Methods and materials for using ataciguat based on alpha subunit composition and enzymatic oxidation state

Abstract

This document provides methods and materials for using ataciguat to treat clinical conditions / disorders / diseases within a mammal. For example, this document provides methods and materials for using ataciguat to treat mammals having clinical conditions / disorders / diseases associated with decreased levels or activity of the targeted sGC subunit or dimer.

Description

[0001] Attorney Docket No.: 07039-2402W01 / 2024-017

[0002] METHODS AND MATERIALS FOR USING ATACIGUAT BASED ON ALPHA SUBUNIT COMPOSITION AND ENZYMATIC OXIDATION STATE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority' to U.S. Application Serial No. 63 / 789,522, filed on April 16, 2026. The disclosure of the prior application is considered part of the disclosure of this application, and is incorporated in its entirety into this application.

[0004] TECHNICAL FIELD

[0005] This document relates to methods and materials involved in treating clinical conditions that are characterized by a reduction in soluble guanylate cyclase (sGC) signaling. For example, this document provides methods and materials for selecting ataciguat for uses targeted to particular sGC subunits, or to sGC dimers containing particular subunits, to treat mammals having clinical conditions that are characterized by a reduction in sGC signaling via the targeted sGC subunits or dimers.

[0006] BACKGROUND

[0007] Soluble guanylate cyclase (sGC) is an enzyme that binds nitric oxide (NO) and exerts effects that include cardioprotection, significantly’ slowing the progression of cardiovascular calcification. There are four types of sGC subunits: alphal (GUCY1A1, also referred to as GUCY1A3, alpha3, Al, and A3), betal (GUCY1B1, also referred to as GUCY1B3, beta3, Bl, and B3), alpha2 (GUCY1A2, also referred to as A2), and beta2 (GUCY1B2, also referred to as B2). For consistency, the subunits will be referred to herein as GUCY1 A3, GUCY1A2, GUCY1B3, and GUCY1B2. The sGC enzyme is a heterodimer that consists of one alpha subunit (GUCY1A3 or GUCY1 A2) and one beta subunit (GUCY1B3 or GUCY1B2). GUCY1A3 / GUCY1B3 heterodimers make up the vast majority of sGC in most tissues, and are responsible for most of the vasoactive effects of multiple sGC agonists. GUCY1 A3 and GUCY1A2 have significantly different subcellular localization patterns, with GUCY1 A3 having a largely cytosolic distribution and GUCY1 A2 localizing in close proximity' to the membrane, thereby providing a more concentrated pool of cGMP in that microenvironment. This is consistent with the fact that theAttorney Docket No.: 07039-2402W01 / 2024-017

[0008] GUCY1 A2 / GUCY1B3 heterodimer is localized near the cell membrane, while the GUCY1A3 / GUCY1B3 heterodimer is cytosolic.

[0009] SUMMARY

[0010] This document provides methods and materials for treating clinical disorders that are characterized by a reduction in sGC signaling (e.g., through genetic inactivation of enzymes in pathways that regulate nitric oxide signaling). For example, this document provides methods and materials for using ataciguat (or a pharmaceutically acceptable salt thereof) in uses targeted to particular sGC subunits, or targeted to sGC dimers containing particular subunits, to treat mammals having clinical conditions that are characterized by a reduction in sGC signaling via the targeted sGC subunits or dimers.

[0011] As demonstrated herein, sGC subunits (GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3) are differentially expressed in different tissues. These results demonstrate that ataciguat (or a pharmaceutically acceptable salt thereof) having a preference for a particular sGC subunit, or for a sGC dimer containing the particular subunit, can be used to treat diseases in tissues where the particular subunit is highly expressed. Having the ability to selectively activate sGC via a particular subunit as described herein can allow patients and clinicians to effectively treat patients who have disorders associated with a deficiency of the particular subunit.

[0012] In a first aspect, this document features a method for treating a mammal having a disorder characterized by a GUCY1 A2 deficiency. The method can include, or consist essentially of, administering, to the mammal, a composition comprising ataciguat (or a pharmaceutically acceptable salt thereof) having a GUCY1A2 preference, wherein one or more symptoms of the disorder within the mammal is reduced. The mammal can be a human. The disorder can be selected from the group consisting of systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, peripheral arterial disease, coronary’ vasospasm, coronary calcification, aortic aneurysm, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary’ hypertension, sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary- hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling inAttorney Docket No.: 07039-2402W01 / 2024-017

[0013] asthma, age-related cognitive decline, gastroparesis, irritable bowel disease, Crohn’s disease, non-alcoholic fatty’ liver disease, cirrhosis of the liver, cholecystitis, sclerosing cholangitis, hepatitis. Huntington’s disease, traumatic brain injury, chronic traumatic encephalopathy, Parkinson’s disease, vascular dementia, attention deficit / hyperactivity disorder, age associated dementia and / or cognitive decline, neurological recovery from stroke / transient ischemic attack, chronic renal failure, acute kidney injury, polycystic renal disease, renal fibrosis, glomerulonephritis, granulomatosis. Goodpasture Syndrome, diabetic nephropathy, lupus nephritis, IgA nephropathy, cystinosis, complement 3 glomerulopathy, focal segmental glomerulosclerosis, interstitial nephritis, Fabry' disease, cardiovascular-kidney-metabolic syndrome, renal stones, overactive bladder, and neurogenic bladder dysfunction.

[0014] In another aspect, this document features a method for avoiding unnecessarily treating a mammal having a disorder characterized by a GUCY1 A3 deficiency. The method can include, or consist essentially of, not administering, to the mammal, a composition comprising ataciguat. The mammal can be a human. The disorder can be selected from the group consisting of sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary' hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, peripheral arterial disease, coronary' vasospasm, coronary calcification, aortic aneurysm, Moyamoya Syndrome, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, gastroparesis, systemic scleroderma opioid-induced constipation, chronic constipation, irritable bowel disease, Crohn’s disease, anesthesia-induced / post-operative constipation, nonalcoholic fatty liver disease, cirrhosis of the liver, gallstones, cholecystitis, chronic acalculus gallbladder disease, sclerosing cholangitis, and hepatitis.

[0015] In another aspect, this document features a method for treating a mammal having a disorder characterized by a GUCY1 A2 / GUCY1B3 deficiency. The method can include, or consist essentially of, administering, to the mammal, a composition comprising ataciguat (or a pharmaceutically acceptable salt thereof) having a GUCY1A2 / GUCY1B3 preference, wherein one or more symptoms of the disorderAttorney Docket No.: 07039-2402W01 / 2024-017

[0016] within the mammal is reduced. The mammal can be a human. The disorder can be selected from the group consisting of systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneury sm, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary' hypertension, sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, age-related cognitive decline, gastroparesis, irritable bowel disease. Crohn’s disease, non-alcoholic fatty liver disease, cirrhosis of the liver, cholecystitis, sclerosing cholangitis, hepatitis, Huntington’s disease, traumatic brain injury, chronic traumatic encephalopathy, Parkinson’s disease, vascular dementia, attention deficit / hyperactivity disorder, age associated dementia and / or cognitive decline, neurological recovery from stroke / transient ischemic attack, chronic renal failure, acute kidney injury', polycystic renal disease, renal fibrosis, glomerulonephritis, granulomatosis, Goodpasture Syndrome, diabetic nephropathy, lupus nephritis, IgA nephropathy, cystinosis, complement 3 glomerulopathy, focal segmental glomerulosclerosis, interstitial nephritis, Fabry disease, cardiovascular-kidney-metabolic syndrome, renal stones, overactive bladder, and neurogenic bladder dysfunction.

[0017] In another aspect, this document features a method for avoiding unnecessarily treating a mammal having a disorder characterized by a GUCY1 A3 / GUCY1B3 deficiency. The method can include, or consist essentially of, not administering, to the mammal, a composition comprising ataciguat. The mammal can be a human. The disorder can be selected from the group consisting of sarcopenia / age-associated muscle loss, chemotherapy-induced muscle wasting, immobilization-induced muscle wasting, microgravity-induced muscle wasting, other muscle atrophy / wasting conditions, systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, peripheral arterial disease, coronary' vasospasm, coronary calcification, aortic aneurysm, Moyamoya Syndrome, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, gastroparesis,Attorney Docket No.: 07039-2402W01 / 2024-017

[0018] systemic scleroderma opioid-induced constipation, chronic constipation, irritable bowel disease, Crohn's disease, anesthesia-induced / post-operative constipation, nonalcoholic fatty liver disease, cirrhosis of the liver, gallstones, cholecystitis, chronic acalculus gallbladder disease, sclerosing cholangitis, and hepatitis.

[0019] In still another aspect, this document features a method for treating a mammal likely to develop a disorder characterized by a GUCY1A2 deficiency. The method can include, or consist essentially of, administering, to the mammal, a composition comprising ataciguat (or a pharmaceutically acceptable salt thereof) having a GUCY1 A2 preference, wherein the likelihood of development of the disorder within the mammal is reduced. The mammal can be a human. The disorder can be selected from the group consisting of systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneury sm, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity-induced muscle wasting, other muscle atrophy / wasting conditions, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, age-related cognitive decline, gastroparesis, irritable bowel disease. Crohn’s disease, non-alcoholic fatty liver disease, cirrhosis of the liver, cholecystitis, sclerosing cholangitis, hepatitis, Huntington’s disease, traumatic brain injury, chronic traumatic encephalopathy, Parkinson’s disease, vascular dementia, attention deficit / hyperacti vity disorder, age associated dementia and / or cognitive decline, neurological recovery from stroke / transient ischemic attack, chronic renal failure, acute kidney injury', polycystic renal disease, renal fibrosis, glomerulonephritis, granulomatosis, Goodpasture Syndrome, diabetic nephropathy, lupus nephritis, IgA nephropathy, cystinosis, complement 3 glomerulopathy, focal segmental glomerulosclerosis, interstitial nephritis, Fabry disease, cardiovascular-kidney-metabolic syndrome, renal stones, overactive bladder, and neurogenic bladder dysfunction.

