1 per se are adaptive in aortic valve disease and protect against fundamental molecular processes that drive osteogenesis, valvular calcification, and stenosis, including suppression of canonical BMP signaling, USF-1 activity, and their nuclear targets.

[0138] Example 2 - Preservation of DDAH1 levels is an adaptive response and protects against valvular calcification

[0139] This Example identifies the preservation DDAH1 levels in diseased blood vessels as an adaptive response, protective against vascular calcification in conduit blood vessels independent of changes in osteogenic signaling or differentiation.

[0140] Results

[0141] Aortic tissue from patients undergoing coronary bypass surgery were acquired to determine changes in enzymes related to ADMA synthesis or metabolism. As a positive control, increased expression of inflammatory and osteogenic genes in diseased aortic vascular tissue was confirmed. Unlike observations from patients with end-stage aortic valve disease, expression of DDAH1 or DDAH2 did not differ between normal and diseased aortic vessel punch tissue, nor did expression of the ADMA transporter SLC7A2 (Figure 7). Changes in expression of arginine methyltransferases paralleled those observed in CAVS, however, with PRMT1 being significantly reduced in aorta from CABG patients compared to disease-free control subjects (Figure 7). While these data depict an overall adaptive strategy to minimize local ADMA synthesis in advanced atherosclerosis in conduit vessels, these data also depict fundamental differences in expression of DDAH1 and SLC7A2 between diseased aortic valve and aortic tissue.

[0142] To determine whether reducing DDAH1 accelerates intimal calcification, aortic tissue from the same LA-D+ / +and LA-D+ / ’ mice, which develop advanced atherosclerotic plaques and calcified intimal lesions following 6 months of Western diet feeding, was examined. Consistent with previous studies, mice with either genotype developedAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0143] advanced atherosclerotic plaques, and there were no significant differences in plaque size between groups. Much like observations in aortic valve, however, loss of one copy of DDAH1 increased intimal plaque calcification in LA-D+‘ mice compared to their LA-D+ / +littermates (Figure 8A). When taken in the context of prior reports that overexpression of DDAH1 can reduce atherosclerotic burden in hyperlipidemic mice (Jacobi et al., Am. J. Pathol., 176:2559-2570 (2010)), this data suggested that losses in DDAH1 are likely to have a predominant impact on atherosclerotic plaque composition. Consistent with previous observations (Roos et al., supra), experimentally reducing DDAH1 did not, however, further impair vascular relaxation to acetylcholine (Figure 8B) or other indices of vasomotor function (Figure 9).

[0144] When combined with the observation that haploinsufficiency of DDAH1 does not further increase atherosclerotic plaque size, these data suggested there may be a “floor effect” where reductions in DDAH1 activity may not be sufficient to drive overt changes in nitric oxide bioavailability or vascular function in the context of severe atherosclerosis. Interestingly, while increases in vascular calcification were associated with increases in canonical BMP signaling (Figure 10A), no increased expression of markers of mature osteoblast like cells (Figure 10B) or pSMADl / 5 / 8 target genes (Figure 10C) were observed, suggesting a functional disconnect between upstream BMP signaling and induction of downstream ectopic osteogenic gene programs. Furthermore — and in contrast to findings in aortic valve — no increases in USF-1 (Figure 11 A) or its target genes osteopontin and alkaline phosphatase (ALPL) in aorta from LA-D+ / ' mice were observed (Figure 1 IB). Similar to observations in aortic valve — there were no significant increases in SPP1 protein levels in aortic tissue (Figure 12A) in LA-D+ / ‘ mice compared to LA-D+ / +mice. Also, in contrast to findings in aortic valve, changes in TRAP protein and mRNA levels were variable and failed to reach statistical significance between genotypes in aortic tissue (Figure 12B-C). Altogether, the failure to increase SPP1 expression and / or protein levels may represent the dysregulation of mechanisms that protect against calcification in aorta (Ge et al., Sci. Rep., 7:40253 (2017); and Lee-Kirsch et al., Semin. Immunopathol. , 37:349-357 (2015)).

[0145] To determine the relative contributions of ADMA and DDAH1 to vascular calcification, an in vitro model system of cultured murine vascular smooth muscle cellsAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0146] (VSMCs) was used. Similar to aortic valve cells, treatment of VSMCs with exogenous ADMA did not alter expression of DDAH1, DDAH2, ADMA transporters (Figure 13 A), or USF-1 protein levels or molecular targets (Figure 13B). Unlike aortic valve interstitial cells, however, inhibition of DDAH1 with PD404182 failed to drive changes in expression or protein levels of DDAH isoforms or USF-1 protein or targets (Figure 13B).

[0147] Collectively, these data suggest that preservation of DDAH1 levels is an adaptive response in diseased blood vessels, and protects against vascular calcification in conduit blood vessels independent of changes in osteogenic signaling or differentiation. These results also demonstrate that sGS agonists can be used to reduce formation of ectopic tissue mineralization within mammals and / or to slow progression of ectopic tissue mineralization within mammals, in some cases, by restoring impaired nitric oxide signaling.

[0148] Example 3 - Treating Ectopic Tissue Mineralization Formation A human identified as having ectopic tissue mineralization formation is administered a composition containing 0.01 mg to 400 mg (e.g., 1 mg to 300 mg, or 10 mg to 250 mg) of ataciguat daily. After administering ataciguat daily for 30 days, ectopic tissue mineralization formation within the human is reduced.

