Compositions and methods for the diagnosis and treatment of microvascular dysfunction and related diseases

Abstract

Endothelin-1 (ET-1), a pro-inflammatory and vasoconstrictor, is a pathogenic molecule involved in a range of cardiovascular diseases. Unlike ET-1 signaling for vasoconstriction in the aorta, we demonstrate herein that the coronary microvascular system exhibits a unique signaling mechanism for vasoconstriction (independent of PKC, CPI-17, and intracellular calcium storage). The pathophysiological levels of ET-1 are related to the ET-1 receptor ET-1. A It preferentially acts on the microvascular system, resulting in prolonged vasoconstriction through the activation of Rho kinase after binding to R. ET A The R antagonist BQ123 only inhibits vasoconstriction against ET-1, but cannot reverse it. In contrast, ROCK inhibitors (e.g., H-1152) effectively reverse arteriolar constriction against ET-1. Therefore, ROCK inhibition is a potent and specific option for treating coronary ischemic disease (vasoconstrictive abnormalities) associated with microvascular dysfunction using overproduced ET-1. ROCK inhibitors may also be used to treat diabetic retinopathy and microvascular diseases associated with microvascular dysfunction from cancer therapy or immunosuppressants. Since H-1152 can also alleviate the basal tone of the microvascular system, ROCK inhibitors are also good diagnostic agents when used during angiography or by non-invasive methods.

Description

[Technical Field]

[0001] Statement on federally sponsored research or development This invention was made with government support under grant numbers R01EY1018420, R01EY023335, and R01EY024624, awarded by the National Institutes of Health. The government has certain rights to this invention.

[0002] Cross-reference of related applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 501,944, filed on 12 May 2023, which is incorporated in its entirety by reference herein. [Background technology]

[0003] Cardiac tissue exhibits a high density of coronary microvessels. Dysfunction of these small vessels can cause symptoms such as angina (chest pain) and may lead to serious consequences such as myocardial infarction or sudden cardiac death. The American College of Cardiology—National Cardiovascular Data Registry and the National Heart, Lung and Blood Institute database—shows that at least 3 to 4 million women and men have signs and symptoms of myocardial ischemia without coronary occlusion (INOCA), also known as myocardial infarction without coronary occlusion (MINOCA), or cardiac syndrome X (CSX). In other cases, “occlusive” coronary artery disease (CAD) is associated with induced ischemia and other measurable parameters, based on recent evidence-based European Society of Cardiology and American College of Cardiology / American Heart Association (ACC / AHA) guidelines, and involves either more than 70% narrowing (stenosis) or 50-70% stenosis of the major coronary arteries. Coronary microvascular dysfunction / disease (CMD), characterized by increased coronary resistance and / or impaired vasodilator function in the coronary microcirculation, underlies blood flow deficiency or ischemia in as many as 60%–90% of INOCA patients, which appears to increase the risk of experiencing major adverse cardiovascular events. Furthermore, microvascular angina coexists among patients with occlusive CAD and among individuals with angina after coronary revascularization, as CMD contributes to myocardial ischemia in many affected patients, and is often a cause of persistent symptoms. Coronary microvascular angina and dysfunction have also been observed in heart transplant recipients. Unfortunately, despite the identification of the pathophysiological and prognostic roles of CMD in several conditions, there is currently no specific treatment for CMD. Moreover, CMD is also a cause of angina in individuals with cardiomyopathy and valvular disease, as well as in cases of acute coronary syndrome such as Takotsubo syndrome. Microvascular dysfunction is associated with a variety of conditions, including aging, hypertension, stroke, cognitive impairment, dementia, depression, Alzheimer's disease, and the onset / progression of diabetic retinopathy, systemic lupus erythematosus (SLE), and pre-eclampsia or eclampsia.Microcirculatory dysfunction is also induced among patients receiving cancer therapy, patients receiving immunosuppressants, or patients abusing substances such as cocaine.

[0004] As mentioned above, microvascular disease is a major cause of commonalities among different clinical phenotypes. However, currently there are no medicines that can effectively treat or prevent microvascular disease. Only a few of the available anti-anginal drugs are slightly useful, and the treatment of CMD remains a major challenge. Despite recent advances in the diagnosis and treatment of heart disease, there is insufficient evidence of improvement in the clinical outcomes of CMD with currently available therapies.

[0005] Before the availability of modern diagnostic equipment, coronary heart disease (CMD) was diagnosed or identified in patients presenting with chest pain after ruling out or excluding the contribution of coronary artery occlusion from physical occlusion (e.g., atherosclerotic plaques) and / or structural changes (arterial stenosis) of large conduit arteries. These large vessel occlusions could be diagnosed by coronary angiography with X-rays and contrast agents to visualize the vessel walls, and coronary blood flow could be assessed via intravascular ultrasound catheters. Even with the most advanced technology available today, coronary arteries can only be evaluated / visualized with a resolution limited to approximately 200 μm in diameter. The signal-to-noise ratio decreases with decreasing vessel size. Because the disease develops in vessels less than 100 μm in diameter, these limitations restrict the application of coronary angiography in the diagnosis of populations with CMD. Methods for directly measuring changes in arterial size in the μm range are not practical. Therefore, indirect assessment of microvascular function (vasodilatory capacity), i.e., microcirculatory resistance index (IMR) and coronary flow reserve (CFR), is used as a quantitative methodology for evaluating the functional responsiveness and capacity of microvessels for a proper diagnosis of CMD. Coronary flow velocity can be measured invasively or non-invasively before and after administration of pharmacological vasodilators such as acetylcholine (testing endothelial function) or adenosine / adenosine analogs (testing endothelial-independent function at maximum dose). Currently, different angiography wires (e.g., Doppler-based velocity wires for thermodilution) are used during invasive angiography, and cutoff values ​​may differ slightly. Generally, a CFR of less than 2.0 or an IMR greater than 25 leads to a diagnosis of CMD.

[0006] Unfortunately, the clinical utility of these diagnostic methods is hampered by serious adverse reactions reported in patients receiving intracoronary acetylcholine infusion, as impaired endothelial function due to disease causes vasoconstriction. Intracoronary adenosine infusion has also been reported to increase the risk of progressive atrioventricular block and the incidence of adenosine-induced ventricular arrhythmias, as these vasodilators are receptor-dependent and consequently activate pathways unrelated to CFR. It is preferable that agents without receptor-dependent properties be used as diagnostic agents for CFR or IMR assessment.

[0007] The repeated failures in developing effective treatments for CMD necessitate a better understanding of its underlying causes. From a physiological standpoint, proper functioning of tissues / organs depends on adequate blood flow (oxygen / nutrients) to those tissues / organs via the microcirculation (arterioles, capillaries, and venules) and the removal of waste products from those tissues / organs. Therefore, the regulation of the activity / responsiveness of these microvessels is crucial, and their dysfunction can contribute to the onset and progression of ischemic disease. Under physiological conditions, the aorta contributes only about 5-7% to total coronary resistance, while coronary arterioles (diameter approximately 100 μm or less) control over 70% of total coronary resistance and are considered the primary regulatory sites of blood flow to the heart. The ability of these small vessels to contract or dilate in response to changes in the metabolic requirements of specific tissues is paramount to cardiovascular (CV) homeostasis and depends on signaling occurring within endothelial and smooth muscle cells. Any perturbation of these signaling processes in resistance arterioles can destabilize blood flow. Therefore, the function of arterioles determines not only the supply of blood flow, but also blood pressure and tissue survival.

[0008] As described above, despite the widespread observation of microvascular dysfunction, the clinical need remains unmet, and important underlying problems(s) have not been identified in basic research. Procedures in the field of microcirculation have not led to approved drugs specifically for the treatment of diseases resulting from arteriolar or venuleal dysfunction. Furthermore, despite the empirical use of commercially available vasodilators and / or antianginal drugs for medical conditions such as coronary microvascular angina and hypertensive retinopathy, therapeutic modes that restore tissue blood flow and microvascular function are relatively ineffective. Therefore, there is an urgent need to diagnose microvascular dysfunction, identify potential therapeutic targets and agents that can effectively reverse microvascular dysfunction during tissue ischemia (i.e., alleviate vasospasm), and restore tissue blood flow in the microvascular domain (i.e., improve vasodilation). [Overview of the project]

[0009] Dysregulation of microvascular blood flow is directly linked to the onset and progression of many cardiovascular diseases, particularly increased microvasoconstriction (ischemia), which can restrict blood flow to support cell / tissue survival. Overproduction of endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory agent, is thought to be a potential cause of severe vasoconstriction and ischemia in various types of tissues. However, studies of ET-1-induced vasoconstriction have been largely limited in microvessels, including coronary arteries. While so-called "coronary arteries" have been used in some studies, confounding factors warrant significant discussion to understand the limitations (see below).

[0010] The results described herein demonstrate that Rho kinase (ROCK) inhibitors, such as H-1152, can effectively reduce coronary and / or peripheral microvasculature and potentially treat coronary microvascular diseases and angina pectoris, including those insensitive to nitrates and calcium channel blockers. The results described herein also suggest that ROCK inhibitors, such as H-1152, may be used to treat diabetic retinopathy associated with retinal microvascular complications. Overall, the results described herein suggest that ROCK inhibitors, such as H-1152, can be used to treat a range of microvascular diseases associated with ET-1-mediated ROCK activation. The results described herein further suggest that receptor-independent vasodilators, such as ROCK inhibitors like H-1152, can be used as diagnostic agents during coronary angiography.

[0011] Accordingly, the present invention provides a method for treating or preventing microvascular dysfunction in a subject in need thereof, comprising administering to the subject an effective amount of a ROCK inhibitor. In some embodiments, microvascular dysfunction may be associated with elevated ET-1 levels, measured, for example, using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA).

[0012] A method for inducing microvasodilation in a subject, comprising administering an effective amount of a ROCK inhibitor to the subject, is also provided herein.

[0013] A method for reversing ET-1-induced vasoconstriction in a subject requiring such reversal, comprising administering an effective amount of a ROCK inhibitor to the subject, is also provided herein.

[0014] Also provided herein are methods for treating or preventing a disease, disorder, or condition associated with microvascular dysfunction of a subject, comprising administering an effective amount of a ROCK inhibitor to the subject.

[0015] In some embodiments, a disease, disorder, or condition is associated with elevated ET-1 levels, measured, for example, using ELISA or RIA.

[0016] In some embodiments, the disease, disorder, or condition is unable to respond to vasodilators such as calcium channel blockers or nitrates.

[0017] In some embodiments, the disease, disorder, or condition may include microvascular angina (also known as cardiac syndrome X, CSX).

[0018] In some embodiments, the disease, disorder, or condition may include coronary microvascular dysfunction / disease (CMD).

[0019] In some embodiments, the disease, disorder, or condition may include microangiopathy or dysfunction among heart transplant recipients. These patients do not complain of chest pain despite severe microvascular findings because all nerve supply to the heart has been surgically interrupted.

[0020] In some embodiments, the disease, disorder, or condition may include drug-resistant hypertension.

[0021] In some embodiments, the disease, disorder, or condition may include drug-induced hypertension such as hypertension, coronary spasm, and / or CMD, induced by the administration of anticancer agents (e.g., chemotherapeutic agents such as 5-fluorouracil, anthracyclines, and bevacizumab).

[0022] In some embodiments, the disease, disorder, or condition may include coronary artery spasm, percutaneous coronary intervention (PCI)-related refractory myocardial ischemia, cerebral vasospasm after subarachnoid hemorrhage, drug-resistant systemic hypertension, drug-induced hypertension (e.g., bevacizumab-induced hypertension), drug-induced cardiotoxicity (e.g., cardiotoxicity induced by chemotherapeutic agents such as 5-fluorouracil and anthracyclines or immunosuppressants such as cyclosporine A and tacrolimus), pulmonary arterial hypertension, diabetes-induced microangiopathy, microangiopathy, asymptomatic stroke, substance abuse (e.g., cocaine)-related myocardial infarction, or any combination thereof. In some embodiments described herein, the method may further include measuring the circulating ET-1 level of the subject to determine an appropriate dosing plan for ROCK inhibitors.

[0023] In some of the embodiments described herein, an effective amount of ROCK inhibitor includes an effective amount for treating or preventing coronary microvascular dysfunction or vascular disease among heart transplant recipients taking immunosuppressants such as tacrolimus.

[0024] Also provided herein is a method for treating cancer in a subject in need, comprising administering an effective dose of cancer therapy in combination with an effective dose of a ROCK inhibitor to the subject. Also provided herein is a method for improving the efficacy of cancer therapy, comprising co-administering an effective dose of a ROCK inhibitor in combination with cancer therapy.

[0025] In some of these embodiments, administering an effective dose of a ROCK inhibitor in combination with cancer therapy includes administering the ROCK inhibitor concurrently with the cancer therapy. In other embodiments, administering an effective dose of a ROCK inhibitor in combination with cancer therapy includes administering the ROCK inhibitor after cancer therapy. In other embodiments, administering an effective dose of a ROCK inhibitor in combination with cancer therapy includes administering the ROCK inhibitor before cancer therapy.

[0026] In certain embodiments, an effective amount of ROCK inhibitor includes an effective amount for treating or preventing cardiovascular disease caused by cancer therapy, such as drug-induced hypertension or drug-induced cardiotoxicity.