[0020] In another aspect, this document features a method for avoiding unnecessarily treating a mammal likely to develop a disorder characterized by a GUCY1 A3 deficiency. The method can include, or consist essentially of, not administering, to the mammal, a composition comprising ataciguat. The mammal can be a human. TheAttorney Docket No.: 07039-2402W01 / 2024-017

[0021] disorder can be selected from the group consisting of sarcopenia / age-associated muscle loss, chemotherapy-induced muscle wasting, immobilization-induced muscle wasting, microgravity-induced muscle wasting, other muscle atrophy / wasting conditions, systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneurysm, Moyamoya Syndrome, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary' hypertension, gastroparesis, systemic scleroderma opioid-induced constipation, chronic constipation, irritable bowel disease, Crohn’s disease, anesthesia-induced / post-operative constipation, nonalcoholic fatty liver disease, cirrhosis of the liver, gallstones, cholecystitis, chronic acalculus gallbladder disease, sclerosing cholangitis, and hepatitis.

[0022] In another aspect, this document features a method for treating a mammal likely to develop a disorder characterized by a GUCY1A2 / GUCY1B3 deficiency. The method can include, or consist essentially of, administering, to the mammal, a composition comprising ataciguat (or a pharmaceutically acceptable salt thereof) having a GUCY1A2 / GUCY1B3 preference, wherein the likelihood of development of the disorder within the mammal is reduced. The mammal can be a human. The disorder can be selected from the group consisting of systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, peripheral arterial disease, coronary' vasospasm, coronary' calcification, aortic aneurysm, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, chronic obstructive pulmonary' disease, idiopathic pulmonary' fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, age-related cognitive decline, gastroparesis, irritable bowel disease, Crohn’s disease, non-alcoholic fatty liver disease, cirrhosis of the liver, cholecystitis, sclerosing cholangitis, hepatitis, Huntington’s disease, traumatic brain injury, chronic traumatic encephalopathy, Parkinson’s disease, vascular dementia, attention deficit / hyperactivity disorder, age associated dementia and / or cognitive decline, neurological recovery’ from stroke / transient ischemic attack, chronic renal failure, acute kidney injury; polycysticAttorney Docket No.: 07039-2402W01 / 2024-017

[0023] renal disease, renal fibrosis, glomerulonephritis, granulomatosis, Goodpasture Syndrome, diabetic nephropathy, lupus nephritis, IgA nephropathy, cystinosis, complement 3 glomerulopathy, focal segmental glomerulosclerosis, interstitial nephritis, Fabry disease, cardiovascular-kidney-metabolic syndrome, renal stones, overactive bladder, and neurogenic bladder dysfunction.

[0024] In yet another aspect, this document features a method for avoiding unnecessarily treating a mammal likely to develop a disorder characterized by a GUCY1A3 / GUCY1B3 deficiency. The method can include, or consist essentially of, not administering, to the mammal, a composition comprising ataciguat. The mammal can be a human. The disorder can be selected from the group consisting of sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneurysm, Moyamoya Syndrome, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, gastroparesis, systemic scleroderma opioid-induced constipation, chronic constipation, irritable bowel disease, Crohn’s disease, anesthesia-induced / post-operative constipation, non-alcoholic fatty liver disease, cirrhosis of the liver, gallstones, cholecystitis, chronic acalculus gallbladder disease, sclerosing cholangitis, and hepatitis.

[0025] 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.

[0026] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, andAttorney Docket No.: 07039-2402W01 / 2024-017

[0027] advantages of the invention will be apparent from the description and drawings, and from the claims.

[0028] DESCRIPTION OF DRAWINGS FIGS. 1A-1F are graphs plotting mRNA levels of the indicated sGC subunits (GUCY1A3, GUCY1A2, GUCY1B3, and GUCY1B2) in various tissues of male and female mice.

[0029] FIG. 2A is a graph plotting the effect of ataciguat (an activator of sGC) on mRNA levels of the osteogenic gene Runx2 in aortic valve tissue (i.e., ectopic ossifying tissue) of mice treated with ataciguat for 3 months. Restoration of nitric oxide signaling by ataciguat significantly reduced mRNA levels of this master regulator of osteoblastogenesis in aortic valve tissue. FIG. 2B is a graph plotting the effect of ataciguat on mRNA levels of Runx2 in femur tissue (i.e., orthotopic ossifying tissue) of mice treated with ataciguat for 3 months. Restoration of nitric oxide signaling did not significantly negatively impact mRNA levels of this master regulator of osteoblastogenesis in skeletal bone from either male or female mice.

[0030] FIG. 3A includes representative images and graphs showing calcium burden in male and female mice treated with WD or WD plus ataciguat. Ataciguat significantly reduced calcific burden in valves from mice of either sex. FIG. 3B is a graph plotting the change in bone volume / trabecular volume in femurs from mice treated with WD or WD plus ataciguat. Consistent with the molecular data from femur in FIG. 2B, long-term treatment w ith ataciguat did not have a negative impact on bone quality.

[0031] FIGS. 4A-4H are graphs plotting mRNA levels of the indicated sGC subunits in various tissues of male or female mice.

[0032] FIG. 5 includes graphs plotting GUCY1A2 mRNA levels in various tissues, relative to GUCY1 A3 mRNA levels in the same tissues.

[0033] FIGS. 6A-6C are graphs plotting relative expression of GUCY1 A3 (FIG. 6A), GUCY1A2 (FIG. 6B), and GUCY1B3 (FIG. 6C) in the indicated tissues, normalized to the level of each subunit in aorta.

[0034] FIG. 7 is a graph plotting phospho-VASPSER2?9expression by mouse aortic valve interstitial cells treated with either siRNA (20 nM) targeting knockdown of GUCY1A2 (siGUCYlA2) or GUCY1A3 (siGUCYlA3) as compared to scrambled siRNA-treated controls (CTL) and by mouse aortic valve interstitial cells treated withAttorney Docket No.: 07039-2402W01 / 2024-017

[0035] ataciguat (ATA; 1 pM) together with either siRNA (20 nM) targeting knockdown of GUCY1A2 (siGUCYl A2 + ATA) or GUCY1A3 (siGUCY!A3 + ATA) as compared to scrambled siRNA-treated controls treated with ataciguat (ATA).

[0036] FIGS. 8A-8D are graphs plotting GUCY1A2 expression (FIG. 8 A), GUCY1A3 expression (FIG. 8B), SP7 expression (FIG. 8C), and SPP1 expression (FIG. 8D) by mouse aortic vascular smooth muscle cells treated with either siRNA (20 pM) targeting knockdown of GUCY1A2 (siGUCYl A2) or GUCY1A3 (siGUCYl A3) as compared to scrambled siRNA-treated controls (NC siRNA).

[0037] FIGS. 9A-9D are graphs plotting GUCY1A2 expression (FIG.9A), GUCY1A3 expression (FIG. 9B), SP7 expression (FIG. 9C), and SPP1 expression (FIG. 9D) by mouse aortic vascular smooth muscle cells treated with ataciguat (ATA; 1 pM) and either siRNA (20 pM) targeting knockdown of GUCY1A2 (siGUCYl A2 + ATA) or GUCY1 A3 (siGUCYl A3 + ATA) as compared to controls treated with scrambled siRNA plus ataciguat (NC + ATA).

[0038] DETAILED DESCRIPTION

[0039] This document provides methods and materials for treating clinical disorders that are characterized by a reduction in sGC signaling (e.g., through genetic inactivation of enzymes in pathways that regulate nitric oxide signaling). For example, this document provides methods and materials for using ataciguat (or a pharmaceutically acceptable salt thereof) in uses targeted to particular sGC subunits, or targeted to sGC dimers containing particular subunits, to treat mammals having clinical conditions that are characterized by a reduction in sGC signaling via the targeted sGC subunits or dimers.

[0040] As described herein, disorders in which sGC signaling is deficient can, in some cases, be attributed to a reduction in signaling through a particular sGC subunit or a particular sGC dimer. In some cases, disorders in which sGC signaling is deficient can be attributed to a reduction in signaling through the GUCY1A3 subunit. As described herein, such disorders (also referred to herein as “GUCY1 A3 -deficient disorders’’) can be treated with a sGC agonist that preferentially targets GUCY1A3 (as opposed to GUCY1A2), which can increase or restore GUCY1A3 activity'. In some cases, disorders in which sGC signaling is deficient can be attributed to a reduction in signaling through the GUCY1 A2 subunit. As described herein, such disorders (also referred to herein asc'GUCYlA2-deficient disorders”) can be treatedAttorney Docket No.: 07039-2402W01 / 2024-017

[0041] with ataciguat (or a pharmaceutically acceptable salt thereof), which preferentially targets GUCY1A2 (as opposed to GUCY1A3), and which can increase or restore GUCY1 A2 activity'. In some cases, disorders in which sGC signaling is deficient can be attributed to a reduction in signaling through the GUCY1A3 / GUCY1B3 heterodimer. As described herein, such disorders (also referred to herein as “GUCYlA3 / GUCYlB3-deficient disorders") can be treated with a sGC agonist that preferentially targets a GUCY1A3 / GUCY1B3 (as opposed to

[0042] GUCY1A2 / GUCY1B3), which can increase or restore GUCY1 A3 / GUCY1B3

[0043] activity7. In some cases, disorders in which sGC signaling is deficient can be attributed to a reduction in signaling through the GUCY1A2 / GUCY1B3 heterodimer. As described herein, such disorders (also referred to herein as “GUCY 1 A2 / GUCY 1 B3-deficient disorders”) can be treated with ataciguat (or a pharmaceutically acceptable salt thereof), which preferentially targets GUCY1A2 / GUCY1B3 (as opposed to GUCY1A3 / GUCY1B3), and which can increase or restore GUCY1A2 / GUCY1B3 activity.