[0149] Example 4 - Preventing Development of Ectopic Tissue Mineralization Formation A human identified as being susceptible to ectopic tissue mineralization formation is administered a composition containing 0.01 mg to 400 mg (e.g., 1 mg to 300 mg, or 10 mg to 250 mg) of ataciguat daily. While receiving daily administration of ataciguat, the human does not develop any new ectopic tissue mineralization formation.

[0150] Example 5 - Role of Ataciguat in Muscle Ossification sGC is a dimer made up of one alpha subunit (GUCY 1 A2 or GUCY 1 A3) and one beta subunit (GUCY1B2 or GUCY1B3). The subunits can heterodimerize to form a receptor that binds to ataciguat and related compounds as described herein. The A2 / B3 and A3 / B3 heterodimers are the primary receptors for ataciguat. Studies were conducted to assess the expression of each subunit in quadricep muscle tissue of mice treated orallyAtorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0151] with ataciguat for 3 months. sGC subunits that heterodimerize to form ataciguat-responsive enzymes (A2 / B3, A3 / B3) were expressed at detectable levels (GUCY 1 A2, Figure 14A; GUCY1A3, Figure 14B; and GUCY1B3, Figure 14D) and were not substantively changed by ataciguat treatment. GUCY1B2 (Figure 14C), which is thought to homodimerize and is not responsive to sGC agonists, was not expressed at detectable levels. Relative expression levels of the four sGC subunits (as compared to GUCY1A3) is shown in Figure 15.

[0152] Expression levels of sGC subunits also were compared between male and female mouse quadricep muscle tissue, with and without treatment with ataciguat. Expression of sGC subunits in quadricep muscle tissue from male and female mice was comparable (Figures 16A-16D).

[0153] In further studies, expression levels of the osteogenic transcription factors RUNX2 (Figure 17A) and Sp7 (Figure 17B) were measured in mouse quadricep muscle tissue after 3 months of oral treatment with ataciguat. Treatment with ataciguat in vivo substantially reduced expression of both RUNX2 and Sp7 in quadricep tissue as compared to control.

[0154] RUNX2 expression also was assessed in male and female mice treated with ataciguat. Basal levels of RUNX2 were higher in male mice compared to female mice (Figure 18), and treatment with ataciguat resulted in a more robust reduction in RUNX2 expression in the quadricep muscle tissue of male mice compared to female mice. This finding was consistent with clinical observations that males are significantly more likely to suffer from heterotopic ossification than females.

[0155] Expression of ID1, a classical target gene of canonical BMP signaling, in aortic valve interstitial cells, in response to increasing doses of ataciguat (IpM, 2 pM, or 5pM) in the absence or presence of 100 pg / mL BMP2, was assessed in vitro. Ataciguat effectively attenuated ID1 expression (canonical BMP signaling) in a dose-dependent manner in unstimulated and BMP2-stimulated conditions, indicating that activation of soluble guanylate cyclase effectively reduces osteogenic pathways known to drive soft tissue calcification (Figure 19).

[0156] When expressed in full-length form, osteopontin (SPP1) can protect against soft tissue calcification. The effect of ataciguat on expression of SPP1 in mouse quadricepsAttorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355

[0157] was assessed, demonstrating that ataciguat induced SPP1 expression (Figure 20). SPP1 expression in male and female mouse quadricep muscle after ataciguat treatment was then assessed, revealing that induction of SPP1 expression by ataciguat was more pronounced in male mice compared to female mice (Figure 21). This result was consistent with the observed reductions in Runx2 associated with ataciguat treatment in male mice (Figure 18).

[0158] CDKN2A is a marker of cellular senescence. Studies were conducted to evaluate the effect of ataciguat on CDKN2A expression in mouse quadricep muscle tissue. The ataciguat treatment had no apparent effect on CDKN2A expression in mouse quadricep muscle tissue overall (Figure 22A), but resulted in small but variable reductions in CDKN2A expression in male mice (Figure 22B).

[0159] OTHER EMBODIMENTS

[0160] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, 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. Attorney Docket No.: 07039-2400W01 / 2021-420 / 2019-355WHAT IS CLAIMED IS:

1. A method for reducing ectopic tissue mineralization formation or slowing ectopic tissue mineralization progression within a tissue of a mammal, wherein said method comprises administering a composition comprising ataciguat or a pharmaceutically acceptable salt thereof to said mammal having ectopic tissue mineralization or likely to develop ectopic tissue mineralization within said tissue, wherein formation of said ectopic tissue mineralization within said tissue is reduced or progression of said ectopic tissue mineralization within said tissue is slowed, wherein said tissue is tendonous tissue, ligamentous tissue, muscle tissue, skeletal muscle tissue, nervous system tissue, or skin tissue.

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

3. The method of any one of claims 1-2, wherein formation of ectopic tissue mineralization within said mammal is delayed, or wherein progression of ectopic tissue mineralization within said mammal is slowed.

4. The method of any one of claims 1-2, wherein formation of ectopic tissue mineralization within said mammal is delayed for at least 6 months, or wherein progression of ectopic tissue mineralization within said mammal is slowed by at least 50 percent.

5. The method of any one of claims 1-4, wherein said mammal is a mammal having said ectopic tissue mineralization.

6. The method of any one of claims 1-5, wherein said mammal is a mammal likely to develop said ectopic tissue mineralization.

7. The method of any one of claims 1-6, wherein said composition comprises said pharmaceutically acceptable salt thereof.

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