[0027] In some of these embodiments, cancer therapy may include the administration of radiotherapy. In other embodiments, cancer therapy may include the administration of chemotherapeutic agents such as DNA alkylating agents, antitumor antibiotics, antimetabolites, tubulin stabilizers, tubulin destabilizers, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors, cyclin-dependent kinase (CDK) inhibitors, tyrosine kinase inhibitors (TKIs), hypoxia-inducible factor 2 (HIF2α) inhibitors, angiogenesis antagonists, immune checkpoint inhibitors (ICIs), monoclonal antibodies and bispecific antibodies, antibody-drug conjugates (ADCs), cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, molecularly modified viral agents, bacterial agents, or exotoxic agents, or any combination thereof. In some examples, chemotherapeutic agents may include cytidine arabinoside, cytarabine, methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin), cisplatin (CDDP), dexamethasone, algrabine, cyclophosphamide, sarcoridine, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, streptozosin, taxol and its derivatives, tamoxifen, dacarbazine, rituximab, daunorubicin, 1-β-D-arabinofuranosylcytosine, fludarabine, docetaxel, FOLFOX4, and others. In some cases, TKIs may include imatinib, dasatinib, nilotinib, bosutinib, ponatinib, pazopanib, sunitinib, sorafenib, regorafenib, cabozantinib, lenvatinib, axitinib, and tivozanib. Anti-angiogenic agents containing antibodies may include aflibercept, fluquintinib, ramucirumab, and bevacizumab. ICIs may include pembrolizumab, nivolumab, and semiprimab as anti-PD-1 antibodies, ipilimumab as an anti-CTLA-4 antibody, and atezolizumab, avelumab, and durvalumab as anti-PD-L1 antibodies.Antibodies and ADCs may include, for example, rituximab, trastuzumab, denosumab, pertuzumab, grofitamab, tisotumab vedotin, sacituzumab govitecan, enfortumab vedotin, gemtuzumab ozogamicin, etc. Proteasome inhibitors may include bortezomib, carfilzomib, ixazomib, or any combination thereof.

[0028] A method is also provided for treating or preventing cancer treatment-induced hypertension in a subject undergoing cancer treatment, comprising administering an effective dose of a ROCK inhibitor to the subject. A method is also provided for treating or preventing substance abuse-related cardiovascular disease in a subject, comprising administering an effective dose of a ROCK inhibitor to the subject.

[0029] In some embodiments, substance abuse-related cardiovascular disease includes cocaine-induced cardiotoxicity. In certain embodiments, substance abuse-related cardiovascular disease includes cocaine-induced myocardial infarction.

[0030] Diagnostic methods are also provided for assessing microvascular function during coronary angiography of a subject (e.g., by calculating CFR and IMR). These methods can be used alone or as part of a conventional coronary angiography protocol in which adenosine (intravenous or intracoronal) is administered to induce hyperemia. These methods may include measuring the subject's coronary blood flow velocity before and after administration of a ROCK inhibitor (e.g., H-1152) in single or multiple doses, and calculating the subject's CFR (i.e., the ratio of the diastolic flow increase due to the ROCK inhibitor to the resting flow without the inhibitor) and / or IMR as indicators of coronary dilator function.

[0031] In healthy subjects, adenosine-based CFR values ​​(CFRad) are generally greater than 2.5, while CFRad values ​​below 2.0 indicate CMD. CFRad values ​​between 2.0 and 2.5 are a gray zone, requiring more clinical information to be considered for diagnosis. To align with current CFRad-based standards, intracoronary administration of ROCK inhibitors such as H-1152 can establish a series of H-1152-based CFRs (CFRh). At fixed doses, patient CFRh is expected to show a similar trend to CFRad. The use of ROCK inhibitors in these diagnostic methods can offer several advantages, including (1) replacing adenosine in patients who cannot tolerate adenosine administration, (2) providing additional interpretation of CFRad scores, particularly those between 2.0 and 2.5, (3) providing additional guidance on possible treatment strategies for treating CMD in patients with CFRad scores between 2.0 and 2.5, and / or (4) in subjects undergoing coronary angiography for clear evidence of ischemia but showing no signs of occlusive disease, bolus ROCK inhibitors can rapidly improve ischemic conditions when time is of the essence. Therefore, intracoronary administration can be useful for both diagnostic and therapeutic purposes.

[0032] In some embodiments, the coronary blood flow velocity of a target can be measured invasively, for example, during coronary angiography or angioplasty. In certain embodiments, the coronary blood flow velocity of a target is measured invasively using, for example, a surgically placed flow probe, Doppler velocity catheter or guidewire, or coronary sinus thermodilution catheter.

[0033] In other embodiments, the coronary blood flow velocity of a target can be measured non-invasively, for example, using magnetic resonance imaging (MRI) or positron emission tomography (PET).

[0034] The target coronary blood flow reserve can be defined as the ratio of peak hypertensive blood flow velocity (i.e., in response to intracoronary vasodilators) to resting peak blood flow velocity (i.e., without vasodilators).

[0035] In some embodiments, if a subject exhibits a change in CFR below the expected level as assessed by administration of a fixed dose of a ROCK inhibitor, the method further includes administering an effective dose of the ROCK inhibitor to the subject to achieve better blood flow. Subjects requiring higher intracoronary ROCK inhibitor doses to achieve better CFR can be classified as candidates for ROCK inhibitor therapy.

[0036] In some of the embodiments described herein, the ROCK inhibitor can selectively inhibit ROCK2 activity rather than ROCK1 activity. In other embodiments described herein, the ROCK inhibitor can selectively inhibit ROCK1 activity rather than ROCK2 activity.

[0037] In some embodiments of the method described herein, the ROCK inhibitor is of formula I, [ka] During the ceremony, L is either O or SO2. R 1 However, it is H, hydroxyl, NH2, NHR', or NR'R', R 2 However, it is H, halogen, hydroxyl, (C1-C6)alkyl, or (C1-C6)alkoxy, R 3 However, it is H, halogen, hydroxyl, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR', NR'R', or CN, R 4 However, it is H, halogen, hydroxyl, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR', NR'R', or CN, R 5 However, it is H, halogen, hydroxyl, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR', NR'R', or CN, R6 is H, (C1-C8)alkyl, (C1-C6)alkylene-R'', (C1-C6)alkylene-C(O)-R', (C1-C6)alkylene-C(O)O-R', (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR', (C1-C6)alkylene-C(O)NR'R', C(O)OR', C(O)R', C(O)NH2, C(O)NHR', C(O)NR'R', C(O)-(C1-C6)alkylene-NH2, C(O)-(C1-C6)alkylene-NHR', C(O)-(C1-C6)alkylene-NR'R', C(O)O-(C1-C6)alkylene-NH2, C(O)O-(C1-C6)alkylene-NHR', or C(O)O-(C1-C6)alkylene-NR'R', R 7 is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR', NR'R', or CN, R 8 is H, halogen, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR', NR'R', or CN, n is 1, 2, 3, or 4, m is, independently for each occurrence, 1, 2, 3, or 4, R' is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C 10 )heterocyclyl, or (C6-C 10 )aryl, R'' is (C3-C8)cycloalkyl, (C5-C 10 )heterocyclyl, or (C6-C 10 )aryl, which can be defined by formula I, or their stereoisomeric forms and / or their tautomeric forms and / or their pharmaceutically acceptable salts.

[0038] In some embodiments of the methods described herein, the ROCK inhibitor can comprise an isoquinoline sulfonamide.

[0039] In some embodiments of the methods described herein, the ROCK inhibitor is of formula IA, [ka] During the ceremony, R 1 However, it is H, hydroxyl, NH2, NHR', or NR'R', R 2 However, it is H, halogen, hydroxyl, (C1-C6)alkyl, or (C1-C6)alkoxy, R 4 However, it is H, halogen, hydroxyl, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR', NR'R', or CN, R6 is H, (C1-C8)alkyl, (C1-C6)alkylene-R'', (C1-C6)alkylene-C(O)-R', (C1-C6)alkylene-C(O)O-R', (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR', (C1-C6)alkylene-C(O)NR'R', C(O)OR', C(O)R', C(O)NH2 , C(O)NHR', C(O)NR'R', C(O)-(C1-C6)alkylene-NH2, C(O)-(C1-C6)alkylene-NHR', C(O)-(C1-C6)alkylene-NR'R', C(O)O-(C1-C6)alkylene-NH2, C(O)O-(C1-C6)alkylene-NHR', or C(O)O-(C1-C6)alkylene-NR'R', R' is (C1-C6) alkyl, (C3-C8) cycloalkyl, (C5-C 10 )heterocyclyl, or (C6-C 10 ) It is an aryl, and can it be defined by formula IA? Alternatively, these may be stereoisomers and / or tautomers and / or pharmaceutically acceptable salts thereof.

[0040] In some embodiments of the methods described herein, the ROCK inhibitor may include belmosdil, dimethylfasudil, fasudil, hydroxyfasudil, netalusdil, ripasudil, AR-12286, AT-13148, BA-1049(R), FCN-016, GSK429286A, H-1152, PHP-201, RKI-1447, SAR407899, Y-27632, Y-30141, Y-32885, Y-39983, their prodrugs, their salts, their hybrid compounds, or any combination thereof.

[0041] In some embodiments of the methods described herein, the ROCK inhibitor may include fasudil (HA-1077), whose structure is shown below. [ka]

[0042] In some embodiments of the methods described herein, the ROCK inhibitor may include glycyl-H 1152, whose structure is shown below. This is also commonly synthesized as glycyl-H 1152 dihydrochloride (right). [ka]

[0043] In some embodiments of the methods described herein, the ROCK inhibitor may include H-1152 (also known as H-1152P), whose structure is shown below. This is also commonly synthesized as H-1152 dihydrochloride (right). [ka]

[0044] In some embodiments of the methods described herein, the ROCK inhibitor may include ripasudil (K-115), whose structure is shown below. [ka]

[0045] In some embodiments of the methods described herein, the ROCK inhibitor may include netalusdil (AR-13503), whose structure is shown below. [ka]

[0046] In some embodiments of the methods described herein, the ROCK inhibitor may include belmosdil (KD025, SLx-2119), whose structure is shown below. [ka]

[0047] In some embodiments of the methods described herein, the ROCK inhibitor may include OPL-0401 (SAR407899), whose structure is shown below. [ka]

[0048] Pharmaceutical compositions and kits are also provided herein, comprising: an effective amount of a ROCK inhibitor for treating or preventing microvascular dysfunction in a subject in need thereof; an effective amount of a ROCK inhibitor for inducing microvasodilation in a subject; an effective amount of a ROCK inhibitor for reversing ET-1-induced vasoconstriction in a subject; an effective amount of a ROCK inhibitor for treating or preventing a disease, disorder, or condition associated with microvascular dysfunction in a subject; and / or an effective amount of a ROCK inhibitor for treating or preventing a substance abuse-related cardiovascular disease in a subject.

[0049] Pharmaceutical compositions and kits comprising an effective amount of a ROCK inhibitor and an effective amount of a chemotherapeutic agent are also provided herein. [Brief explanation of the drawing]