[0044] The sGC term ‘'determinative chain,” as used herein, refers to a sGC subunit having reduced or lost activity in a particular disorder. For example, if a disorder can be characterized by reduced or lost activity7of GUCY1A3, then GUCY1A3 is the “determinative chain” for that disorder. If a disorder can be characterized by reduced or lost activity of GUCY1 A2, then GUCY1A2 is the “determinative chain” for that disorder. Similarly, the term '‘determinative dimer,” as used herein, refers to a sGC dimer having reduced or lost activity7in a particular disorder. For example, if a

[0045] disorder can be characterized by reduced or lost activity of GUCY1A3 / GUCY1B3, then GUCY1 A3 / GUCY1B3 is the “determinative dimer” for that disorder. If a

[0046] disorder can be characterized by reduced or lost activity of GUCY1 A2 / GUCY1B3, then GUCY1 A2 / GUCY1B3 is the “determinative dimer” for that disorder. It is to be noted that alterations of either subunit of the “determinative dimer” (GUCY1A2, GUCY1A3, GUCY1B3, or a combination of alterations in those subunits) can be responsible for the reduced or lost activity.

[0047] Non-limiting examples of disorders and their associated determinative chains and dimers are listed in TABLES 1A and IB:

[0048] TABLE 1A: GUCY lA2-deficient / GUCY 1A2 / GUCY IBS-deficient disorders Cardiac muscle-related diseases

[0049]

[0050] Attorney Docket No.: 07039-2402W01 / 2024-017

[0051] systolic heart failure

[0052] diastolic heart failure

[0053] ventricular fibrosis

[0054] cardiac steatosis

[0055] atrial dysfunction

[0056] Vascular smooth muscle-related diseases

[0057] peripheral arterial disease

[0058] coronary vasospasm

[0059] coronary calcification

[0060] aortic aneurysm (thoracic or abdominal)

[0061] occlusive vascular disease

[0062] vascular rarefaction

[0063] hypertension

[0064] pulmonary hypertension

[0065] other conditions with pathological vascular remodeling and / or excessive vascular tone Skeletal muscle-related diseases

[0066] sarcopenia / age-associated muscle loss

[0067] chemotherapy-induced muscle wasting

[0068] immobilization-induced muscle wasting

[0069] microgravity-induced muscle wasting

[0070] other muscle atrophy / wasting conditions

[0071] Pulmonary disorders

[0072] chronic obstructive pulmonary' disease

[0073] idiopathic pulmonary fibrosis

[0074] pulmonary hypertension

[0075] acute asthmatic bronchoconstriction

[0076] chronic airway remodeling in asthma

[0077] Age-related cognitive decline

[0078] Gastrointestinal and hepatobiliary disorders

[0079] gastroparesis

[0080] irritable bowel disease

[0081] Crohn’s disease

[0082] non-alcoholic fatty liver disease (also referred to as liver steatosis)

[0083] cirrhosis of the liver

[0084] cholecystitis

[0085] sclerosing cholangitis

[0086] hepatitis

[0087] other conditions with elevated liver enzymes

[0088] Neurodegenerative conditions

[0089] Huntington’s disease

[0090] traumatic brain injury

[0091] chronic traumatic encephalopathy

[0092]

[0093] Parkinson’s diseaseAttorney Docket No.: 07039-2402W01 / 2024-017

[0094] vascular dementia

[0095] attention deficit / hyperactivity disorder

[0096] age associated dementia and / or cognitive decline

[0097] neurological recovery from stroke / transient ischemic attack

[0098] Renal and bladder disorders

[0099] chronic renal failure

[0100] acute kidney injury

[0101] polycystic renal disease

[0102] renal fibrosis

[0103] glomerulonephritis

[0104] granulomatosis

[0105] Goodpasture Syndrome (autoimmune attack and inflammation of the kidney) diabetic nephropathy

[0106] lupus nephritis

[0107] IgA nephropathy

[0108] Cystinosis

[0109] complement 3 glomerulopathy

[0110] focal segmental glomerulosclerosis

[0111] interstitial nephritis

[0112] Fabry disease

[0113] cardiovascular-kidney-metabolic syndrome

[0114] renal stones

[0115] overactive bladder

[0116]

[0117] neurogenic bladder dysfunction

[0118] TABLE IB: GUCY lA3-deficient / GUCY1A3 / GUCY IBS-deficient disorders Skeletal muscle-related diseases

[0119] sarcopenia / age-associated muscle loss

[0120] chemotherapy-induced muscle wasting

[0121] immobilization-induced muscle wasting

[0122] microgravity-induced muscle wasting

[0123] other muscle atrophy / wasting conditions

[0124] Cardiac muscle-related diseases

[0125] systolic heart failure

[0126] diastolic heart failure

[0127] ventricular fibrosis

[0128] cardiac steatosis

[0129] atrial dysfunction

[0130] Pulmonary disorders

[0131] chronic obstructive pulmonary' disease

[0132] idiopathic pulmonary fibrosis

[0133] pulmonary hypertension

[0134]

[0135] acute asthmatic bronchoconstrictionAttorney Docket No.: 07039-2402W01 / 2024-017

[0136] chronic airway remodeling in asthma

[0137] Vascular smooth muscle-related diseases

[0138] peripheral arterial disease

[0139] coronary vasospasm

[0140] coronary calcification

[0141] aortic aneurysm (thoracic or abdominal)

[0142] Moyamoya Syndrome

[0143] occlusive vascular disease

[0144] vascular rarefaction

[0145] hypertension

[0146] pulmonary hypertension

[0147] other conditions with pathological vascular remodeling and / or excessive vascular tone Gastrointestinal and hepatobiliary disorders

[0148] gastroparesis

[0149] systemic scleroderma opioid-induced constipation

[0150] chronic constipation

[0151] irritable bowel disease

[0152] Crohn's disease

[0153] anesthesia-induced / post-operative constipation

[0154] non-alcoholic fatty liver disease (also referred to as liver steatosis)

[0155] cirrhosis of the liver

[0156] gallstones

[0157] cholecystitis

[0158] chronic acalculus gallbladder disease

[0159] sclerosing cholangitis

[0160] hepatitis

[0161]

[0162] other conditions with elevated liver enzymes

[0163] Striated and smooth muscle-related disorders

[0164] In some cases, this document provides methods and materials for treating mammals having striated or smooth muscle-related disorders that are associated with a reduction in sGC signaling. For example, this document provides methods for targeted restoration of sGC activity in a mammal having a striated or smooth muscle-related disorder. The methods can include administering to the mammal ataciguat to preferentially target GUCY1 A2 to maximize therapeutic efficacy in a diseasedependent manner while minimizing off-target side effects in other tissues.

[0165] GUCY1A2 expression is comparable to GUCY1A3 expression in cardiac muscle, whereas GUCY1A3 expression is substantially higher than GUCY1A2 expression in skeletal muscle. In some cases, ataciguat, which can be selected asAttorney Docket No.: 07039-2402W01 / 2024-017

[0166] preferentially targeting GUCY1 A2 subunits can be used to treat mammals having a variety of striated and / or smooth muscle-related clinical conditions.

[0167] Further, increased oxidative stress is observed in a broad range of diseases in which striated or smooth muscle function is impaired. In some cases, the use of ataciguat to preferentially target the oxidized and / or heme-free states of heterodimeric sGC can confer greater selectivity and efficacy compared to compounds that preferentially target the reduced form of sGC (e.g., riociguat) in states of chronic striated or smooth muscle dysfunction.

[0168] Examples of acute and / or chronic striated cardiac muscle-related clinical conditions that can be treated using the methods and materials provided herein include, without limitation, systolic heart failure (right or left sided), diastolic heart failure (right or left sided), ventricular fibrosis (right or left sided), cardiac steatosis, and atrial dysfunction. Examples of acute and / or chronic striated skeletal muscle-related clinical conditions that can be treated using the methods and materials provided herein include, without limitation, sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity-induced muscle wasting, and other muscle atrophy / wasting conditions. Examples of acute / or chronic vascular smooth muscle-related clinical conditions that can be treated using the methods and materials provided herein include, without limitation, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneurysm (thoracic or abdominal), occlusive vascular disease, vascular rarefaction, hypertension, pulmonary' hypertension, and other conditions with pathological vascular remodeling and / or excessive vascular tone.

[0169] Pulmonary Diseases

[0170] In some cases, this document provides methods and materials for treating mammals having pulmonary disorders that are associated with a reduction in sGC signaling. For example, this document provides methods for targeted restoration of sGC activity in a mammal having a pulmonary disorder. The methods can include administering to the mammal ataciguat to preferentially target GUCY1 A2 to maximize therapeutic efficacy in a disease-dependent manner while minimizing off-target side effects in other tissues.

[0171] While GUCY1A2 expression is slightly lower than GUCY1A3 expression in homogenized lung tissue, GUCY1 A2 and GUCY1 A3 are expressed at comparableAttorney Docket No.: 07039-2402W01 / 2024-017

[0172] levels in nasopharynx, bronchial airways, and distal lung tissue. Ataciguat, which appears to exert protective effects in tissues and cells with high levels of oxidized / heme free GUCY1A2, can profoundly reduce BMP signaling in fibroblasts and in smooth muscle without negatively impacting blood pressure or vascular smooth muscle tone, suggesting a role for activation of various states (reduced / oxidized / heme free) of GUCY1A2 in the prevention of chronic tissue remodeling and interstitial disease, where reaching therapeutic efficacy without systemic vasodilation is highly favorable. Given the increase in oxidative stress observed in a broad range of diseases in which pulmonary’ vascular function or bronchial / bronchiolar function is impaired, the use of ataciguat that preferentially targets the oxidized and / or heme-free states of sGC with either heterodimeric subunit (either GUCY1A2 or GUCY1A3 heterodimerized with GUCY1B3) can confer greater selectivity and efficacy compared to compounds that preferentially target the reduced form of the enzyme (e.g., riociguat).

[0173] Examples of acute and / or chronic clinical pulmonary disorders that can be treated using the methods and materials provided herein include, without limitation, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, and chronic airway remodeling in asthma.

[0174] Cognitive Decline

[0175] In some cases, this document provides methods and materials for treating mammals having cognitive decline that is associated with a reduction in sGC signaling. For example, this document provides methods for targeted restoration of sGC activity in a mammal having cognitive decline. The methods can include administering to the mammal ataciguat to preferentially target GUCY1 A2 to maximize therapeutic efficacy in a disease-dependent manner while minimizing off-target side effects in other tissues.

[0176] Losses in NO (the ligand that activates sGC) can accelerate structural and functional changes associated with neurodegeneration and aging. Restoring sGC signaling may slow' or halt progression of cognitive decline. Ataciguat (which can activate oxidized sGC) appears to exert its greatest phenotypic effects in tissues where expression of GUCY1A2 is highest, and ataciguat is more effective at driving favorable shifts in ion channel function in cardiomyocytes compared to sGC agonistsAttorney Docket No.: 07039-2402W01 / 2024-017

[0177] that preferentially activate GUCY1A3. Further, rare loss-of-function mutations in GUCY1A2 are associated with severe cognitive impairment. Given the association between increases in oxidative stress and impaired NO signaling and cognitive / neural dysfunction in a variety of model systems, there is therapeutic utility in using ataciguat, which can preferentially activate the oxidized and / or heme-free states of sGC, particularly the oxidized and heme-free states of the GUCY1A2 / GUCY1B3 heterodimer, to effectively restore its activity and halt progression of cognitive decline with aging.

[0178] Gastrointestinal and Hepatobiliary Diseases

[0179] In some cases, this document provides methods and materials for treating mammals having gastrointestinal and / or hepatobiliary diseases that are associated with a reduction in sGC signaling. For example, this document provides methods for targeted restoration of sGC activity' in a mammal having a gastrointestinal disease. The methods can include administering to the mammal ataciguat to preferentially target GUCY1A2 to maximize therapeutic efficacy in a disease-dependent manner while minimizing off-target side effects in other tissues.

[0180] GUCY1A2 expression is thought to be is dramatically higher than GUCY1A3 in small intestine and duodenum, and high but comparable with GUCY1A3 in stomach and rectum. GUCY1A3 expression is much higher than GUCY1A2 expression in colon. Patients treated with ataciguat (which appears to exert protective effects in tissues and cells with high levels of oxidized / heme free GUCY1A2) had slightly higher self-reports of gastrointestinal side effects compared to placebo (e.g., symptoms consistent with increased GI motility); such changes may be more evident in patients having clinically-meaningful GI conditions (e.g., severe constipation) in which local oxidative stress is increased and greater therapeutic efficacy would be reached.

[0181] The moderate but comparable expression of GUCY1A2 and GUCY1A3 in liver provides a unique opportunity to use ataciguat to target GUCY 1 A2 to treat hepatic diseases. Further, given the increase in oxidative stress observed in a broad range of diseases in which gastrointestinal motility is impaired, the use of ataciguat to preferentially target the oxidized and / or heme-free states of sGC with either heterodimeric subunit (either GUCY1A2 or GUCY1A3 heterodimerized with GUCY1B3) is likely to confer greater selectivity and efficacy compared toAttorney Docket No.: 07039-2402W01 / 2024-017

[0182] compounds that preferentially target the reduced form of the enzyme (e.g., riociguat) in states of chronic gastrointestinal dysfunction.

[0183] Examples of acute and chronic clinical gastrointestinal indications that can be treated using the methods and materials provided herein include, without limitation, gastroparesis, systemic scleroderma opioid-induced constipation, chronic constipation, irritable bowel disease. Crohn's disease, and anesthesia-induced / post-operative constipation. Examples of acute and chronic clinical hepatobiliary conditions that can be treated using the methods and materials provided herein include, without limitation, non-alcoholic fatty' liver disease (also referred to as liver steatosis), cirrhosis of the liver, gallstones, cholecystitis, chronic acalculus gallbladder disease, sclerosing cholangitis, hepatitis, and other conditions in which liver enzymes are elevated due to hepatic inflammation or other clinical conditions.

[0184] Neurodegenerative Disease

[0185] In some cases, this document provides methods and materials for treating mammals having neurodegenerative disorders that are associated with a reduction in sGC signaling. For example, this document provides methods for targeted restoration of sGC activity7in a mammal having a neurodegenerative disorder. The methods can include administering to the mammal ataciguat to preferentially target GUCY1A2 to maximize therapeutic efficacy in a disease-dependent manner while minimizing off-target side effects in other tissues.

[0186] GUCY1A2 is selectively enriched in hippocampus, basal ganglia, cerebral cortex, and cerebellum compared to GUCY1A3, with GUCY1A3 not being detectable in some brain regions. Ataciguat can be used to exert protective effects in tissues and cells with high levels of oxidized / heme-free GUCY1A2 and can have profoundly favorable effects on action potential duration and cellular repolarization compared to other agonists that exert robust physiological effects in tissues with high levels of expression of GUCY1A3 (with those agonists having a lesser impact on action potential duration in cells with higher levels of GUCY1 A2). Given the increase in oxidative stress observed in a broad range of diseases in which neurocognitive function is impaired, the use of ataciguat to preferentially target the oxidized and / or heme-free states of sGC with either heterodimeric subunit (either GUCY1 A2 or GUCY1A3 heterodimerized with GUCY1B3) can confer greater selectivity andAttorney Docket No.: 07039-2402W01 / 2024-017

[0187] efficacy compared to compounds that preferentially target the reduced form of the enzyme (e.g., riociguat).

[0188] Examples of acute and chronic neurodegenerative clinical indications that can be treated using the methods and materials provided herein include, without limitation, Huntington's disease, traumatic brain injury7, chronic traumatic encephalopathy, Parkinson’s disease, vascular dementia, attention deficit / hyperactivity disorder, age associated dementia and / or cognitive decline, and neurological recovery from stroke / transient ischemic attack.

[0189] Renal and Bladder Disorders

[0190] In some cases, this document provides methods and materials for treating mammals having renal and / or bladder disorders that are associated with a reduction in sGC signaling. For example, this document provides methods for targeted restoration of sGC activity in a mammal having a renal disorder. The methods can include administering to the mammal ataciguat to preferentially target GUCY1A2 to maximize therapeutic efficacy in a disease-dependent manner while minimizing off-target side effects in other tissues.

[0191] Selective enrichment of GUCY1A2 has been observed in kidneys from mice with chronic hyperlipidemia compared to GUCY1A3. with the absolute levels of GUCY1A2 being much lower than those observed in cardiovascular tissues (suggesting an increased propensity for side effects if an agonist activates sGC to comparable amounts in both tissues). Ataciguat can be used to exert protective effects in tissues and cells with high levels of oxidized / heme-free GUCY1A2 and can have profoundly favorable effects on action potential duration and cellular repolarization compared to other agonists known to exert robust physiological effects in tissues with high levels of expression of GUCY1 A3 (with those agonists having a lesser impact on action potential duration in GUCYfA2-high cells). The increase in oxidative stress observed in a broad range of diseases in which renal function is impaired indicates that the use of ataciguat to preferentially target the oxidized and / or heme-free states of sGC with either heterodimeric subunit (either GUCY1A2 or GUCY1A3 heterodimerized with GUCY1B3) may confer greater selectivity7and efficacy compared to compounds that preferentially target the reduced form of the enzyme (e.g., riociguat) unless used in very early stages of disease (e.g.. for preventative treatment before increases in oxidative stress occur). In some cases, urine-basedAttorney Docket No.: 07039-2402W01 / 2024-017

[0192] screening for redox state can be used to identify appropriate treatment regimens of ataciguat for mammals having, or being at risk for, acute or chronic renal disease. Further, urine microvesicles can be screened to determine which GUCY1 A isoform is enriched and to determine its oxidation state.

[0193] Examples of renal and bladder disorders that can be treated using the methods and materials provided herein include, without limitation, chronic renal failure, acute kidney injury, polycystic renal disease, renal fibrosis, glomerulonephritis, granulomatosis. Goodpasture Syndrome (autoimmune attack and inflammation of the kidney), diabetic nephropathy, lupus nephritis, IgA nephropathy, cystinosis, complement 3 glomerulopathy, focal segmental glomerulosclerosis, interstitial nephritis. Fabry disease, cardiovascular-kidney-metabolic syndrome, facilitation of passing renal stones, overactive bladder, and neurogenic bladder dysfunction.