[0050] [Figure 1A] The video microscopy techniques used to evaluate microvascular function in the examples are shown. A is a schematic diagram illustrating the video microscopy technique used to evaluate microvascular function (i.e., dilation and contraction) using basal tension. [Figure 1B] The video microscopy techniques used to evaluate microvascular function in the examples are shown. B shows human retinal arterioles pressurized with cannulas inserted in both a stable basal tension state (B) and a maximally dilated state (C) in a calcium-free solution. [Figure 1C] The video microscopy techniques used to evaluate microvascular function in the examples are shown. C shows human retinal arterioles pressurized with cannulas inserted in both a stable basal tension state (B) and a maximally dilated state (C) in a calcium-free solution. [Figure 2A] This plot characterizes coronary arteriolar constriction in response to ET-1. It directly compares arterial constriction at high (10 nM) and low (0.1 nM) ET-1 concentrations. The roles of ET-1 receptor antagonists, extracellular and intracellular calcium are studied. [Figure 2B] This plot characterizes coronary arteriolar constriction in response to ET-1. It directly compares arterial constriction at high (10 nM) and low (0.1 nM) ET-1 concentrations. The roles of ET-1 receptor antagonists, extracellular and intracellular calcium are studied. [Figure 2C] This plot characterizes coronary arteriolar constriction in response to ET-1. It directly compares arterial constriction at high (10 nM) and low (0.1 nM) ET-1 concentrations. The roles of ET-1 receptor antagonists, extracellular and intracellular calcium are studied. [Figure 2D] This plot characterizes coronary arteriolar constriction in response to ET-1. It directly compares arterial constriction at high (10 nM) and low (0.1 nM) ET-1 concentrations. The roles of ET-1 receptor antagonists, extracellular and intracellular calcium are studied. [Figure 3A]This plot characterizes the effect of differential extracellular Ca2+ influx via L-type or T-type calcium channels on coronary artery constriction in response to ET-1 and the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). [Figure 3B] This plot characterizes the effect of differential extracellular Ca2+ influx via L-type or T-type calcium channels on coronary artery constriction in response to ET-1 and the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). [Figure 3C] This plot characterizes the effect of differential extracellular Ca2+ influx via L-type or T-type calcium channels on coronary artery constriction in response to ET-1 and the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). [Figure 3D] This plot characterizes the effect of differential extracellular Ca2+ influx via L-type or T-type calcium channels on coronary artery constriction in response to ET-1 and the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). [Figure 4A] This plot characterizes the mechanism of coronary artery constriction in relation to ET-1, specifically the roles of phospholipase C and myosin light chain kinase (MLCK). [Figure 4B] This plot characterizes the mechanism of coronary artery constriction in relation to ET-1, specifically the roles of phospholipase C and myosin light chain kinase (MLCK). [Figure 4C] This plot characterizes the mechanism of coronary artery constriction in relation to ET-1, specifically the roles of phospholipase C and myosin light chain kinase (MLCK). [Figure 5A] This plot shows the roles of PKC and MLCK in coronary artery constriction in relation to PDBu-induced PKC activation. [Figure 5B] This plot shows the roles of PKC and MLCK in coronary artery constriction in relation to PDBu-induced PKC activation. [Figure 6A]This plot characterizes the mechanism of coronary artery constriction in relation to ET-1, specifically the roles of PKC and ROCK. [Figure 6B] This plot characterizes the mechanism of coronary artery constriction in relation to ET-1, specifically the roles of PKC and ROCK. [Figure 6C] This plot characterizes the mechanism of coronary artery constriction in relation to ET-1, specifically the roles of PKC and ROCK. [Figure 6D] This plot characterizes the mechanism of coronary artery constriction in relation to ET-1, specifically the roles of PKC and ROCK. [Figure 7] This plot characterizes the mechanism of coronary artery constriction in relation to ET-1, specifically the role of myosin light chain phosphatase compared to ROCK. [Figure 8A] This paper provides molecular-level evidence of MYPT1 and MLC phosphorylation by ET-1 via ROCK activation. [Figure 8B] This paper provides molecular-level evidence of MYPT1 and MLC phosphorylation by ET-1 via ROCK activation. [Figure 8C] This paper provides molecular-level evidence of MYPT1 and MLC phosphorylation by ET-1 via ROCK activation. [Figure 8D] This paper provides molecular-level evidence of MYPT1 and MLC phosphorylation by ET-1 via ROCK activation. [Figure 9A] This study demonstrates the phosphorylation of MLCs in ET-1 cultured coronary artery smooth muscle cells and the reciprocal regulation of that phosphorylation by H-1152. [Figure 9B] This study demonstrates the phosphorylation of MLCs in ET-1 cultured coronary artery smooth muscle cells and the reciprocal regulation of that phosphorylation by H-1152. [Figure 10A] This paper describes the characteristics of ROCK isoform expression in different species and shows their expression in porcine coronary arterioles compared to cardiomyocytes and the left anterior descending (LAD) artery. [Figure 10B]This paper describes the characteristics of ROCK isoform expression in different species and shows their expression in porcine coronary arterioles compared to cardiomyocytes and the left anterior descending (LAD) artery. [Figure 10C] This paper describes the characteristics of ROCK isoform expression in different species and shows their expression in porcine coronary arterioles compared to cardiomyocytes and the left anterior descending (LAD) artery. [Figure 10D] This paper describes the characteristics of ROCK isoform expression in different species and shows their expression in porcine coronary arterioles compared to cardiomyocytes and the left anterior descending (LAD) artery. [Figure 10E] This paper describes the characteristics of ROCK isoform expression in different species and shows their expression in porcine coronary arterioles compared to cardiomyocytes and the left anterior descending (LAD) artery. [Figure 10F] This paper describes the characteristics of ROCK isoform expression in different species and shows their expression in porcine coronary arterioles compared to cardiomyocytes and the left anterior descending (LAD) artery. [Figure 11A] We demonstrate differential expression of CPI-17 in the coronary artery network, specifically illustrating vessel size-dependent CPI-17 expression. [Figure 11B] We demonstrate differential expression of CPI-17 in the coronary artery network, specifically illustrating vessel size-dependent CPI-17 expression. [Figure 11C] We demonstrate differential expression of CPI-17 in the coronary artery network, specifically illustrating vessel size-dependent CPI-17 expression. [Figure 11D] We demonstrate differential expression of CPI-17 in the coronary artery network, specifically illustrating vessel size-dependent CPI-17 expression. [Figure 12A] This characterizes the effect of ROCK inhibitors on resting vascular tone, which is independent of endothelial nitric oxide (NO). [Figure 12B] This characterizes the effect of ROCK inhibitors on resting vascular tone, which is independent of endothelial nitric oxide (NO). [Figure 13A] This plot shows vasomotor heterogeneity between coronary arteries and retinal arteries. [Figure 13B]This plot shows vasomotor heterogeneity between coronary arteries and retinal arteries. [Figure 14] This is a schematic diagram illustrating the intracellular mechanism by which ET-1 and PKC activator PDBu induce coronary artery constriction. [Figure 15] This plot shows that dasatinib, an anti-cancer tyrosine kinase inhibitor, causes constriction of small ocular resistance arteries, and that this response can be reversed by H-1152. [Modes for carrying out the invention]

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification, including definitions, shall prevail.

[0052] Divalent linking substituents are described in various places in this specification. Where a structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups.

[0053] The term "n-membered" (where n is an integer) typically refers to the number of ring-forming atoms in a moiety that has n ring-forming atoms. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydronaphthalene is an example of a 10-membered cycloalkyl group.

[0054] As used herein, the phrase "optionally substituted" means either unsubstituted or substituted. As used herein, the term "substituted" means that a hydrogen atom has been removed and replaced by a substituent. It should be understood that substitutions in a given atom are limited by their valence.

[0055] Throughout the definition, "C n-m The term "C" indicates a range including the endpoints, where n and m are integers indicating the number of carbon atoms. For example, C 1-4 , C 1-6 These are some examples.

[0056] When used herein, "C" is used alone or in combination with other terms. n-m The term "alkyl" refers to a saturated hydrocarbon group that may be linear or branched, having n to m carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, and sec-butyl, and higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, and 1,2,2-trimethylpropyl. In some embodiments, alkyl groups contain 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0057] When used herein, "C n-m An "alkenyl" refers to an alkyl group having one or more double carbon-carbon bonds and n to m carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, and sec-butenyl. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0058] When used herein, "C n-m"Alkynyl" refers to an alkyl group having one or more triple carbon-carbon bonds and n to m carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, propyne-1-yl, and propyne-2-yl. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0059] When used herein, "C" is used alone or in combination with other terms. n-m The term "alkylene" refers to a divalent alkyl linking group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethane-1,2-diyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, and 2-methyl-propane-1,3-diyl. In some embodiments, the alkylene portion contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0060] When used herein, "C" is used alone or in combination with other terms. n-m The term "alkoxy" refers to a group of the formula -O-alkyl (wherein the alkyl group has n to m carbon atoms). Exemplary alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), and tert-butoxy. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0061] When used herein, "C n-m The term "alkylamino" refers to a group of the formula -NH(alkyl) (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0062] When used herein, "C n-mThe term "alkoxycarbonyl" refers to a group of the formula -C(O)O-alkyl (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0063] When used herein, "C n-m The term "alkylcarbonyl" refers to a group of the formula -C(O)-alkyl (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0064] When used herein, "C n-m The term "alkylcarbonylamino" refers to a group of the formula -NHC(O)-alkyl (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0065] When used herein, "C n-m The term "alkylsulfonylamino" refers to a group of the formula -NHS(O)2-alkyl (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0066] As used herein, the term "aminosulfonyl" refers to the group of the formula -S(O)2NH2.

[0067] When used herein, "C n-m The term "alkylaminosulfonyl" refers to a group of the formula -S(O)2NH(alkyl) (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0068] When used herein, "Ji (C) n-mThe term "alkyl)aminosulfonyl" refers to a group of the formula -S(O)2N(alkyl)2 (wherein each alkyl group independently has n to m carbon atoms). In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0069] As used herein, the term "aminosulfonylamino" refers to the group of the formula -NHS(O)2NH2.

[0070] When used herein, "C n-m The term "alkylaminosulfonylamino" refers to a group of the formula -NHS(O)2NH(alkyl) (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0071] When used herein, "Ji (C) n-m The term "alkyl)aminosulfonylamino" refers to a group of the formula -NHS(O)2N(alkyl)2 (wherein each alkyl group independently has n to m carbon atoms). In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0072] As used herein, the term "aminocarbonylamino," whether used alone or in combination with other terms, refers to the group of the formula -NHC(O)NH2.

[0073] When used herein, "C n-m The term "alkylaminocarbonylamino" refers to a group of the formula -NHC(O)NH(alkyl) (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0074] When used herein, "Ji (C) n-mThe term "alkyl)aminocarbonylamino" refers to a group of the formula -NHC(O)N(alkyl)2 (wherein each alkyl group independently has n to m carbon atoms). In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0075] When used herein, "C n-m The term "alkylcarbamyl" refers to a group of the formula -C(O)-NH(alkyl) (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0076] As used herein, the term "thio" refers to the group of formula -SH.

[0077] When used herein, "C n-m The term "alkylsulfinyl" refers to a group of the formula -S(O)-alkyl (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0078] When used herein, "C n-m The term "alkylsulfonyl" refers to a group of the formula -S(O)2-alkyl (wherein the alkyl group has n to m carbon atoms). In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0079] As used herein, the term "amino" refers to the group of formula -NH2.

[0080] As used herein, the term “aryl,” used alone or in combination with other terms, refers to an aromatic hydrocarbon group that may be monocyclic or polycyclic (for example, having two, three, or four fused rings). n-mThe term "aryl" refers to an aryl group having n to m ring carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracenyl, phenantrenyl, indanyl, and indenyl. In some embodiments, the aryl group has 6 to about 20 carbon atoms, 6 to about 15 carbon atoms, or 6 to about 10 carbon atoms. In some embodiments, the aryl group is substituted or unsubstituted phenyl.

[0081] As used herein, the term "carbamyl" refers to the group of formula -C(O)NH2.

[0082] As used herein, the term "carbonyl," whether used alone or in combination with other terms, refers to a -C(=O)- group, which may be written as C(O).

[0083] When used herein, "Ji (C) n-m The term "alkyl)amino" refers to a group of the formula -N(alkyl)2 (wherein the formula, each of the two alkyl groups independently has n to m carbon atoms). In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0084] When used herein, "Ji (C) n-m The term "alkyl)carbamyl" refers to a group of the formula -C(O)N(alkyl)2 (wherein the formula, each of the two alkyl groups independently has n to m carbon atoms). In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0085] As used herein, the term "halo" refers to F, Cl, Br, or I. In some embodiments, the halo is F, Cl, or Br. In some embodiments, the halo is F or Cl.

[0086] When used herein, "C n-mThe term "haloalkoxy" refers to a group of the formula -O-haloalkyl having n to m carbon atoms. An example of a haloalkoxy group is OCF3. In some embodiments, the haloalkoxy group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0087] When used herein, "C" is used alone or in combination with other terms. n-m The term "haloalkyl" refers to an alkyl group having 1 halogen atom to 2s+1 halogen atoms, which may be the same or different, where "s" is the number of carbon atoms in the alkyl group, and the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0088] As used herein, “cycloalkyl” refers to a non-aromatic cyclic hydrocarbon containing a cyclized alkyl and / or alkenyl group. Cycloalkyl groups may include monocyclic or polycyclic (e.g., having two, three, or four fused rings) groups and spiro rings. Cycloalkyl groups may have three, four, five, six, seven, eight, nine, or ten ring-forming carbons (C 3-10) may have. The ring-forming carbon atoms of the cycloalkyl group can optionally be substituted with oxo or sulfide (e.g., C(O) or C(S)). Cycloalkyl groups also include cycloalkylides. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, etc. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, or adamantyl. In some embodiments, the cycloalkyl group has 6 to 10 ring-forming carbon atoms. In some embodiments, the cycloalkyl group is adamantyl. The definition of a cycloalkyl group also includes moieties having one or more aromatic rings fused to (i.e., having a common bond with) a cycloalkyl ring, such as benzo derivatives or thienyl derivatives such as cyclopentane and cyclohexane. Cycloalkyl groups containing fused aromatic rings can be bonded via any ring-forming atoms, including the ring-forming atoms of the fused aromatic ring.

[0089] As used herein, “heteroaryl” refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any of the ring-forming nitrogen atoms in the heteroaryl portion can be N-oxides. In some embodiments, the heteroaryl has 5 to 10 ring atoms and 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl has 5 to 6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5-membered or 6-membered heteroaryl ring. A 5-membered heteroaryl ring is a heteroaryl having a ring with 5 ring atoms, where one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary five-membered heteroaryl rings are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A six-membered heteroaryl ring is a heteroaryl having a ring with six ring atoms, where one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary six-membered heteroaryl rings are pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

[0090] As used herein, “heterocycloalkyl” refers to a non-aromatic monocyclic or polycyclic heterocycle having one or more ring-forming heteroatoms selected from O, N, or S. Heterocycloalkyls include monocyclic 4-membered, 5-membered, 6-membered, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups may also include spiro rings. Examples of heterocycloalkyl groups include pyrrolidine-2-one, 1,3-isoxazolidine-2-one, pyranyl, tetrahydroplan, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, and benzazapene. The ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted with oxo or sulfide (e.g., C(O), S(O), C(S), or S(O)2). Heterocycloalkyl groups can be bonded via ring-forming carbon atoms or ring-forming heteroatoms. In some embodiments, heterocycloalkyl groups contain 0 to 3 double bonds. In some embodiments, heterocycloalkyl groups contain 0 to 2 double bonds. The definition of a heterocycloalkyl group also includes moieties having one or more aromatic rings condensed (i.e., having a common bond with them) to a cycloalkyl ring, such as benzo or thienyl derivatives like piperidine, morpholine, and azepine. Heterocycloalkyl groups containing condensed aromatic rings can be bonded via any ring-forming atoms, including the ring-forming atoms of the condensed aromatic ring. In some embodiments, a heterocycloalkyl group has 4 to 10, 4 to 7, or 4 to 6 ring atoms, along with one or two heteroatoms independently selected from nitrogen, oxygen, or sulfur, and has one or more oxidized ring members.

[0091] In certain contexts, definitions or embodiments refer to specific rings (e.g., azetidine rings, pyridine rings, etc.). Unless otherwise indicated, these rings can be bonded to any ring member, provided that the valence of the atoms is not exceeded. For example, an azetidine ring may be bonded to any position on the ring, while a pyridine-3-yl ring is bonded to the 3-position.

[0092] As used herein, the term “compound” is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structure depicted. Unless otherwise specified, a compound identified by name or structure as a specific tautomer form is intended to include other tautomer forms.

[0093] The compounds provided herein also include tautomer forms. Tautomer forms result from the exchange of single bonds with adjacent double bonds, in addition to the associated transfer of protons. Tautomer forms include prototropic tautomers, which are isomeric protonation states having the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imoid acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions in a heterocyclic system, such as 1H- and 3H-imidazoles, 1H-, 2H-, and 4H-1,2,4-triazoles, 1H- and 2H-isoindole, and 1H- and 2H-pyrazoles. Tautomer forms may be in equilibrium or sterically fixed to one form by appropriate substitution.