[0194] It is to be noted that in some cases, a particular disorder can be associated with a deficiency in GUCY1A2 (or GUCY1A2 / GUCY1B3) or a deficiency in GUCY1A3 (or GUCY1 A3 / GUCY1B3). Thus, in some cases, a person having a particular disorder (e.g., systolic heart failure) may have a GUCY1A2 deficiency or a GUCY1A2 / GUCY1B3 deficiency, while a different person having the same disorder may have a GUCY1A3 deficiency or a GUCY1A3 / GUCY1B3 deficiency. In other cases, a particular disorder (e.g., neurodegeneration) can be associated with a deficiency GUCY1A2 (or GUCY1A2 / GUCY1B3), and not with a deficiency in GUCY1 A3 (or GUCY 1 A3 / GUCY1 B3).

[0195] To determine whether a disorder associated with reduced sGC signaling is likely to be a GUCY1 A2-deficient disorder or a GUCY1 A3-deficient disorder (or a GUCYlA2 / GUCYlB3-deficient disorder or a GUCYlA3 / GUCYlB3-deficient disorder), the level of each sGC subunit and heterodimer type can be assessed (e.g., in healthy tissue corresponding to the tissue that is affected by the disorder). If the level of one of the sGC subunits is higher than the level of the other subunit, it is likely that the disorder can be treated with agonists targeting the subunit having the higher level of expression. Further, based on the biological underpinnings of the disease and their interactions with different sGC subunits, targeting one subunit over the other to suppress known pathological disease drivers can yield greater therapeutic efficacy. For example, if bone morphogenetic protein signaling is a key molecular driver of disease and is effectively suppressed by restoration of GUCYlA2-dependent sGC activity, treating with ataciguat can yield greater therapeutic efficacy than aAttorney Docket No.: 07039-2402W01 / 2024-017

[0196] GUCYlA3-selective agonist. Moreover, if the level of the GUCY1A2 -heterodimer is higher than the level of the GUCY1 A3 -heterodimer, then it is likely that the disorder can be treated with ataciguat targeting the heterodimer having the higher level of expression.

[0197] As described herein, diseases characterized by aGUCYlA2 deficiency (also referred to herein as 'GUCY I A2-deficient diseases'’) can be treated by administering ataciguat having a GUCY1 A2 preference, while diseases characterized by a GUCY1A3 deficiency (also referred to herein as “GUCYlA3-deficient diseases”) can be treated in some cases by not administering ataciguat. In some cases, the ability to use ataciguat in a particular therapeutic indication can be assessed. For example, ataciguat can be evaluated to confirm or characterize its ability to activate the oxidized (e.g., diseased) or reduced (e.g., healthy) state of the sGC enzyme. For example, ataciguat can be evaluated to confirm or characterize its ability to activate the oxidized (e.g., diseased) or reduced (e.g., healthy) state of the sGC enzy me. The sGC enzyme resides in one of three physiological states: (1) a "reduced" state in which it is responsive to endogenous nitric oxide or sGC ‘'stimulators” (pharmacological ligands that increase enzymatic activity of the heme-containing, reduced form of the enzy me and can synergize with endogenous nitric oxide), (2) an “oxidized” state in which sGC is oxidized, still contains heme, and is no-longer responsive to endogenous nitric oxide but can be responsive to sGC “activators” (pharmacological ligands that increase activity of the oxidized form of the enzyme and generally do not synergize with endogenous nitric oxide), or (3) a “heme-free” state in which the heme group is displaced from the oxidized sGC enzymatic complex, rendering it non-responsive to endogenous nitric oxide but possibly still responsive to sGC “activators” (pharmacological ligands that increase activity of the heme-free form of the enz me and generally do not synergize with endogenous nitric oxide).

[0198] In some cases, the ability to use ataciguat can be assessed using, for example, biochemical assays of purified GUCY1A2 / GUCY1B3 or GUCY1A3 / GUCY1B3 heterodimers in cell-based assays with a variety of exogenous stressors (e.g., treatment with ODQ or paraquat to induce sGC oxidation) and measuring cGMP generation or phospho-VASPSER2?9. In some cases, the ability to use ataciguat can be assessed using in vivo animal models with a variety of exogenous stressors (e.g., treatment with ODQ or paraquat to induce sGC oxidation) and measuring either tissueAttorney Docket No.: 07039-2402W01 / 2024-017

[0199] cGMP levels, phospho-VASPSER2?9levels, or other physiological readouts. In some cases, approaches for assessing ataciguat’ s selectivity for the GUCY1A2 / GUCY1B3 heterodimer (which can inhibit calcification, inhibit BMP signaling, and improve cognitive function) vs. the GUCY1A3 / GUCY1B3 heterodimer (which is vasoactive) can include:

[0200] (a) Using biochemical assays with purified GUCY1A2 / GUCY1B3 heterodimers or purified GUCY1A3 / GUCY1B3 heterodimers to assess the ability of ataciguat to generate cGMP;

[0201] (b) In the biochemical assays of (a), using treatment with oxidizing agents, reducing agents, or heme-sufficient / heme-deficient conditions to create reduced, oxidized, or heme-free states of the sGC heterodimers and allow characterization of ataciguat for both the heterodimeric pairing and its unique state in health or disease;

[0202] (c) Knocking down or overexpressing the GUCY1 A3 or GUCY1A2 subunit in cells and assessing the ability of ataciguat to generate cGMP, to generate phospho-VASP (indicative of sGC activation), or to inhibit BMP signaling;

[0203] (d) Knocking down or overexpressing the GUCY1A3 or GUCY1A2 subunit in cardiomyocytes in vitro and assessing the ability' of ataciguat to generate cGMP, to generate phospho-VASP, or to shorten the QT interval; and

[0204] (e) Assessing the ability of ataciguat to generate cGMP / phospho-VASP and / or inhibit BMP signaling in animal models in vivo, with a focus on tissues known to have differential regulation of isoform expression (e.g., valve vs. aorta vs. bone vs. brain). This may include the use of GUCY1 A3- or GUCY1 A2-deficient mice.

[0205] Such approaches for screening can allow for greater local therapeutic efficacy by targeting subunit expression in specific tissues (and subsequent biological signaling changes) as well as oxidation state of sGC in varying stages and / or types of disease.

[0206] As described herein, ataciguat can be administered to a mammal (e.g., a human) to treat a disorder characterized by a deficiency of a particular sGC subunit or dimer within the mammal. For example, ataciguat having a GUCY1A2 preference can be administered to a mammal (e.g., a human) having a disorder characterized by a GUCY1A2 deficiency to treat (e.g., reduce one or more symptoms of) the disorder. In another example, ataciguat can be avoided from being administered to a mammal (e.g., a human) having a disorder characterized by a GUCY1A3 deficiency. In another example, ataciguat having a GUCY1 A2 / GUCY1B3 preference can be administered toAttorney Docket No.: 07039-2402W01 / 2024-017

[0207] a mammal (e.g., a human) having a disorder characterized by a GUCY1A2 / GUCY1B3 deficiency to treat (e.g., reduce one or more symptoms of) the disorder. In another example, ataciguat can be avoided from being administered to a mammal (e.g., a human) having a disorder characterized by a GUCY1 A3 / GUCY1B3 deficiency. A deficiency in the heterodimer can be caused by a reduction in either component of the enzyme. For example, deficiency in a GUCY1 A2 / GUCY1B3 heterodimer can be caused by either a lack of GUCY1A2 or a lack of GUCY1B3, as loss of either subunit reduces the total amount of heterodimeric protein available for binding endogenous or exogenous ligands. Similarly, deficiency in a GUCY1A3 / GUCY1B3 heterodimer can be caused by either a lack of GUCY1A3 or a lack of GUCY1B3, as loss of either subunit reduces the total amount of heterodimeric protein available for binding endogenous or exogenous ligands.

[0208] Any type of mammal having or likely to develop a disease characterized by a deficiency of a particular sGC subunit and / or a particular sGC dimer can be treated as described herein. Examples of mammals that can have or be likely to develop a disease characterized by a deficiency of a particular sGC subunit and / or a particular sGC dimer and that 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 a disease characterized by a deficiency of a particular sGC subunit and / or a particular sGC dimer can be treated as described herein. In some cases, a human likely to develop a disease characterized by a deficiency of a particular sGC subunit and / or a particular sGC dimer can be treated as described herein.

[0209] In some cases, ataciguat can be administered to a mammal having a disease characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer (or likely to develop a disease characterized by such a deficiency) to treat the disease or to reduce the likelihood of development of the disease. 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 a disease characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1 A2 / GUCY1B3 dimer) to reduce or delay development of the disease within the mammal. In some cases, the methods and materials described herein can be effective to delay the development of a disease characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer within a mammal by, for example, at least 6 months (e.g., at least about 7Attorney Docket No.: 07039-2402W01 / 2024-017

[0210] 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, or at least about 5 years). In some cases, the development of a disease characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1 A2 / GUCY1B3 dimer 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.

[0211] In some cases, ataciguat can be administered to a mammal having a disease characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer, to slow progression of the disease or to reduce one or more symptoms of the disease. For example, ataciguat can be administered to a mammal (e.g.. a human) in need thereof (e.g., a human having a disease characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1 A2 / GUCY1B3 dimer) to slow progression of the disease within the mammal. In some cases, the methods and materials described herein can be effective to slow progression of the disease 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 a disease within a mammal being treated as described herein (e.g., by administration of ataciguat) can be reduced to no observable progression for as long as the mammal is administered ataciguat. In some cases, ataciguat can be administered to a mammal (e.g.. a human) in need thereof (e.g.. a human having a disease characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer) to reduce one or more symptoms of the disease within the mammal. In some cases, the methods and materials described herein can be effective to reduce one or more symptoms of the disease within a mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, one or more symptoms of a disease within a mammal being treated as described herein (e.g., by administration of ataciguat) can be reduced to no observable level for as long as the mammal is administered ataciguat.

[0212] Any appropriate form of ataciguat (5-chloro-2-[[(5-chloro-2-thienyl)sulfonyl]amino]-N-[4-(4-morpholinylsulfonyl)phenyl]-benzamide;

[0213] HMR1766) can be administered to a mammal (e.g., a human) having (or likely to develop) a disease characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer as described herein.Attorney Docket No.: 07039-2402W01 / 2024-017

[0214] In some cases, a mammal (e.g., a human) having (or likely to develop) a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer can be administered or instructed to self-administer ataciguat. For example, a mammal (e.g., a human) having (or likely to develop) a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1 A2 / GUCY1B3 dimer can be administered a composition containing ataciguat to reduce one or more symptoms of the disorder. In some cases, a mammal (e.g., a human) having (or likely to develop) a disorder characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1 A2 / GUCY1B3 dimer can be instructed to selfadminister a composition containing ataciguat to slow progression of the disorder.

[0215] In some cases, when a composition containing ataciguat is administered to a mammal (e.g., a human) having or likely to develop a disorder characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1 A2 / GUCY1B3 dimer, 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.

[0216] 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).

[0217] 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 orAttorney Docket No.: 07039-2402W01 / 2024-017

[0218] suspending medium. In some cases, a bland fixed oil can be used such as synthetic mono- or di-glycerides.

[0219] 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 a disorder characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer 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.

[0220] 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 a disorder characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1 A2 / GUCY1B3 dimer 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®), dimethylsulfoxide (DMSO), sucrose, lactose, starch (e.g., starch glycolate), cellulose, 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, serumAttorney Docket No.: 07039-2402W01 / 2024-017

[0221] 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, disodium 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 bi oatai I ability 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. 2006 / 0094744 and 2006 / 0079502.

[0222] In some cases, a composition containing ataciguat can be designed to be in unit-dose 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.

[0223] Ataciguat (e.g., a composition containing 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 one or more symptoms of a disorder characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1 A2 / GUCY1B3 dimer).

[0224] An effective amount of ataciguat can be administered to a mammal as described herein can be any amount that reduces one or more symptoms of a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer within the mammal and / or slows progression of a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer within the mammal, without producing significant toxicity to the mammal. For example, an effective amount of ataciguat can be from about 0.01 mg of ataciguat / kg of body weight to about 80 mg of ataciguat / kg of body weight (e.g., from about 0.1 mg / kg to about 70 mg / kg, from about 0.1 mg / kg to about 60 mg / kg, from about 0.1 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.1 mg / kg to about 30 mg / kg, from about 0.1 mg / kg to about 20 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.075 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, or from about 2 mg / kg to about 10 mg / kg). In some cases, between about 0.5 mg and 200 mg (e.g., between about 50 mg and 180 mg, between about 50 mg and 150 mg, between about 50 mg and 125 mg, between about 60 mg and 200 mg, between about 75 mg and 200 mg, between about 100 mg and 200 mg, between aboutAttorney Docket No.: 07039-2402W01 / 2024-017

[0225] 75 mg and 150 mg, or between about 100 mg and 150 mg) of ataciguat can be 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 can be made accordingly. 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 can require an increase or decrease in the actual effective amount administered.

[0226] A composition containing ataciguat can be administered to a mammal (e.g., a human) having or likely to develop a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer at any appropriate frequency. The frequency of administration can be any frequency that reduces one or more symptoms of a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer within the mammal and / or slows progression of a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer within the mammal, without producing significant toxicity7to 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 severity7of the condition can require an increase or decrease in administration frequency.

[0227] A composition containing ataciguat can be administered to a mammal (e.g.. a human) having or likely to develop a disorder characterized a deficiency of aAttorney Docket No.: 07039-2402W01 / 2024-017

[0228] GUCY1 A2 subunit and / or a GUCY1 A2 / GUCY1B3 dimer within the mammal for any appropriate duration. An effective duration for administering a composition containing ataciguat can be any duration that reduces one or more symptoms of a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer within the mammal and / or slows progression of a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer 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 a condition 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 being treated.

[0229] In some cases, methods for treating a mammal (e.g., a human) having or likely to develop a disorder characterized by a deficiency of a GUCY1 A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer as described herein (e.g., by administering ataciguat) can include administering to the mammal ataciguat as the sole active ingredient(s) to reduce one or more symptoms of the disorder within the mammal, or to reduce the likelihood that the mammal will develop the disorder. For example, a composition containing ataciguat can include ataciguat as the sole active ingredient in the composition that is effective to reduce one or more symptoms of the disorder within the mammal and / or to slow progression of the disorder within the mammal.

[0230] In some cases, methods for treating a mammal (e.g., a human) having or likely to develop a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer 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 beAttorney Docket No.: 07039-2402W01 / 2024-017

[0231] administered to the mammal first followed by ataciguat. Examples of PDE5 A inhibitors include, without limitation, sildenafil, vardenafil, tadalafil, EMD 360527, DA 8159, UK-343-664 (Walker et al., Xenobiolica. 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., 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-di-hydro-pyrazol-l-yl)-benzoic acid; Senzaki et al., FASEBJ., 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]-pyrimi din-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, and FR22934 (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.

[0232] In some cases, a course of treatment and the severity and / or progression of a disorder characterized by a deficiency of a GUCY1A2 subunit and / or a GUCY1A2 / GUCY1B3 dimer can be monitored. Any appropriate method can be used to determine whether or not treatment with ataciguat as described herein is effectiveAttorney Docket No.: 07039-2402W01 / 2024-017

[0233] to reduce a symptom of such a disorder or is effective to slow progression of such a disorder within the mammal being treated.

[0234] The invention will be further described in the following example, which does not limit the scope of the invention described in the claims.

[0235] EXAMPLE

[0236] Studies were conducted to determine the expression levels of the different sGC subunits in various mouse tissues. The relative levels of expression of each of the predominant heterodimers (A2 / A3 / B2 / B3, all expressed relative to the expression of A3) was comparable between bone tissue (pulverized femur) and aortic valve (excised tissue) (FIGS. 1A-1C). Expression of the A2 subunit was very low compared to the A3 subunit in bone, with the ratio of A2 / A3 being relatively high / enriched in aortic valve tissue (FIGS. 1A-1C). When comparing levels of subunit expression across tissues, the absolute levels of A3 expression were dramatically higher in cardiovascular tissues than in bone tissue in both male and female mice (FIG. ID), with expression of A3 being highest in aorta. Similar to the expression patterns of A3, the absolute levels of expression of A2 were dramatically higher in cardiovascular tissues compared to bone tissue, and A2 expression was highest in aortic valve (FIG. IE). Absolute expression levels of B3 largely paralleled expression levels of A3, and were dramatically higher than those observed in bone tissue (FIG. IF).

[0237] FIGS. 2A-2B show the effects of ataciguat on expression of Runx2, a master regulator of osteoblastogenesis, in aortic valve tissue and in bone tissue from control mice (Idlr 7apoBl0(l 100mice fed a Western diet for 9 months) or ataciguat-treated mice (Idlr ’ 7apoB100 / 100mice fed a Western diet for 6 months and then fed a Western diet + ataciguat for 3 additional months). Expression of Runx2 was significantly reduced in aortic valve tissue from mice that were treated with ataciguat (FIG. 2A).

[0238] Notably, expression of Runx2 in bone tissue was not significantly affected by treatment with ataciguat (FIG.2B).

[0239] sGC can be reactivated with ataciguat for treatment of, for example, soft tissue calcification (e g., in the heart valves, aorta, muscle, and / or kidney), and for treatment of long QT syndromes. Although ataciguat can reduce soft tissue calcification, it does not appear to negatively impact bone, as demonstrated by DEXA scanning in patients receiving ataciguat in a Phase 2 trial and in preclinical mouse work using uCTAttorney Docket No.: 07039-2402W01 / 2024-017

[0240] scanning of the femur. See, also, FIG. 3A (effects of ataciguat on valve calcification) and FIG. 3B (effects of ataciguat on bone architecture).

[0241] To investigate the mechanism of ataciguat’s selectivity, mRNA levels of the four sGC isoforms (GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3) were measured in various tissues. Across all tissues evaluated, GUCY1B3 expression was highest, followed by robust expression of GUCY1A3 (FIGS. 4A-4H). This observation was consistent with the fact that these two isoforms are the most abundant in cells and form the most abundant 1A3 / 1B3 heterodimer found in cells. mRNA levels of GUCY1B2 in all tissue studied were very low and often undetectable (FIGS.

[0242] 4A-4H), consistent with the fact that 1B2 appears to form a relatively inactive 1B2 / 1B2 homodimer of poorly understood function.