[0094] In some embodiments, the compounds described herein may contain one or more chiral centers and therefore may appear as racemates and racemic mixtures, enantiomer-rich mixtures, single enantiomers, individual diastereomers and diastereomer mixtures (including, for example, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (+)(dextrorotatory) forms, (-)(levorotatory) forms, racemic mixtures thereof, and other mixtures thereof). Additional chiral carbon atoms may be present in substituents such as alkyl groups. All such isomeric forms of these compounds, as well as mixtures thereof, are expressly included in this description. The compounds described herein may also contain, or further contain, bonds whose rotation is restricted due to the presence of rings or double bonds (e.g., carbon-carbon bonds, carbon-nitrogen bonds such as amide bonds) whose rotation is restricted around a particular bond. Thus, all cis / trans and E / Z isomers and rotational isomers are expressly included in this description. Unless otherwise stated or indicated, the chemical name of a compound encompasses a mixture of all possible stereochemical isomers of that compound.

[0095] Optical isomers are known to those skilled in the art and can be obtained in their pure form by standard procedures, including, but not limited to, diastereomer salt formation, kinetic resolution, and asymmetric synthesis. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981), Wilen, SH, et al., Tetrahedron 33:2725 (1977), Eliel, ELStereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962), and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p.268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972), each incorporated herein by reference in its entirety. It is also understood that the compounds described herein include all possible positional isomers and mixtures thereof that are known to those skilled in the art and can be obtained in pure form by standard separation procedures, including but not limited to column chromatography, thin-layer chromatography, and high-performance liquid chromatography.

[0096] Unless otherwise specified, the compounds provided herein may also include all isotopes of atoms appearing in the intermediate or final compound. Isotopes include atoms that have the same atomic number but different mass numbers. Unless otherwise stated, atoms are isotopes or radioactive isotopes (e.g., deuterium, 11 C], [ 18 If an atom is designated as [F], it is understood that the atom contains its isotope or radioactive isotope in an amount at least greater than the natural abundance of that isotope. For example, if an atom is designated as "D" or "deuterium", it is understood that the position contains deuterium in an abundance at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% of deuterium).

[0097] All compounds and their pharmaceutically acceptable salts can be found together with other substances such as water and solvents (e.g., hydrates and solvates), or they can be isolated.

[0098] In some embodiments, the preparation of the compound may involve the addition of an acid or base to influence, for example, the formation of a salt form such as a catalyst for a desired reaction or an acid addition salt.

[0099] Exemplary acids can be inorganic or organic acids and may include, but are not limited to, strong and weak acids. Some exemplary acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, but are not limited to, acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

[0100] Examples of bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some examples of strong bases include, but are not limited to, hydroxides, alkoxides, metal amides, metal hydrides, metal dialkylamides, and arylamines. Alkoxides include lithium, sodium, and potassium salts of methyl, ethyl, and t-butyl oxides; metal amides include sodium amide, potassium amide, and lithium amide; metal hydrides include sodium hydride, potassium hydride, and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, trimethylsilyl, and cyclohexyl-substituted amides.

[0101] In some embodiments, the compounds or salts thereof provided herein are substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial isolation may include, for example, a composition in which the compounds provided herein are concentrated. Substantial isolation may include a composition containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds or salts thereof provided herein. Methods for isolating compounds and their salts are common in the art.

[0102] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0103] This application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salt” means a derivative of a disclosed compound in which the parent compound is modified by converting an existing acidic or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Examples of pharmaceutically acceptable salts in this application include conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts in this application can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acidic or base form of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or in a mixture thereof, generally preferred in non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (MeCN). A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

[0104] As used herein, the term “subject” refers to any animal, including mammals. For example, the term “subject” includes, but is not limited to, mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human.

[0105] In some embodiments, subjects may include “subjects at risk,” i.e., subjects who are at a higher risk of developing diseases or disorders associated with microvascular dysfunction compared to the general population. Such higher risk may be due to another condition the subject has or to a family history of the subject.

[0106] How to use A method for diagnosing, treating, and / or preventing microvascular dysfunction in a subject in need thereof, comprising administering to the subject an effective amount of a ROCK inhibitor. Also provided herein is a method for diagnosing, treating, and / or preventing a disease, disorder, or condition associated with microvascular dysfunction in a subject, comprising administering to the subject an effective amount of a ROCK inhibitor.

[0107] As used herein, the term “local blood flow” refers to blood flow that permeates local tissues through several arterioles, rather than through global or systemic perfusion, to support local or regional needs for oxygen and nutrients.

[0108] The term "microvascular dysfunction," as used interchangeably with "microvascular disease" in publications herein, refers to a heterogeneous range of conditions resulting from microvascular dysfunction affecting the function of the microvascular system, particularly arterioles, or specifically resulting in reduced local blood flow or maximal tissue perfusion due to undesirable vasodilation, particularly through arterioles less than 100 micrometers (μm) in diameter. Microvascular dysfunction is also intended to encompass undesirable vasoconstriction of blood vessels, such as vasospasm, which restricts blood flow perfusion to local tissues.

[0109] The term "coronary flow reserve" (CFR) reflects the maximum volume of microvessels in the heart that expand from a resting state after stimulation with pharmacological vasodilators or under metabolic stress. Using currently available techniques, several studies in patients without microvascular disease have found that a normal CFR is greater than 2.5.

[0110] The term "microcirculatory resistance index" (IMR) is a guidewire-based measurement that allows for the quantitative assessment of the minimum microcirculatory resistance within a target coronary artery region. It is obtained after invasive procedures to measure both coronary pressure and coronary blood flow. Using currently available techniques, several studies in patients without microvascular disease have found that a normal IMR is less than 25.

[0111] In some embodiments, microvascular dysfunction may be associated with elevated levels of endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory agent, which can be measured, for example, using enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). ET-1, a 21-amino acid peptide, is a pro-inflammatory pathogen and a potent endogenous vasoconstrictor. ET-1 synthesis and release are enhanced in a wide range of cardiovascular and non-cardiac heart diseases, including treatment-resistant hypertension, neuroinflammation, obesity, pre-eclampsia, autoimmune diseases, cancer, sickle cell disease, diabetic retinopathy, SLE, and nephropathy, and are involved in their pathogenesis. In patients with hypertension or ischemic heart disease, plasma ET-1 levels are 2-4 times higher than the normal range of 1-3 pg / ml. Elevated circulating ET-1 levels are generally associated with poor clinical outcomes and survival rates in patients with myocardial infarction and are considered an independent predictor of myocardial no-flow, impaired left ventricular function, and long-term mortality. Small arterioles can respond preferentially to ET-1 compared to their upstream arteries, and this response can be enhanced under disease conditions where vasodilatory function is impaired. Therefore, the microvascular system is more vulnerable to ET-1 attack and preferentially suffers from enhanced vasoconstriction and impaired vasodilation, resulting in microvascular dysregulation and tissue ischemia, which exacerbates disease progression.

[0112] In some embodiments, the vessels affected by microvascular dysfunction, or the vessels from which vasodilation is induced, are arterioles or venules. In some embodiments, unless specifically mentioned, microvessels refer to the arteriole network. In some embodiments, the vessels are coronary arterioles. In some embodiments, arterioles have a diameter of less than approximately 100 μm. In some embodiments, the vessels from which vasodilation is induced exhibit basal tension. In some embodiments, the term “basal tension” for microvessels as used herein refers to arterioles exhibiting a taut (i.e., partially constricted) state of contraction without the use of exogenous pharmacological constrictors such as norepinephrine, thromboxane A2, prostaglandin E2, KCl, and ET-1, which are preconstrictors. Arterial vessels described herein as aortic vessels (diameter greater than 300 μm) do not exhibit significant basal tension. These vessels used in in vitro vasodilation studies may have been preconstricted with pharmacological vasoconstrictors that confound vasoconstrictor signaling pathways.

[0113] Several conditions, including aging, amyloidosis, chronic thromboembolic pulmonary hypertension, dementia, diabetes, substance abuse (such as tobacco or cocaine abuse), heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), systemic hypertension, hypertrophic obstructive cardiomyopathy, idiopathic cardiomyopathy, inflammatory diseases, ischemic cardiomyopathy, no-reflow phenomenon, obesity, obstructive sleep apnea, peripheral neuropathy, schizophrenia, stress-related cardiomyopathy, systemic lupus erythematosus, systemic sclerosis, tumor angiogenesis, and vasospasm, are closely associated with microvascular dysfunction. Additional conditions associated with vasoconstrictive abnormalities / enhancements include angina pectoris, congestive heart failure, transplanted heart, erectile dysfunction, pre-eclampsia, migraine, stroke, and Raynaud's phenomenon. In some embodiments, the disease, disorder, or condition is associated with elevated ET-1 levels, measured using ELISA or RIA.

[0114] In some embodiments, the disease, disorder, or condition may include coronary artery spasm, percutaneous coronary intervention (PCI)-related refractory myocardial ischemia, cerebral vasospasm after subarachnoid hemorrhage, drug-resistant systemic hypertension, drug-induced hypertension (e.g., bevacizumab-induced hypertension), drug-induced cardiotoxicity (e.g., cardiotoxicity induced by chemotherapeutic agents such as 5-fluorouracil and doxorubicin or immunosuppressants such as cyclosporine A and tacrolimus), pulmonary arterial hypertension (PAH), drug-induced PAH (e.g., dasatinib), diabetes-induced microangiopathy, microangiopathy, asymptomatic stroke, drug abuse (e.g., cocaine)-related myocardial infarction, or any combination thereof.

[0115] In certain embodiments, the disease, disorder, or condition may include microvascular angina (also known as cardiac syndrome X, CSX).

[0116] In certain embodiments, the disease, disorder, or condition may include coronary microvascular dysfunction / disease (CMD).

[0117] In some embodiments, the disease, disorder, or condition may include drug-resistant hypertension.

[0118] In some embodiments, the disease, disorder, or condition may include drug-induced hypertension, such as hypertension induced by the administration of anticancer drugs.

[0119] In some embodiments, the disease, disorder, or condition is unresponsive to at least one existing medicament for the treatment of hypertension. In some embodiments, the disease, disorder, or condition is unresponsive to vasodilators such as calcium channel blockers or nitrates. The terms “unresponsive,” “non-responsive,” “refractory,” or “resistant,” as used interchangeably herein, mean that existing medicaments are little to no effect, thereby failing to achieve satisfactory results with respect to the treatment of microvascular dysfunction. The terms “partially responsive” and “poorly responsive” mean that existing drugs have some effect, but that effect is partial, and a more effective treatment is still desired.

[0120] In some embodiments described herein, the method may further include measuring the circulating ET-1 level of the subject to determine an appropriate dosing plan for a ROCK inhibitor.

[0121] Inducing vasodilation in response to increased vascular tone of the microvessel system may be clinically desirable in a variety of situations, as detailed above, including treating certain diseases or disorders, such as those involving microvascular dysfunction caused by ET-1. Additional situations may include preventing or reducing the risk of developing certain diseases (e.g., heart disease) or improving certain conditions. An example of such use is the need to maintain sufficient local blood flow within the heart to reduce the risk of developing cardiac disease.

[0122] Accordingly, the Specified Publication also provides a method for inducing microangiectasia in a subject, comprising administering to the subject an effective amount of a ROCK inhibitor to improve local blood flow. As used herein, the term “microangiectasia” refers to the widening of the blood vessels of the microvascular system, the opposite of vasoconstriction, which is the narrowing of blood vessels. Microangiectasia results from the relaxation of smooth muscle cells within the blood vessel walls, specifically the smaller arterioles. Because arterioles contribute to the majority of vascular resistance that regulates local blood flow in organs and tissues, the dilation of these microvessels improves local blood flow.

[0123] A method for reversing ET-1-induced vasoconstriction in a subject requiring such reversal, comprising administering an effective amount of a ROCK inhibitor to the subject. As used herein, the term “vasoconstriction” refers to narrowing of blood vessels resulting from the constriction of the muscular layer of blood vessels, specifically the small arterioles, which reduces tissue blood flow.

[0124] In some of the embodiments described above, ROCK inhibitors may be administered in combination with additional active agents, such as agents for treating hypertension or for controlling blood pressure. Examples of such additional active agents include, for example, diuretics (e.g., thiazides), beta-blockers (e.g., atenolol), calcium channel blockers (e.g., dihydropyridine, phenylalkylamines, and benzothiazepines), angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, zofenopril, perindopril, trandolapril, captopril, enalapril, lisinopril, and ramipril), and angiotensin II receptor blockers (e.g., valsartan, telmisartan, losartan, irbesartan, azilsartan, and olmesartan).

[0125] In some of the embodiments described above, administering the ROCK inhibitor described herein in combination with an additional active agent to increase blood flow to ischemic tissue allows for administration of lower doses of the additional active agent, and even sub-thermal doses, thereby preventing or mitigating side effects. As used herein, the term “sub-therapeutic dose” means less than the amount of the additional active agent required to produce a therapeutic effect when administered alone.

[0126] Many cancer therapies induce systemic hypertension with tissue ischemia and promote coronary microvascular complications. While we do not wish to be bound by theory, it is thought that these cancer therapies cause arteriolar constriction by promoting ET-1-dependent activation of ROCK or by directly activating ROCK signaling. Therefore, ROCK inhibitors can effectively reverse the side effects of anticancer drugs. Hence, ROCK inhibitors can be administered not only to treat hypertension and tissue ischemia, but also to enhance the effectiveness of chemotherapy and radiotherapy, which are less effective under hypoxia / ischemia.

[0127] Accordingly, the Specified also provides a method for treating cancer in a subject in need, which includes administering to the subject an effective dose of cancer therapy in combination with an effective dose of a ROCK inhibitor.

[0128] Also provided herein are methods for improving the effectiveness of cancer therapy, which include co-administering an effective amount of ROCK inhibitor in combination with cancer therapy.

[0129] In some of these embodiments, administering an effective dose of a ROCK inhibitor in combination with cancer therapy includes administering the ROCK inhibitor concurrently with the cancer therapy. In other embodiments, administering an effective dose of a ROCK inhibitor in combination with cancer therapy includes administering the ROCK inhibitor after the administration of cancer therapy. In other embodiments, administering an effective dose of a ROCK inhibitor in combination with cancer therapy includes administering the ROCK inhibitor before the administration of cancer therapy.

[0130] In certain embodiments, an effective amount of ROCK inhibitor includes an effective amount for treating or preventing cardiovascular disease caused by cancer therapy, such as drug-induced hypertension or drug-induced cardiotoxicity.

[0131] In some of these embodiments, cancer therapy may include the implementation of radiotherapy. As used herein, radiotherapy refers to the use of high-energy radiation for the treatment of cancer. High-energy radiation for use in radiotherapy may be provided, among other things, by X-rays, gamma rays, and neutrons. Different methods of radiotherapy are well known in the art and are suitable for use with the methods of the present invention. These methods include, but are not limited to, external beam radiation, brachytherapy, intensity-modulated radiation therapy (IMRT), brachytherapy, whole-body irradiation, and stereotactic radiotherapy.

[0132] In other embodiments, cancer therapy may include the administration of chemotherapeutic agents (i.e., chemotherapy). As used herein, chemotherapy means the administration of one or more chemicals or drugs for the treatment of cancer. Suitable chemotherapeutic agents for use in embodiments of the methods herein may be any chemical known to be useful in the treatment of cancer, such as DNA alkylating agents, antitumor antibiotics, antimetabolites, tubulin stabilizers, tubulin destabilizers, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, and molecularly modified viral agents, bacterial agents, or exotoxic agents.

[0133] Examples of agents particularly suitable for use in the method of the present invention include cytidine arabinoside, cytarabine, methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin), cisplatin (CDDP), dexamethasone, algrabine, cyclophosphamide, sarcoridine, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin D, mitomycin C, hydrogen peroxide, oxaliplatin, Irinotecan, topotecan, leucovorin, carmustine, streptozocin, taxol and its derivatives, tamoxifen, dacarbazine, rituximab, daunorubicin, 1-β-D-arabinofuranosilcytosine, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, fludarabine, docetaxel, bevacizumab, trastuzumab, FOLFOX4, bortezomib, carfilzomib, ixazomib, or any combination thereof.

[0134] A method is also provided for treating or preventing cancer treatment-induced hypertension in a patient undergoing cancer treatment, the method comprising administering an effective amount of a ROCK inhibitor to the patient.

[0135] A method for treating or preventing a target substance abuse-related cardiovascular disease is also provided, comprising administering an effective amount of a ROCK inhibitor to the subject.

[0136] In some embodiments, substance abuse-related cardiovascular disease includes cocaine-induced cardiotoxicity. In certain embodiments, substance abuse-related cardiovascular disease includes cocaine-induced myocardial infarction.

[0137] In some embodiments, substance abuse (e.g., cocaine) may cause coronary artery spasm and / or CMD, accompanied by evidence suggesting increased ET-1 expression.

[0138] Also provided is a method for treating or preventing coronary microvascular dysfunction and / or vascular disease in a subject who has received or is scheduled to receive an organ or tissue transplant, the method comprising administering to the subject an effective amount of a ROCK inhibitor. In some embodiments, the method may include administering to the subject an effective amount of a ROCK inhibitor and an effective amount of an immunosuppressant (e.g., tacrolimus and / or cyclosporine A). In some embodiments, the organ or tissue transplant may include a heart transplant.

[0139] Diagnostic methods are also provided to assess microvascular function during coronary angiography as an additional test to current coronary angiography protocols in which adenosine (intravenous or intracoronal) or other vasodilators are administered to induce hyperemia. These methods may include measuring the coronary blood flow velocity of a subject before and after administration of a ROCK inhibitor (e.g., H-1152) (multiple doses may be used if desired), and calculating the subject's CFR (i.e., the ratio of the flow increase due to the ROCK inhibitor to the resting flow without the inhibitor) and IMR as indicators of coronary diastolic function. While CFR values ​​based on intracoronary adenosine (CFRad) are generally greater than 2.5 in healthy subjects, CFRad values ​​less than 2.0 indicate CMD. CFRad values ​​between 2.0 and 2.5 are a gray zone, requiring more clinical information to be considered for diagnosis. To conform to current CFRad-based standards, intracoronal administration of a ROCK inhibitor such as H-1152 establishes a set of H-1152-based CFRs (CFRh). At a fixed dose, the target CFRh is expected to show a similar trend to that of CFRad.

[0140] In some embodiments, if a subject exhibits a change in the subject's CFR below expected level induced by the administration of a ROCK inhibitor, the method further includes administering an effective dose of the ROCK inhibitor to the subject to induce a desired increase in blood flow. In certain specific examples, the change below expected level includes CFR and IMR scores corresponding to a CFRad of less than 2.0 or an IMRad of greater than 25, using current standards generated based on adenosine administration. Cutoff values ​​for CFRh or IMRh under hyperemia after administration of H-1152 or other ROCK inhibitors will be established through clinical trials.

[0141] In some embodiments, the coronary blood flow velocity of a target can be measured invasively, for example, during coronary angiography or angioplasty. In certain embodiments, the coronary blood flow velocity of a target is measured invasively using, for example, a surgically placed flow probe, Doppler velocity catheter or guidewire, or coronary sinus thermodilution catheter.

[0142] In other embodiments, the coronary blood flow velocity of the target can be measured non-invasively, for example, using MRI or PET.

[0143] The target coronary blood flow reserve is calculated as the ratio of the peak diastolic blood flow velocity induced by pharmacological vasodilators to the resting peak diastolic blood flow velocity without pharmacological vasodilators.

[0144] As used herein, the term “effective dose” refers to the amount of an active compound or drug that elicits a desired biological or pharmaceutical response in a tissue, system, animal, individual, or human, as determined by a researcher, veterinarian, physician, or other clinician. The effective dose will vary depending not only on the route of administration but also on the possibility of co-administration with other drugs.

[0145] Methods of administration include, but are not limited to, oral, parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, mucosal (e.g., intranasal, buccal, vaginal, rectal, intraocular, sublingual), intrathecal, intravitreous, topical, intradermal, and enteral administration.

[0146] The method of administration may be systemic or local. In some embodiments, the composition is formulated for administration by injection. In some embodiments, the composition is formulated for administration by intravenous injection. In some embodiments, the composition is formulated for administration via an intracoronary route. In some embodiments, the composition is formulated for administration through a catheter (IV catheter, peripherally inserted central catheter, central line, etc.) and a port (implantable port or port-a-cas). In some embodiments, the composition is administered by intramuscular injection.

[0147] The following examples of carriers, methods of administration, and dosage forms are listed as known possibilities that may be selected for use with the present invention. However, those skilled in the art will understand that any given formulation and method of administration selected should first be examined to determine whether it achieves the desired results.

[0148] This composition may be formulated for injection, e.g., bolus injection, or parenteral administration by continuous infusion. Formulations for injection may be provided in unit dosage forms, for example, in ampoules or multi-dose containers, along with added preservatives. This composition may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the active ingredient may be in powder form for preparation with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0149] This compound can be formulated in rectal compositions such as suppositories or retained enemas containing, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0150] The determination of the dose of the active ingredient to be used for human use is based on commonly used practices in the art and will ultimately be determined by the clinical trial physician. The expected approximate equivalent dose for administration to humans can be calculated based on the in vivo experimental evidence disclosed below herein using a known formula (e.g., Reagan-Show et al. (2008) Dose translation from animal to human studies revisited. The FASEB Journal 22:659-661). According to this paradigm, the adult human equivalent dose (mg / kg body weight) is equal to the dose given to mice (mg / kg body weight) multiplied by 0.081.

[0151] In some embodiments, the composition is administered in a single dose, which is hereafter referred to as “acute administration” in this specification.

[0152] In some embodiments, the composition is administered in more than one dose, such as multiple doses, for example, more than once a day, twice or three times a day, or less than once a day, such as once every two days, once every three days, or once a week. These methods of administration are referred to as "chronic administration" below in this specification.

[0153] Pharmaceutical compositions and kits Pharmaceutical compositions and kits are also provided herein, comprising an effective amount of a ROCK inhibitor for treating or preventing microvascular dysfunction in a subject in need thereof, an effective amount of a ROCK inhibitor for inducing microvasodilation in a subject, an effective amount of a ROCK inhibitor for reversing ET-1-induced vasoconstriction in a subject, and / or an effective amount of a ROCK inhibitor for treating or preventing a disease, disorder, or condition associated with microvascular dysfunction in a subject.

[0154] Pharmaceutical compositions and kits comprising an effective amount of a ROCK inhibitor and an effective amount of a chemotherapeutic agent are also provided herein.

[0155] Pharmaceutical compositions can be prepared as described herein or elsewhere and can be administered by various routes depending on whether local or systemic treatment is desired and on the area to be treated. Administration may be local (including transdermal, epidermal, ocular, and mucosal, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or blowing of powder or aerosol, including nebulizer, intratracheal or intranasal), retinal (intravitreal), oral (including enteral), or parenteral. Parenteral administration may include intravenous, intra-arterial, intracoronal, subcutaneous, intraperitoneal, intramuscular, or by injection or infusion, or intracranial (e.g., intrathecal or intraventricular administration). Parenteral administration may be in the form of a single bolus or, for example, by a continuous perfusion pump. In some embodiments, the compounds provided herein or their pharmaceutically acceptable salts are suitable for parenteral administration. In some embodiments, the compounds provided herein are suitable for intravenous administration. In some embodiments, the compounds provided herein are suitable for oral administration. In some embodiments, the compounds provided herein are suitable for topical administration.

[0156] Pharmaceutical compositions and formulations for topical administration may include, but are not limited to, transdermal patches, ointments, lotions, creams, gels, infusions, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc., may be required or desirable. In some embodiments, the pharmaceutical compositions provided herein are suitable for parenteral administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for intravenous administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for oral administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for topical administration.

[0157] Pharmaceutical compositions are also provided that contain, as an active ingredient, one or more pharmaceutically acceptable carriers (e.g., excipients). When preparing the pharmaceutical compositions provided herein, the active ingredient is typically mixed with the excipient, diluted by the excipient, or encapsulated in such carrier, for example, in the form of a capsule, sachet, paper, or other container. Where the excipient functions as a diluent, it can be a solid, semi-solid, or liquid material acting as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions can be, for example, in the form of tablets, pills, powders, licks, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injection solutions, and sterile packaged powders.

[0158] Some examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulation may also include, but is not limited to, lubricants such as talc, magnesium stearate, and mineral oil; humectants; emulsifiers and suspending agents; preservatives such as methyl benzoate and propyl hydroxybenzoate; sweeteners; flavoring agents; or combinations thereof.

[0159] This active compound may be effective across a wide range of dosages and is generally administered at an effective dose. However, it should be understood that the actual amount of the compound administered is usually determined by a physician, depending on the condition being treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.

[0160] The compositions provided herein may be administered at least once daily to at least once weekly (including every other day). Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other pre-existing conditions, may influence the dosage and timing required to effectively treat the subject. Furthermore, treatment of the subject with a therapeutically effective dose of the compounds described herein may include a single treatment or a series of treatments.

[0161] The dosage, toxicity, and therapeutic efficacy of the compounds provided herein are, for example, based on LD50. 50 (A lethal dose for 50% of the population) and ED 50 This can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including determining the dose that is therapeutically effective for 50% of the population. The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which is the LD50. 50 / ED 50 This can be expressed as a ratio. Compounds exhibiting a high therapeutic index are preferred. Compounds exhibiting toxic side effects can be used, but care should be taken to design a delivery system that targets such compounds to the site of the affected tissue, minimizing potential damage to uninfected cells and thereby mitigating side effects.

[0162] Rho kinase (ROCK) inhibitors As used herein, the terms “Rho-related protein kinase,” “Rho kinase,” or “ROCK” have their common meanings in the art. ROCK is a member of the serine-threonine protein kinase family. ROCK has been identified as an effector molecule of RhoA, a small GTP-binding protein (G protein) that plays a crucial role in several cellular signaling pathways. ROCK exists in two isoforms: ROCK1 and ROCK2. ROCK1 and ROCK2 exhibit a high degree of homology, with a total amino acid sequence identity of 65%.

[0163] The methods described herein may involve the administration of a ROCK inhibitor. Similarly, the compositions and kits described herein may contain a ROCK inhibitor.

[0164] As used herein, the term “ROCK inhibitor” refers to a natural or synthetic compound that inhibits ROCK1 and / or ROCK2 activity. In some embodiments, the ROCK inhibitor may be selective.

[0165] Selective ROCK inhibitors can exhibit increased inhibitory activity against a particular ROCK isoform compared to other ROCK isoforms. For example, a “selective” Rho kinase 1 (ROCK1) inhibitor may exhibit at least 2, 5, 10, 20, 50, 100, or 200-fold higher inhibitory activity against a specific isozyme of ROCK1 compared to other members of the ROCK family (e.g., IC50). 50 , K i ROCK1 inhibitors have at least 2, 5, 10, 20, 50, 100, or 200-fold higher inhibitory activity (e.g., IC) against a specific isozyme of ROCK2 compared to other members of the ROCK family. 50 , K i This refers to ROCK2 inhibitors that have (or are determined by the calculation of other affinity or effect measures).

[0166] For example, in some embodiments, a ROCK inhibitor can selectively inhibit ROCK1 activity rather than ROCK2 activity. In other embodiments, a ROCK inhibitor can selectively inhibit ROCK2 activity rather than ROCK1 activity. In some embodiments, a ROCK inhibitor can inhibit both ROCK1 and ROCK2 activity with similar ability.