[0243] Surprisingly, mRNA levels of GUCY1 A2 were highest in tissues that showed robust phenotypic changes in response to treatment with ataciguat (e.g., reductions in calcification in aortic valve, improvements in function and QT in left ventricle, and reductions in osteogenic signaling in aorta), as shown in FIG. 6B. Conversely, mRNA levels of GUCY1A2 were extremely low in tissues that did not show substantive changes (e.g., femur). More specifically, expression of the GUCY1 A2 isoform was 30-50 fold higher in the cardiovascular tissues than in femur, and in several subgroups (e.g., male / female of various tissues), GUCY1A2 mRNA levels were nearly identical to the GUCY1A3 mRNA levels (FIG. 5). GUCY1A3 and GUCY1B3 each displayed different expression levels across the tissues that were evaluated (FIGS. 6A and 6C).

[0244] Taken together, the results described herein suggested at least the following: 1. Targeting activation of the membrane-localized GUCY1A2 / GUCY1B3 heterodimer can effectively attenuate soft tissue calcification / pathological BMP signaling, prolonged QT, and other anomalies in tissues that express high levels of GUCY1A2.

[0245] 2. Effective clinical applications of ataciguat, as well as its phenotypic outcomes and potentially unwanted side effects, can be predicted based at least in part on expression levels of GUCY1A2 and / or by relative expression levels of GUCY1A2 / GUCY1A3 in a variety of tissues.

[0246] 3. Selective restoration of GUCY1A2 / GUCY1B3 heterodimers can prevent evolution of unwanted age-related traits, and can complement existing therapies that aim to improve body composition / reduce fat mass.Attorney Docket No.: 07039-2402W01 / 2024-017

[0247] 4. Ataciguat that preferentially activates the GUCY1A2 / GUCY1B3 heterodimer may have greater therapeutic utility as compared to those that preferentially activate the more abundant GUCY1 A3 / GUCY1B3 heterodimer.

[0248] 5. There are numerous applications / indications for which there may be clinical / therapeutic utility' for the GUCYlA2-selective / preferential agonist ataciguat (both oxidized and reduced forms of the 1 A2 / 1B3 heterodimer), which can also serve to minimize undesired side effects (e.g., hypotension) associated with activation of the more abundant 1A3 / 1B3 heterodimer.

[0249] In additional studies, mouse aortic valve interstitial cells (n = two independent cell lines) were treated with either siRNA (20 nM) targeting knockdown of GUCY1A2 (siGUCYlA2) or GUCY1A3 (siGUCYlA3) and compared to scrambled siRNA-treated control cells. Mouse aortic valve interstitial cells (n = two independent cell lines) also were treated with ataciguat (ATA; 1 pM) together with either siRNA (20 nM) targeting knockdown of GUCY1A2 (siGUCYlA2 + ATA) or GUCY1A3 (siGUCYlA3 + ATA) and compared to scrambled siRNA-treated control cells treated with ataciguat. The siGUCYlA2 molecule, Mm01253540_ml (ThermoFisher, Inc.), targeting exons 7 and 8 was used to knock down GUCY1 A2, while the RNA sequence, Mm01220285_ml (ThermoFisher, Inc), targeting exon 6 was used to knock down GUCY1A3. Western blot analysis was performed to measure the level of phospho-VASPSER239expression by the siRNA-treated cells as compared to the control cells.

[0250] Knockdown of GUCY1A2 and knockdown of GUCY1A3 substantially reduced phospho-VASPSER239expression as compared to the baseline for control cells but only knockdown of GUCY1A2 attenuates ataciguat-induced levels of phospho-VASPSER239expression, suggesting context dependence of the contributions of these subunits in health, disease, and therapeutic targeting (FIG. 7).

[0251] In another study, mouse aortic vascular smooth muscle cells (n = 3 independent cell lines) were treated with either siRNA (20 pM) targeting knockdown of GUCY1A2 (S1GUCY1A2) or GUCY1A3 (S1GUCY1A3) and compared to scrambled siRNA-treated control cells. The siGUCYl A2 molecule, Mm01253540_ml (ThermoFisher, Inc.), targeting exons 7 and 8 was used to knock down GUCY1A2, while the RNA sequence, Mm01220285_ml (ThermoFisher, Inc), targeting exon 6 was used to knock down GUCY1A3. Cells were transfected with the respective siRNA molecule for 24 hours, then treated with drug for 24 hours, at whichAttorney Docket No.: 07039-2402W01 / 2024-017

[0252] point cells were harvested for protein or mRNA analyses. Quantitative real-time RT-PCR was initially performed to measure the level of GUCY1A2 expression, GUCY1A3 expression, SP7 expression, and SPP1 expression by the siRNA-treated cells as compared to the control cells. SP7 is a polypeptide involved in osteogenic differentiation of cells, and upregulation of SP7 expression by aortic vascular smooth muscle cells can demonstrate that cells are assuming a more osteogenic / pro-calcific phenotype. SPP1 is a polypeptide involved in protection against osteogenesis, and downregulation of SPP1 expression by aortic vascular smooth muscle cells can demonstrate a permissive change allowing cells to become more osteoblast-like.

[0253] Treatment with each siRNA resulted in selective knockdow n of each subunit with varying degrees of compensatory upregulation of the other subunit (FIGS. 8A and 8B). Mild upregulation of GUCY1A2 expression was observed with GUCY1A3 knockdown (FIG. 8A), and substantial upregulation of GUCY1A3 expression was observed with GUCY1A2 knockdow n (FIG. 8B). Upregulation of SP7 expression was most robust with GUCY1A2 knockdown, as was downregulation of SPP1 expression (FIGS. 8C and 8D). Similar results with smaller magnitudes were observed with GUCY1A3 knockdown (FIGS. 8C and 8D). These results demonstrate the dynamic interplay betw een losses in GUCY subunit expression and their compensatory changes with losses in one subunit and transcriptional responses.

[0254] These results also demonstrate that the loss of expression of one GUCY subunit may significantly increase osteogenic differentiation (as suggested by the upregulation of SP7 and downregulation of SPP1) despite compensatory changes in the other, and that in certain conditions, restoration of expression and / or activity of one GUCY subunit (GUCY1 A2 in particular) may elicit a preferred protective response.

[0255] In another study, mouse aortic vascular smooth muscle cells (n = three independent cell lines) were treated simultaneously with ataciguat (ATA; 1 pM) and with either siRNA (20 pM) targeting knockdown of GUCY1 A2 (siGUCYl A2) or GUCY 1 A3 (siGUCYl A3) and compared to control cells treated with scrambled siRNA plus ataciguat (1 pM). The siGUCYl A2 molecule, MmO125354O_ml (ThermoFisher, Inc.), targeting exons 7 and 8 was used to knock dow n GUCY1 A2, while the RNA sequence, Mm01220285_ml (ThermoFisher, Inc), targeting exon 6 was used to knock down GUCY 1 A3.

[0256] After 24 hours of transfection followed by 24 hours of drug treatment, qRT-PCR analyses were performed to measure the level of GUCY1A2 expression.Attorney Docket No.: 07039-2402W01 / 2024-017

[0257] GUCY1A3 expression, SP7 expression, and SPP1 expression by the siRNA plus ataciguat-treated cells as compared to control cells treated with ataciguat only.

[0258] Treatment with each siRNA resulted in selective knockdown of each subunit with negligible compensatory upregulation of the other subunit given the co-treatment with ataciguat (FIGS. 9A and 9B). Upregulation of SP7 expression was most robust with GUCY1A2 knockdown even in the presence of ataciguat, with knockdown of GUCY1A3 resulting in significant upregulation of SP7 but to a notably lesser magnitude (FIG. 9C). In contrast to unstimulated conditions, reductions in SPP1 expression were nearly identical with knockdown of GUCY1A2 or GUCY1A3 when ataciguat was present (FIG. 9D). These results demonstrate that preferential targeting of restoration of GUCY1 A2 activity may be more efficacious at reducing osteogenic signaling and tissue calcification compared to targeting GUCY1 A3. Furthermore, it also highlights an interplay between the expression levels of each subunit, where losses in GUCY1 A3 resulting in compensatory' increases in GUCY1 A2, which may also provide more substrate for therapeutic agonist binding in some conditions and ultimately making GUCY1 A2 a highly attractive target for agonists.

[0259] OTHER EMBODIMENTS

[0260] 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

Attorney Docket No.: 07039-2402W01 / 2024-017WHAT IS CLAIMED IS:

1. A method for treating a mammal having a disorder characterized by a GUCY1A2 deficiency, wherein said method comprises administering, to said mammal, a composition comprising ataciguat or a pharmaceutically acceptable salt thereof, wherein one or more symptoms of said disorder within said mammal is reduced.

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

3. The method of claim 1 or claim 2, wherein said disorder is selected from the group consisting of systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneury sm, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, sarcopenia / age-associated muscle loss, chemotherapy-induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, chronic obstructive pulmonary' disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, age-related cognitive decline, gastroparesis. irritable bowel disease, Crohn's disease, non-alcoholic fatty liver disease, cirrhosis of the liver, cholecystitis, sclerosing cholangitis, hepatitis, Huntington’s disease, traumatic brain injury, chronic traumatic encephalopathy, Parkinson’s disease, vascular dementia, attention deficit / hyperactivity disorder, age associated dementia and / or cognitive decline, neurological recovery from stroke / transient ischemic attack, chronic renal failure, acute kidney injury', polycystic renal disease, renal fibrosis, glomerulonephritis, granulomatosis, Goodpasture Syndrome, diabetic nephropathy, lupus nephritis, IgA nephropathy, cystinosis, complement 3 glomerulopathy, focal segmental glomerulosclerosis, interstitial nephritis. Fabry’ disease, cardiovascular-kidney-metabolic syndrome, renal stones, overactive bladder, and neurogenic bladder dysfunction.