[0167] ROCK inhibitors are well known in the art. For example, isoquinoline derivatives, particularly fasudil, are typical ROCK inhibitors. Fasudil (hexahydro-l-(5-isoquinolylsulfonyl)-lH-l,4-diazepime) (also known as HA-1077) is an isoquinoline sulfonamide derivative and is the only clinically available ROCK inhibitor for treating cerebral vasospasm, often resulting from subarachnoid hemorrhage. However, this drug is not approved in the United States or Europe. It was jointly developed by Asahi Kasei in Japan and the Faculty of Pharmacology at Nagoya University. Hydroxyfasudil is the active metabolite of fasudil in vivo and has a higher affinity for ROCK than fasudil. Another isoquinoline derivative, H-1152 (also known as H-1152P), is optimized based on fasudil. Another type of ROCK inhibitor, Y-27632, inhibits both ROCK1 and ROCK2 by competitively binding to the ATP binding pocket. Optimization of these compounds has resulted in a more potent ROCK inhibitor, Y-39983, which is beneficial for the treatment of glaucoma (Kubo T, Yamaguchi A, Iwata N, The therapeutic effects of Rho-ROCK inhibitors on CNS disorders. Ther Clin Risk Manag 2008;4(3):605-15). A ROCK2-specific inhibitor, SLx-2119, has recently been developed (Boerma M, Fu Q, Wang J, Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin).Blood Coagul Fibrinolysis 2008;19(7):709-18). A series of fasudil analogs were synthesized, and their selectivity and inhibitory activity against ROCK were evaluated (Satoh N, Toyohira Y, Itoh H, Stimulation of norepinephrine transporter function by fasudil, a Rho kinase inhibitor, in cultured bovine adrenal medullary cells. Naunyn Schmiedebergs Arch Pharmacol 2012;385(9):921-31; Nakabayashi S, Nagaoka T, Tani T, Retinal arteriolar responses to acute severe elevation in systemic blood pressure in cats: role of endothelium-derived factors. Exp Eye Res 2012;103:63-70; Sun X, Minohara M, Kikuchi H, The selective Rho-kinase inhibitor Fasudil is protective and therapeutic in experimental autoimmune encephalomyelitis. J Neuroimmunol 2006;180(l-2):126-34; Yu JZ, Ding J, Ma CG, Therapeutic potential of experimental autoimmune encephalomyelitis by Fasudil, a Rho kinase inhibitor. J Neurosci Res 2010;88(8):1664-72, Hou SW, Liu CY, Li YH, Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis, acting possibly through anti-inflammatory effect.CNS Neurosci Ther 2012;18(11):909-17、LoGrasso PV,Feng Y.Rho kinase(ROCK) inhibitors and their application to inflammatory disorders.Curr Top Med Chem 2009;9(8):704-23、Engel J Jr.A proposed diagnostic scheme for people with epileptic seizures and with epilepsy:report of the ILAE Task Force on Classification and Terminology.Epilepsia 2001;42(6):796-803、Fisher RS,van Emde Boas W,Blume W,Epileptic seizures and epilepsy:definitions proposed by the International League Against Epilepsy(ILAE) and the International Bureau for Epilepsy(IBE).Epilepsia 2005;46(4):470-2.Inan S,Buyukafsar K.Antiepileptic effects of two Rho-kinase inhibitors,Y-27632 and fasudil,in mice.Br J Pharmacol 2008;155(1):44-51、Meihui Chen,Anmin Liu,Ying Ouyang,Yingjuan Huang,Xiaojuan Chao,Rongbiao Pi Fasudil and its analogs:a new powerful weapon in the long war against central nervous system disorders? Expert Opin.Investig.Drugs 2013;22:537-550.)。.

[0168] Two ROCK inhibitors (ripasudil and netarsudil) have been approved as eye drops for the treatment of glaucoma and ocular hypertension in Japan and the United States, respectively. The fourth commercially available ROCK inhibitor, besrusudil, has been approved for the treatment of chronic graft-versus-host disease. None of these medications have been approved for the treatment of microvascular diseases.

[0169] Other examples of ROCK inhibitors include those described in International Patent Publications Nos. WO 98 / 06433, WO 00 / 09162, WO 00 / 78351, WO 01 / 17562, WO 02 / 076976, European Patent No. 1256574, International Patent Publications Nos. WO 02 / 100833, WO 03 / 082808, WO 2004 / 009555, WO 2004 / 024717, WO 2004 / 108724, WO 2005 / 003101, WO 2005 / 035501, WO 2005 / 035503, WO 2005 / 035506, WO 2005 / 058891, WO 2005 / 074642, WO 2005 / 074643, WO 2005 / 080934, WO 2005 / 082367, WO 2005 / 082890, WO 2005 / 097790, WO 2005 / 100342, WO 2005 / 103050, WO 2005 / 105780, WO 2005 / 108397, WO 2006 / 044753, WO 2006 / 051311, WO 2006 / 057270, WO 2006 / 058120, WO 2006 / 072792, WO 2011 / 107608 A1, and WO 2007 / 026920 A2, each of which is incorporated herein by reference.

[0170] Other examples of ROCK inhibitors are described in Feng, Y. et al. Rho Kinase (ROCK) Inhibitors and Their Therapeutic Potential. J Med Chem. 2016;59(6):2269 - 2300.

[0171] In some embodiments, the ROCK inhibitor can be an isoquinoline / isoquinolinone-based ROCK inhibitor. In some embodiments, the ROCK inhibitor is selected from the group consisting of fasudil, hydroxyfasudil, dimethylfasudil, fasudil-based hybrid compounds, H-1152, H-1152P, H-1152 dihydrochloride, lipasudil, netarsudil, bermosudil, SAR407899, their prodrugs, their salts, or combinations thereof.

[0172] In some embodiments, the ROCK inhibitor can be selected from the group consisting of AR-12286, AT-13148, BA-1049(R), FCN-016, GSK429286A, PHP-201, RKI-1447, Y-27632, Y-30141, Y-32885, Y-39983, their prodrugs, their salts, or combinations thereof.

[0173] In some embodiments, the ROCK inhibitor is of Formula I,

Chemical formula

[0174] In some embodiments, the ROCK inhibitor may include isoquinoline sulfonamide.

[0175] In some embodiments, the ROCK inhibitor is of formula IA, [ka] During the ceremony, R 1 However, it is H, hydroxyl, NH2, NHR', or NR'R', R 2 However, it is H, halogen, hydroxyl, (C1-C6)alkyl, or (C1-C6)alkoxy, R 4 However, it is H, halogen, hydroxyl, (C1-C6)alkyl, (C1-C6)alkoxy, NH2, NHR', NR'R', or CN, R6 is H, (C1-C8)alkyl, (C1-C6)alkylene-R'', (C1-C6)alkylene-C(O)-R', (C1-C6)alkylene-C(O)O-R', (C1-C6)alkylene-C(O)NH2, (C1-C6)alkylene-C(O)NHR', (C1-C6)alkylene-C(O)NR'R', C(O)OR', C(O)R', C(O)NH2 , C(O)NHR', C(O)NR'R', C(O)-(C1-C6)alkylene-NH2, C(O)-(C1-C6)alkylene-NHR', C(O)-(C1-C6)alkylene-NR'R', C(O)O-(C1-C6)alkylene-NH2, C(O)O-(C1-C6)alkylene-NHR', or C(O)O-(C1-C6)alkylene-NR'R', R' is (C1-C6) alkyl, (C3-C8) cycloalkyl, (C5-C 10 )heterocyclyl, or (C6-C 10 ) It is an aryl, and can it be defined by formula IA? Alternatively, these may be stereoisomers and / or tautomers and / or pharmaceutically acceptable salts thereof.

[0176] In some embodiments, the ROCK inhibitor can include fasudil, hydroxyfasudil, dimethylfasudil, H-1152, Y-27632, Y-30141, Y-32885, Y-39983, lipasudil, netarsudil, RKI-1447, GSK429286A, belumosudil, AT-13148, BA-1049(R), or any combination thereof.

[0177] In some embodiments, the ROCK inhibitor can include fasudil, H-1152, lipasudil, RKI-1447, Y-27632, GSK429286A, Y-30141, or any combination thereof.

[0178] In some embodiments, the ROCK inhibitor can include H-1152, fasudil, dimethylfasudil, Y-27632, or any combination thereof.

[0179] In some embodiments, the ROCK inhibitor can include fasudil (HA-1077), the structure of which is shown below.

Chemical formula

[0180] In some embodiments, the ROCK inhibitor can include glycyl-H 1152, the structure of which is shown below.

Chemical formula

[0181] In some embodiments, the ROCK inhibitor can include H 1152 or a salt thereof, for example, H-1152 dihydrochloride, the structure of which is shown below.

Chemical formula

[0182] In some embodiments, the ROCK inhibitor may include lipasudil, whose structure is shown below. [ka]

[0183] In some embodiments, the ROCK inhibitor may include belmosdil, the structure of which is shown below. [ka]

[0184] In some embodiments, the ROCK inhibitor may include netaludil, whose structure is shown below. [ka]

[0185] In some embodiments, the ROCK inhibitor may include berosudil, whose structure is shown below. [ka]

[0186] In some embodiments, the ROCK inhibitor may include BA-1049, whose structure is shown below. [ka]

[0187] In some embodiments, the ROCK inhibitor may include SAR407899, whose structure is shown below. [ka]

[0188] In some embodiments, the ROCK inhibitor may include hydroxyphasudil, the structure of which is shown below. [ka]

[0189] In some embodiments, the ROCK inhibitor may include dimethylphasudyl, whose structure is shown below. [ka]

[0190] In some embodiments, the ROCK inhibitor may include Y-27632, whose structure is shown below. [ka]

[0191] In some embodiments, the ROCK inhibitor may include Y-30141, whose structure is shown below. [ka]

[0192] In some embodiments, the ROCK inhibitor may include Y-32885, whose structure is shown below. [ka]

[0193] In some embodiments, the ROCK inhibitor may include Y-39983, whose structure is shown below. [ka]

[0194] In some embodiments, the ROCK inhibitor may include RKI-1447, whose structure is shown below. [ka]

[0195] In some embodiments, the ROCK inhibitor may include GSK429286A, whose structure is shown below. [ka]

[0196] In some embodiments, the ROCK inhibitor may include AT-13148, whose structure is shown below. [ka]

[0197] In some embodiments, the ROCK inhibitor may include PHP-201 (sobesudil), whose structure is shown below. [ka]

[0198] In some embodiments, the ROCK inhibitor may include a hybrid fasudil derivative defined by the following formula, where R is H or methyl and n is 1 or 2. Such compounds are described, for example, in J.Med.Chem.2022,65,3,1867-1882. [ka] [Examples]

[0199] The present invention will be described in more detail using specific examples. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to produce essentially the same results.

[0200] Example 1: Rho kinase (ROCK) inhibition for the treatment of microvascular diseases All tissues require blood circulation to function properly by providing sufficient oxygen / nutrients and removing harmful waste. The microvascular network performs this crucial function by regulating blood flow to tissues via arterioles, maintaining fluid / substance exchange within capillaries, and draining blood through venules. Tiny arterioles (less than 100 μm in diameter) are the "gatekeepers" that control and regulate tissue blood flow through vasoconstriction and vasodilation. Excessive vasoconstriction and prolonged dysregulation, seen in many cardiovascular diseases, can lead to tissue ischemia / hypoxia and subsequent organ damage. In the heart, the clinical manifestations of this problem are chest pain (angina) and heart failure. However, there are currently no FDA-approved effective medications to treat microvascular dysfunction-related ischemia.

[0201] In many forms of cardiovascular disease (CVD) associated with inflammation as well as ischemia, ET-1 levels are elevated in either local tissues or systemic plasma. In this embodiment, we demonstrate that ET-1 is the underlying cause of preferential arteriolar constriction, which is not effectively mitigated by currently available pharmaceuticals. Based on our data, we elaborate on several clinical observations. These observations include that clinically relevant concentrations of calcium channel blockers (e.g., nifedipine) and direct nitric oxide-releasing nitrates do not necessarily alleviate microvascular angina, and that the clinical development of ET-1 receptor antagonists does not necessarily demonstrate clinical benefit in the CMD field. More importantly, we also demonstrate that the vasoconstrictive effect of ET-1 is mediated by ROCK activation, and that inhibition of ROCK (e.g., using specific inhibitors such as H-1152) consistently results in vasodilation, eliminating the vascular adverse effects of ET-1 on microvessels. Our data support the possibility that ROCK inhibitors such as H-1152 could be a solution to this unmet medical need.

[0202] overview ET-1-mediated ROCK signaling and activation of the protein kinase C (PKC) pathway are associated with the development of various cardiovascular diseases, including vascular pathophysiology. While ET-1-mediated PKC activation has been reported at the systemic circulation level (i.e., vessels with a diameter greater than 300 μm), it remains unclear whether PKC signaling is responsible for ET-1-mediated vasoconstriction in the resistance arterioles of the microvascular domain where tissue blood flow is predominantly regulated (i.e., vessels with a diameter less than 100 μm). In this example, we describe our method for investigating coronary arteriolar microvessels that spontaneously exhibit resting basal tension without vasoconstrictors. Subsequently, we present data establishing an ET-1 signaling mechanism via ROCK activation independent of PKC signaling in coronary arteriolar constriction. These data support the idea that ET-1-related vascular dysfunction can be specifically treated, particularly at the microvascular level, using ROCK inhibitors such as H-1152.

[0203] material and method A video microscopy technique for evaluating microvascular function. Microvessels are isolated, cannula-inserted, and pressurized for in vitro studies to directly investigate microvascular activity and responsiveness without confounding effects from changes in systemic parameters such as pressure, flow rate, metabolic activity, and neurohumoral control. This technique also allows for independent control of pressure and flow rate, which is not possible with in vivo preparations.

[0204] Figure 1A shows the in vivo microscopy technique used to evaluate arteriole function. Arterioles (30–100 μm in diameter) were isolated from the target organ and then cannulated using a pair of glass micropipettes (i.e., inflow and outflow pipettes). The arterioles were pressurized to 60 cm H2O (approximately 44 mmHg) using a dual reservoir system to mimic the pressure environment observed in microcirculation in vivo. Saline solution (PSS) was maintained at 36–37°C in the vascular chamber, and the vessels were gradually constricted to a stable level of 50–60% of their maximum diameter within 60 minutes. This state of vasoconstriction at rest is called "basal tension."

[0205] The spontaneous expression of basal tone under PSS without the addition of pharmacological vasoconstrictors is one of the essential / necessary criteria for verifying the viability of vessels in our preparation (see the inset block on the left of the diagram in Figure 1A). This feature is an inherent property of microvessels found in living animals, allowing vasodilation to occur when tissue needs to replenish blood flow, or further constriction when tissue is overperfused. This unique behavior is not seen in large conduit vessels that exhibit little basal tone for blood flow regulation. Another viability criterion is that isolated microvessels must exhibit vasodilation to endothelium-dependent vasodilators such as bradykinin or serotonin to ensure functional integrity of the endothelium. The function of untreated vascular smooth muscle is verified by vasodilation to the smooth muscle-specific vasodilator sodium nitroprusside. Under our experimental conditions, isolated microvessels exhibit natural properties and behaviors similar to those observed in vivo.

[0206] Using this setup, the lumen pressure can be changed without altering the flow by moving both reservoirs simultaneously in the same direction (up or down) to the desired hydrostatic pressure. To induce flow without altering the lumen pressure, the reservoirs are set to the desired pressure as described above, and then moved in opposite directions to create a longitudinal pressure gradient across the vascular segment. The degree of flow (or shear stress) is determined by the amount of pressure gradient applied to the vessels. In some studies, the intraluminal pressure of the vessels was recorded using a servo-null micropressure technique to ensure that the lumen pressure remained constant during flow induction (Figure 1A). The agonist (or antagonist) was prepared in the vehicle solution and administered either cumulatively in 20 μL volumes or as a single application to the vascular chamber in 2 mL volumes. A fresh solution was passed through the vascular chamber every 20–30 minutes to prevent the osmotic pressure and chemical concentration of the bath solution from changing over time due to water evaporation.

[0207] Animals. Domestic pigs (8-12 weeks old, 7-10 kg) purchased from Barfield Farms (Rogers, TX) were sedated with terazole (4.4 mg / kg, intramuscular) and xylazine (2.2 mg / kg, intramuscular) and anesthetized with 2-4% isoflurane. Heparin (1,000 units / kg) was administered intravascularly to prevent coagulation. The heart and eyes were removed and immediately placed in a saline chamber on ice for further vascular dissection and isolation. The physiology, histology, and pathophysiology of the cardiovascular system, including coronary and ocular circulation, have been well-established to be similar to those of humans. Data obtained from this pig model better represent human vascular biology and are applicable to translational medicine.

[0208] Isolation and cannula insertion of microvessels. Single coronary or retinal arterioles (0.6–1.0 mm in length) were carefully dissected in a tissue chamber containing physiological saline (PSS: mmol / L units, NaCl 145.0, KCl 4.7, CaCl2 2.0, MgSO4 1.17, NaH2PO4 1.2, glucose 5.0, pyruvate 2.0, EDTA 0.02, and MOPS buffer 3.0) using a pair of Dumont microscopy forceps (Fine Science Tools (Foster City, CA)) under a stereomicroscope (model SZX12, Olympus (Melville, NY)). After careful removal of remaining connective tissue, the arterioles were transferred to a Lucite vascular chamber containing PSS with 1% albumin (USB (Cleveland, OH)) for cannula insertion. A cannula was inserted into one end of the arteriole using a glass micropipette filled with PSS-albumin solution, and the outside of the arteriole was securely fixed to the pipette with 11-0 ophthalmic suture (Alcon (Fort Worth, TX)). A cannula was inserted into the other end of the vessel using a second micropipette and fixed with suture. After cannula insertion, the vessel and pipette were moved to the stand of an inverted microscope (Model CKX41, Olympus) connected to a video camera (Sony DXC-190, Labtek (Campbell, CA)), a video micrometer (Cardiovascular Research Institute, Texas A&M Health Science Center (College Station, TX)), and a PowerLab data acquisition system (AD Instruments (Colorado Springs, CO)), and the inner diameter was continuously measured and recorded throughout the experiment (Figure 1A). The cannula insertion pipette was connected to an independent pressure reservoir. By adjusting the reservoir height, the vessels were pressurized to an intraluminal pressure of 55–60 cmH2O (40–44 mmHg) without flow. This pressure level was used based on the pressure range recorded in arterioles in vivo. Arterioles with collateral branches and leakage were excluded from further study, and all arteries used exhibited spontaneous basal tension. At the end of each functional experiment, EDTA (1 mmol / L)-Ca2+ Vascular dilation was achieved by using 0.1 mmol / L sodium nitroprusside in free PSS, and the maximum diameter was obtained at an intraluminal pressure of 55-60 cmH2O.

[0209] Immunoblotting and immunohistochemistry. Unless otherwise noted, vascular proteins were isolated from intact porcine conduit coronary arteries (left anterior descending branch) and downstream arteries (less than 100 μm in diameter). To study protein kinase phosphorylation, microvessels were pressurized and pharmacological interventions were applied under the same protocol as when studying vasomotor function. The vessels were then flash-frozen for further protein / enzyme analysis. In some studies, vascular cells from primary cultures were obtained from freshly isolated vessels. Cultured cells were lysed in radioimmunoprecipitation assay lysis buffer. Tissue was disrupted in two disruption cycles using a pellet pestle (Kontes). The lysate supernatant was collected after circulating at 12,000 g rpm for 20 minutes at 4°C. For protein assays, the same amount of protein (5-10 μg / well) was loaded, further separated using a 4-15% gradient SDS-PAGE gel (Bio-Rad (Hercules, CA)), and then transferred to a nitrocellulose membrane (Bio-Rad) for immunoblotting. For pressurized vascular studies, 2-4 arterioles were collected, rapidly flash-frozen in liquid nitrogen, and then lysed for protein isolation. Unless otherwise noted, protein samples were loaded and run on SDS-PAGE gels, and protein signal intensity was normalized with smooth muscle actin (SMA, Sigma-Aldrich (St. Louis, MO)). The following antibodies: ROCK1, ROCK2, pCPI-17, CPI-17, GAPDH, tropomyosin, and HRP-labeled anti-rabbit and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). The pMYPT1-Thr850 antibody was purchased from Millipore (Billerica, MA). The pMLC Ser19 antibody and MLC were purchased from Cell Signaling Technology (Danvers, MA). The SuperSignal West Pico chemiluminescent substrate was purchased from Thermo Scientific (Waltham, MA). For immunohistochemical analysis, isolated vascular and cardiac tissues were subjected to frozen sections (10 μm thick) and immunostaining. The fluorescently labeled secondary antibody was purchased from Jackson Laboratory (Bar Harbor, Maine).

[0210] Analysis. Vasomotor responses were analyzed as the ratio of diameter change (in response to the agonist) to maximum dilating capacity (exposure of vessels to 0.1 mmol / L sodium nitroprusside in the absence of extravascular calcium) as "maximum dilating %", or as "% of resting diameter" after normalizing vessel diameter using resting diameter. Data were presented as mean ± standard error. Where necessary, the statistical significance of the intervention outcomes was determined using one-way or two-way ANOVA with Tukey post-hoc analysis. The significance of changes in protein expression was analyzed using Student's t-test. P < 0.05 was considered statistically significant.

[0211] Technical Note: Due to the technical challenges in studying microvessels, such as the complexity of their network and the limited availability of tissues for molecular characterization, progress in microvessel research has been either linear or stagnant over the past decade. Consequently, knowledge regarding the mechanisms of microvascular dysregulation has been primarily inferred from studies of large conduits or cultured vessels / non-vascular cells. Furthermore, microvessels are susceptible to physical manipulation and require meticulous care during isolation and preparation to maintain viability and vasomotor function, such as the expression of spontaneous basal tonic. Notably, these studies are conducted in microvessels less than 100 μm in diameter, where blood flow is predominantly regulated. Therefore, the data presented herein are from microvessels exhibiting spontaneous basal tonic (i.e., without the addition of pharmacological pre-constrictors).

[0212] Other laboratories commonly use pharmacological vasoconstrictors such as thromboxane analogs (U46619), prostaglandin F2α, KCl, and ET-1 to induce "artificial basal tension" in vessels that cannot spontaneously exhibit basal tension. These pre-constrictors can provide inaccurate information regarding vascular reactivity and responsiveness, and may also induce confounding signaling molecules because additional pathways are induced / mixed / overlapped, blocking or altering the original vascular behavior and signaling. This concern is particularly relevant in aortic studies because the aorta does not exhibit significant levels of basal tension, or in microvessels that cannot exhibit basal tension. Therefore, pre-constrictors are consequently added to study vasodilation mechanisms. Here, in our preparations, arterioles (less than 100 μm in diameter) isolated from the heart, brain, retina, skeletal muscle, or mesentery exhibit spontaneous basal tension without the use of pharmacological constrictors, and these vessels exhibit normal dilation in response to endothelial-dependent agonists (bradykinin or serotonin) that mimic the vasomotor behavior observed in vivo. These criteria are essential for all studies we have conducted.

[0213] As mentioned above, because vascular behavior and function differ fundamentally at the cellular and molecular levels, applying information from large vessels to microvessels may not be appropriate. These differences may highlight why the treatment of microvascular diseases has failed by using commercially available drugs not intended for this purpose. Understanding the molecular mechanisms corresponding to the microvascular functions exhibited is crucial for designing effective therapies to address these unmet clinical needs.

[0214] Characterization of coronary artery constriction in response to ET-1: The roles of calcium and ET-1 receptors The vasomotor effect of ET-1 is due to its receptor (ET A Subtype and ET B This is due to the combination of subtypes. Many studies have shown that arterial constriction, including that of the coronary arteries, is due to ET AAfter receptor activation, intracellular calcium (Ca) is released from the sarcoplasmic reticulum. 2+ It has been shown that this is mediated by the release of ) ). Furthermore, ET-1 induces the release of endothelial nitric oxide (NO) or prostacyclin, thereby mediated by endothelial ET B Vasodilation can also be exerted through receptor activation. However, these findings originate from large conduit vessels without spontaneous basal tone that required pre-constriction with pharmacological agonists (e.g., high concentrations of KCl, U46619, PDBu, etc.). It is unclear whether the same vasomotor mechanisms observed in large conduit vessels can be applied to or adopted in small coronary arterioles. To address these questions, porcine coronary arterioles (less than 100 μm in diameter) were isolated and pressurized for functional studies in the presence of spontaneous basal tone. These studies were used to study intracellular Ca in the vasomotor response to ET-1. 2+ Extracellular Ca compared 2+ The system was designed to determine the role of this receptor and identify the receptor subtype responsible for this response.

[0215] As shown in Figure 2A, we first characterized the coronary arteriole response to ET-1. This is the first investigation of the contraction of such thin, isolated coronary arterioles (<100 μm) to ET-1 with spontaneous resting basal tone (R). Control arterioles isolated from pigs were given PSS-albumin (normal 2.0 mmol / L Ca at luminal pressure of 60 cmH2O). 2+ In the presence of a solution containing Ca, stable basal tension (spontaneous contraction from a maximum diameter of 73±3 μm to 40±4 μm) was induced. ET-1 induced concentration-dependent coronary artery constriction at a threshold concentration of 0.1 pmol / L. The coronary arteries nearly closed the lumen by constricting to approximately 10%–15% of the resting diameter in response to the highest concentration tested (10 nmol / L). Another group of coronary arteries showed Ca 2+ In the presence of a PSS-albumin solution containing Ca, similar basal muscle tension levels (resting diameter 42±5μm) were observed, followed by a vascular bath. 2+After switching to a free solution (containing 1 mmol / L of EDTA in PSS), the arterioles were dilated from their resting diameter to a maximum diameter of 84 ± 5 ​​μm. These dilated arterioles (extracellular Ca) 2+ In the absence of extracellular Ca (10 nmol / L), the arterioles did not constrict in response to ET-1, except at the highest concentration. The data indicate that extracellular Ca is necessary for coronary arteries to express spontaneous basal tension and to respond to ET-1 at the tested concentrations. 2+ This indicates that inflow is necessary.

[0216] It should be noted that other studies on coronary arteriole constriction by other laboratories have shown a threshold constriction response to ET-1 levels greater than 1 nmol / L. Our coronary arterioles exhibit at least 100 times higher sensitivity and responsiveness to ET-1 than previously recorded. Normal plasma ET-1 concentrations in vivo are approximately 1–3 pmol / L, and in vivo blocking of this endogenous ET-1 using its specific receptor blocker resulted in decreased coronary resistance and increased coronary blood flow, indicating that this endogenous ET-1 level (1–3 pmol / L) induces coronary arteriole constriction. Our in vitro data demonstrated that 1 pmol / L of ET-1 was sufficient to induce slight coronary arteriole constriction (Figure 2A). This result also indicates that the arterioles in our study maintained viability and responsiveness to ET-1, as observed in intact hearts in vivo. Most importantly, previous studies of ET-1 at concentrations above 1 nmol / L are nonphysiological, and even suprapathophysiological, considering that estimated interstitial ET-1 concentrations can be five times higher than plasma concentrations, and reported plasma ET-1 levels in patients with coronary artery disease or other conditions rarely exceed 50 pmol / L.