4. The method of any one of claims 1 to 3, wherein said composition comprises said pharmaceutically acceptable salt thereof.Attorney Docket No.: 07039-2402W01 / 2024-0175. A method for avoiding unnecessarily treating a mammal having a disorder characterized by a GUCY1A3 deficiency, wherein said method comprises not administering, to said mammal, a composition comprising ataciguat.

6. The method of claim 5, wherein said mammal is a human.

7. The method of claim 5 or claim 6, wherein said disorder is selected from the group consisting of sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneurysm, Moyamoya Syndrome, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, gastroparesis, systemic scleroderma opioid-induced constipation, chronic constipation, irritable bowel disease, Crohn’s disease, anesthesia-induced / post-operative constipation, non-alcoholic fatty liver disease, cirrhosis of the liver, gallstones, cholecystitis, chronic acalculus gallbladder disease, sclerosing cholangitis, and hepatitis.

8. The method of any one of claims 5 to 7, wherein said disorder is sarcopenia / age-associated muscle loss.

9. A method for treating a mammal having a disorder characterized by a GUCY1A2 / GUCY1B3 deficiency, wherein said method comprises administering, to said mammal, a composition comprising ataciguat or a pharmaceutically acceptable salt thereof, wherein one or more symptoms of said disorder within said mammal is reduced.

10. The method of claim 9, wherein said mammal is a human.

11. The method of claim 9 or claim 10, wherein said disorder is selected from the group consisting of systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneurysm, occlusive vascular disease, vascularAttorney Docket No.: 07039-2402W01 / 2024-017rarefaction, hypertension, pulmonary hypertension, sarcopenia / age-associated muscle loss, chemotherapy-induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, age-related cognitive decline, gastroparesis. irritable bowel disease, Crohn’s disease, non-alcoholic fatty liver disease, cirrhosis of the liver, cholecystitis, sclerosing cholangitis, hepatitis, Huntington’s disease, traumatic brain injury, chronic traumatic encephalopathy, Parkinson’s disease, vascular dementia, attention deficit / hyperactivity disorder, age associated dementia and / or cognitive decline, neurological recovery from stroke / transient ischemic attack, chronic renal failure, acute kidney injury, polycystic renal disease, renal fibrosis, glomerulonephritis, granulomatosis, Goodpasture Syndrome, diabetic nephropathy, lupus nephritis, IgA nephropathy, cystinosis, complement 3 glomerulopathy, focal segmental glomerulosclerosis, interstitial nephritis, Fabry disease, cardiovascular-kidney -metabolic syndrome, renal stones, overactive bladder, and neurogenic bladder dysfunction.

12. The method of any one of claims 9 to 11, wherein said composition comprises said pharmaceutically acceptable salt thereof.

13. A method for avoiding unnecessarily treating a mammal having a disorder characterized by a GUCY1A3 / GUCY1B3 deficiency, wherein said method comprises not administering, to said mammal, a composition comprising ataciguat.

14. The method of claim 13, wherein said mammal is a human.

15. The method of claim 13 or claim 14, wherein said disorder is selected from the group consisting of sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, peripheral arterial disease, coronary vasospasm, coronary calcification, aorticAttorney Docket No.: 07039-2402W01 / 2024-017aneurysm, Moyamoya Syndrome, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, gastroparesis, systemic scleroderma opioid-induced constipation, chronic constipation, irritable bowel disease, Crohn’s disease, anesthesia-induced / post-operative constipation, non-alcoholic fatty liver disease, cirrhosis of the liver, gallstones, cholecystitis, chronic acalculus gallbladder disease, sclerosing cholangitis, and hepatitis.

16. The method of any one of claims 13 to 15, wherein said disorder is sarcopenia / age-associated muscle loss.

17. A method for treating a mammal likely to develop a disorder characterized by a GUCY1A2 deficiency, wherein said method comprises administering, to said mammal, a composition comprising ataciguat or a pharmaceutically acceptable salt thereof, wherein the likelihood of development of said disorder within said mammal is reduced.

18. The method of claim 17, wherein said mammal is a human.

19. The method of claim 17 or claim 18. wherein said disorder is selected from the group consisting of systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneurysm, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary’ hypertension, sarcopenia / age-associated muscle loss, chemotherapy-induced muscle wasting, immobilization-induced muscle wasting, microgravity-induced muscle wasting, other muscle atrophy / wasting conditions, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, age-related cognitive decline, gastroparesis. irritable bowel disease, Crohn’s disease, non-alcoholic fatty liver disease, cirrhosis of the liver, cholecystitis, sclerosing cholangitis, hepatitis, Huntington’s disease, traumatic brain injury7, chronic traumatic encephalopathy, Parkinson’s disease, vascular dementia, attention deficit / hyperactivity disorder, age associated dementia and / or cognitive decline, neurological recovery from stroke / transient ischemic attack, chronic renal failure, acute kidney injury7, polycystic renal disease, renal fibrosis, glomerulonephritis, granulomatosis, Goodpasture Syndrome, diabetic nephropathy, lupus nephritis, IgAAttorney Docket No.: 07039-2402W01 / 2024-017nephropathy, cystinosis, complement 3 glomerulopathy, focal segmental glomerulosclerosis, interstitial nephritis. Fabry disease, cardiovascular-kidney-metabolic syndrome, renal stones, overactive bladder, and neurogenic bladder dysfunction.

20. The method of any one of claims 17 to 19. wherein said composition comprises said pharmaceutically acceptable salt thereof.

21. A method for avoiding unnecessarily treating a mammal likely to develop a disorder characterized by a GUCY1A3 deficiency, wherein said method comprises not administering, to said mammal, a composition comprising ataciguat.

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

23. The method of claim 21 or claim 22, wherein said disorder is selected from the group consisting of sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneurysm, Moyamoya Syndrome, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, gastroparesis, systemic scleroderma opioid-induced constipation, chronic constipation, irritable bowel disease, Crohn’s disease, anesthesia-induced / post-operative constipation, non-alcoholic fatty liver disease, cirrhosis of the liver, gallstones, cholecystitis, chronic acalculus gallbladder disease, sclerosing cholangitis, and hepatitis.

24. The method of any one of claims 21 to 23, wherein said disorder is sarcopenia / age-associated muscle loss.

25. A method for treating a mammal likely to develop a disorder characterized by a GUCY1A2 / GUCY1B3 deficiency, wherein said method comprises administering, to said mammal, a composition comprising ataciguat or a pharmaceutically acceptableAttorney Docket No.: 07039-2402W01 / 2024-017salt thereof, wherein the likelihood of development of said disorder within said mammal is reduced.

26. The method of claim 25, wherein said mammal is a human.

27. The method of claim 25 or claim 26. wherein said disorder is selected from the group consisting of systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneurysm, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary’ hypertension, sarcopenia / age-associated muscle loss, chemotherapy-induced muscle wasting, immobilization-induced muscle wasting, microgravity-induced muscle wasting, other muscle atrophy / wasting conditions, chronic obstructive pulmonary' disease, idiopathic pulmonary' fibrosis, pulmonary- hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, age-related cognitive decline, gastroparesis. irritable bowel disease, Crohn's disease, non-alcoholic fatty liver disease, cirrhosis of the liver, cholecystitis, sclerosing cholangitis, hepatitis, Huntington’s disease, traumatic brain injury’, chronic traumatic encephalopathy, Parkinson’s disease, vascular dementia, attention deficit / hyperactivity disorder, age associated dementia and / or cognitive decline, neurological recovery from stroke / transient ischemic attack, chronic renal failure, acute kidney injury', polycystic renal disease, renal fibrosis, glomerulonephritis, granulomatosis, Goodpasture Syndrome, diabetic nephropathy, lupus nephritis, IgA nephropathy, cystinosis, complement 3 glomerulopathy, focal segmental glomerulosclerosis, interstitial nephritis. Fabry’ disease, cardiovascular-kidney-metabolic syndrome, renal stones, overactive bladder, and neurogenic bladder dysfunction.

28. The method of any one of claims 25 to 27. wherein said composition comprises said pharmaceutically’ acceptable salt thereof.

29. A method for avoiding unnecessarily treating a mammal likely to develop a disorder characterized by a GUCY1A3 / GUCY1B3 deficiency, wherein said method comprises not administering, to said mammal, a composition comprising ataciguat.

30. The method of claim 29, wherein said mammal is a human.Attorney Docket No.: 07039-2402W01 / 2024-01731. The method of claim 29 or claim 30, wherein said disorder is selected from the group consisting of sarcopenia / age-associated muscle loss, chemotherapy -induced muscle wasting, immobilization-induced muscle wasting, microgravity -induced muscle wasting, other muscle atrophy / wasting conditions, systolic heart failure, diastolic heart failure, ventricular fibrosis, cardiac steatosis, atrial dysfunction, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary hypertension, acute asthmatic bronchoconstriction, chronic airway remodeling in asthma, peripheral arterial disease, coronary vasospasm, coronary calcification, aortic aneurysm. Moyamoya Syndrome, occlusive vascular disease, vascular rarefaction, hypertension, pulmonary hypertension, gastroparesis, systemic scleroderma opioid-induced constipation, chronic constipation, irritable bowel disease, Crohn’s disease, anesthesia-induced / post-operative constipation, non-alcoholic fatty liver disease, cirrhosis of the liver, gallstones, cholecystitis, chronic acalculus gallbladder disease, sclerosing cholangitis, and hepatitis.

32. The method of any one of claims 29 to 31, wherein said disorder is sarcopenia / age-associated muscle loss.

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