[0217] To avoid an increase in the volumetric osmolar concentration of the bath solution, it is necessary to refresh the solution in the vascular chamber every 30 - 40 minutes, which may cause an interaction effect on vasomotor activity. It should be noted that the change in diameter was recorded 5 minutes after exposure to ET-1 at each concentration. Therefore, to establish this concentration-dependent vasoconstrictor response, a 5-minute exposure to ET-1 at each concentration was selected.

[0218] Figure 2B is a plot showing the time course of coronary arteriole constriction in response to ET-1 (0.1 nmol / L and 10 nmol / L) from the resting (R) diameter (0.1 nmol / L ET-1: 45 ± 1 μm, 10 nmol / L ET-1: 51 ± 3 μm). It takes about 10 - 20 minutes for vasoconstriction to fully develop, and the contraction lasts for at least 1 hour. The data indicate that ET-1 induces a slow but sustained coronary arteriole contraction. In all remaining studies, arteriole diameter was recorded at the 20-minute point after ET-1 exposure unless otherwise stated.

[0219] As shown in Figure 2C, the role of extracellular Ca 2+ and intracellular Ca 2+ in the vasoconstriction to ET-1 was determined using 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of IP3 signaling and store-operated Ca 2+ release from the sarcoplasmic reticulum via Ca 2+ channels. First, the coronary arterioles were exposed to a Ca 2+ free solution (containing 1 mmol / L EDTA in PSS) to exclude the contribution of extracellular Ca 2+ to vasoconstriction. The vessels lost their basal tone (resting diameter at rest 45 ± 5 μm, n = 5) and dilated maximally in the absence of bath Ca 2+ . * is P < 0.05 relative to the percentage of the resting diameter at R. Administration of 0.1 nmol / L ET-1 ("zero" time point) was unable to induce vasoconstriction. In contrast, in a second group of vessels (resting diameter at rest 44 ± 6 μm, n = 8), administration of 10 nmol / L ET-1 induced a progressive vasoconstriction that stabilized within 15 - 20 minutes and extracellular Ca 2+resulted in a diameter reduction of approximately 40% in the absence of # was P < 0.05 relative to the resting diameter percent observed at the time of addition of ET-1 (0 minutes). In a third group of blood vessels (resting diameter 40 ± 5 μm, n = 7), 2-APB (100 μmol / L) was added to Ca 2+ administered in the presence of free PSS abolished vasoconstriction to 10 nmol / L ET-1. These results indicate that high concentrations of ET-1 (10 nmol / L) induce an additional vasoconstrictor mechanism by activating the release of intracellular Ca 2+ . These ultra-high concentrations of ET-1 (above 1 nmol / L) are commonly used in studies of ET-1 signaling for vasoconstriction or other bioactivities either in vivo or in vitro in thick conduit blood vessels or so-called "thin resistance arterioles" including cell cultures. Thus, these activated "extra" pharmacological activities can confound the interpretation of the physiological / pathological actions of ET-1 because the activated release of intracellular Ca 2+ can trigger and be involved in numerous signaling events and bioactivities independent of the initial extracellular Ca 2+ -dependent vasoconstriction. To accurately determine the effect of ET-1 on the microcirculation (diameter less than 100 μm) and to clarify the underlying mechanisms, we emphasize the importance of using concentrations of ET-1 below 0.1 nmol / L for physiological and pathophysiological interpretations.

[0220] As shown in Fig. 2D, the ET-1 receptors involved in coronary arteriolar constriction to ET-1 were evaluated using the specific ET A receptor antagonist BQ123 and the ET B receptor antagonist BQ788. In the absence of ET-1 receptor antagonists, ET-1 (0.1 nmol / L) caused a progressive contraction of the control blood vessels (39 ± 3 μm) and stabilized at approximately 60% of the resting (R) diameter within 15 - 20 minutes. *The P<0.05 ratio was observed relative to the resting diameter percentage at the time of ET-1 addition (0 min). Pretreatment of coronary arterioles with BQ123 (1 μmol / L) for 20 minutes did not change the resting diameter (43 ± 3 μm), but vasoconstriction in response to ET-1 (0.1 nmol / L) disappeared. Administration of BQ788 (0.1 μmol / L) to the blood vessels did not affect either the resting diameter (41 ± 4 μm) or ET-1 induced vasoconstriction. In a separate study, salafotoxin S6c (ET) B The efficacy of BQ788 was verified by blocking vasoconstriction in response to receptor agonists. These results showed that ET-1-induced coronary artery constriction was inhibited by ET A mediated by receptor activation, ET B This indicates that the role of the receptors is minimal, even if it is in these small coronary arteries. On the other hand, in the upstream aorta, endothelial ET B The role of receptors is more pronounced. These results indicate specific vasomotor signaling in the small coronary arteries, distinct from that in the larger upstream parent vessels.

[0221] In summary, Ca from the extracellular space 2+ The influx is the cause of vasoconstriction induced by physiological / pathophysiological concentrations of ET-1 (0.1 nmol / L). Even with hyperpharmacological concentrations of ET-1 (greater than 0.1 nmol / L), Ca from internal stores (i.e., the sarcoplasmic reticulum) is released. 2+ It appears to induce the release of Ca2+, which in turn induces vasoconstriction. Therefore, different ET-1 concentrations induce different signaling pathways for vasoconstriction. Physiological / pathophysiological concentrations of ET-1 (≤0.1 nmol / L) trigger the release of Ca2+ from internal stores. 2+ It does not induce release. Vasoconstriction induced by ET-1 is ET A It is mediated solely by receptor activation. Unlike large conduital arteries, endothelial ET is used for vasodilation. BReceptor activation is not evident in coronary arterioles. Because in vivo ET-1 concentrations (low pmol / L range) are much lower than those used in numerous previous studies in the aorta or arterioles (greater than 1 nmol / L), the vasoconstrictive mechanisms identified in these studies are not applicable to microvessels, considering not only physiological but also pathophysiological factors.

[0222] Differential extracellular Ca in coronary artery constriction in response to ET-1 and PKC activation 2+ inflow Research has shown that the aorta contracts in response to PDBu, a direct PKC activator, and extracellular Ca 2+ It has been shown to be partially dependent on extracellular Ca for PDBu-induced contraction of coronary arteries. 2+ The relative contribution of ET-1 and PDBu is the same Ca for coronary artery constriction. 2+ It is also unclear whether inflow channels can be activated. These questions are important in helping to understand the mechanism of ET-1 activation compared to PKC activation for coronary artery constriction, as activation of these signaling pathways contributes to the development of various cardiovascular diseases. Since their inhibitors are commonly used in the treatment of patients with systemic hypertension, L-type and T-type electrophalangeal Ca 2+ The focus shifted to the channel.

[0223] As shown in Figure 3A, isolated coronary arteries exhibit spontaneous basal tension, and these can be controlled with different inhibitors (Ca 2+ Free solution, L-type Ca 2+ Channel blocker nifedipine, or T-type calcium 2+ After 20 minutes of exposure to the channel blocker NNC 55-0396), ET-1 (0.1 nmol / L) was administered (at "zero" time). This approach was used to investigate whether pretreatment with these inhibitors could prevent ET-1-induced vasoconstriction. The data showed that coronary arterioles (resting diameter 45 ± 5 μm) lost their resting basal tone (R) and Ca 2+ This indicates that the body was unable to respond to ET-1 under free conditions, which suggests that basal tension and sustained Ca2+ vasoconstriction were not present.2+ Extracellular Ca when maintaining inflow and exerting vasoconstriction in response to ET-1 2+ This demonstrates the important role of L-type and T-type Ca in maintaining basal tension. In normal PSS, resting basal tension (diameter 44±4 μm) is inhibited by 1 μmol / L nifedipine and 1 μmol / L NNC 55-0396, which is responsible for maintaining basal tension. 2+ Ca from the channel 2+ It shows an inflow. However, Ca 2+ Unlike the free solution, ET-1-induced vasoconstriction is due to Ca 2+ Expression continued in the presence of channel blockers, and inhibition by NNC 55-0396 was moderately higher. These data suggest that Ca mediated through voltage-gated channels is impaired. 2+ This shows that even if inflow occurs at the onset of ET-1-induced vasoconstriction, it plays only a minor role. In Figure 3A, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of ET-1 addition (0 min).

[0224] As shown in Figure 3B, isolated coronary arterioles exhibit spontaneous basal tension, which is then exposed to ET-1 to induce vasoconstriction, followed by treatment with different inhibitors (Ca 2+ We tested whether applying nifedipine (free, NNC 55-0396) to blood vessels could reverse this vasoconstriction. Using this approach, we investigated whether these inhibitors could reverse vasoconstriction that is already fully expressed to mimic prolonged vasoconstriction in disease states. The results showed that ET-1 (0.1 nmol / L) induced coronary artery arteriole constriction by reducing their resting (R) diameter by approximately 40%. This vasoconstriction (from a resting diameter of 42 ± 2 μm) reduces the blood vessels' Ca 2+Exposure to the free solution completely maintained the vasoconstriction and reached its maximum diameter. In contrast, nifedipine (resting diameter 46±3 μm) and NNC 55-0396 (resting diameter 42±2 μm) both partially reversed vasoconstriction at 1 μmol / L, with inhibition by NNC 55-0396 being better. These data suggest sustained Ca 2+ Inflow is essential to maintain ET-1-induced vasoconstriction, and T-type Ca 2+ This suggests that channels may play some role in this regard. On the other hand, L-type Ca 2+ Aside from a clear inhibition of resting vasoconstriction, the channels do not appear to play a significant role in maintaining vasoconstriction, as nifedipine's effect on vasoconstriction is minimal. * For R, P < 0.05 relative to the resting diameter percentage. In Figure 3B, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of ET-1 addition (0 min).

[0225] As shown in Figure 3C, Ca in PDBu-induced vasoconstriction 2+ Inflow and L-type and T-type Ca 2+ The role of the channels was investigated and compared with the ET-1 response. Isolated coronary arterioles expressed spontaneous basal tension, and these were subsequently treated with different inhibitors (Ca 2+ After exposure to free L-nifedipine (NNC 55-0396) for 20 minutes, PDBu (0.1 μmol / L) was administered (at the "zero" point). Ca 2+ In the free bath solution, arterioles (resting diameter 47±2 μm) dilated to their maximum diameter and were unable to constrict in response to PDBu. This result indicates that vasoconstriction in response to PKC activation is similar to that with ET-1, as is the case with extracellular Ca. 2+ This indicates that inflow is necessary. Similarly, normal Ca in PSS 2+Pretreatment with nifedipine (1 μmol / L, resting diameter 39 ± 4 μm) or NCC 55-0396 (1 μmol / L, resting diameter 40 ± 5 μm) at lower levels caused blood vessels to lose their basal tone and eliminate vasoconstriction in response to PDBu (0.1 μmol / L). These data were obtained by Ca 2+ Not only does it demonstrate the efficacy of the channel inhibitors nifedipine and NNC 55-0396, but it also shows that L-type and T-type Ca25 mediates the onset of basal tension and the initiation of vasoconstriction in response to PDBu. 2+ It also shows channel opening. This vasoconstriction mechanism appears to be different from that induced by ET-1. In Figure 3C, * For R, P < 0.05 relative to the resting diameter percentage.

[0226] Ca in maintaining PDBu-induced vasoconstriction 2+ Inflow and L-type and T-type Ca 2+ To investigate the role of the channels, coronary arterioles were constricted with PDBu (0.1 μmol / L) during basal tension, and then treated with different inhibitors (Ca 2+ We investigated whether nifedipine (free, NNC 55-0396) could reverse vasoconstriction when applied to blood vessels. As shown in Figure 3D, PDBu caused vasoconstriction by reducing the resting basal diameter (R, 40±3μm) by approximately 50%. This vasoconstriction was due to Ca 2+ The results were reversed by the free solution, 1 μmol / L nifedipine, and 1 μmol / L NNC 55-0396. These findings were consistent with L-type Ca 2+ Channels and T-type Ca 2+ Sustained Ca through both channels 2+ This indicates that the influx contributes significantly to the maintenance of sustained vasoconstriction induced by PKC activation, in a manner distinct from that induced by ET-1. These data compare plasma membrane Ca for vasoconstriction compared to ET-1 compared to PDBu. 2+ This shows differential activation of the channel. In Figure 3D, * For R, P < 0.05 relative to the resting diameter percentage, #The P<0.05 ratio was observed relative to the resting diameter percentage at the time of PDBu addition (0 min).

[0227] In summary, ET-1-induced coronary artery constriction is due to extracellular Ca 2+ It requires inflow, T-type Ca 2+ Channel opening can partially mediate this process. On the other hand, L-type Ca 2+ The channel plays little role in coronary artery constriction in response to ET-1, but mediates vasoconstriction induced by PKC activation. Interestingly, L-type Ca 2+ It should be noted that the channel contributed little to PKC-induced vasoconstriction in large conduit arteries isolated from pigs. This highlights the heterogeneity of vasomotor mechanisms between large and small vessels. While both ET-1 and PKC activation can cause various cardiovascular diseases, the differences in their vasoconstriction mechanisms in response to ET-1 and PKC stimulation strongly emphasize the importance of using targeted therapeutic strategies. To achieve high efficacy in disease treatment, it is necessary to identify the virulence factors involved and determine the size of the vessels involved.

[0228] Mechanism of coronary artery constriction in response to ET-1: Roles of phospholipase C and myosin light chain kinase (MLCK) Despite the activation of phospholipase C (PLC) and myosin light chain (MLC) kinase (MLCK) identified in large vessel studies, the signaling mechanism responsible for coronary artery constriction in response to ET-1 has not been established. Activation of PLC after receptor activation and intracellular Ca 2+ The subsequent phosphorylation (activation) of MLC after the increase in Ca, and Ca for MLCK activation. 2+- The formation of the calmodulin complex is a central dogma pathway that contributes to vasoconstriction in large conduit vessels in response to various agonists, including ET-1. To determine whether this classical vasoconstrictive pathway applies to coronary arteriole constriction in response to ET-1, porcine coronary arterioles (<100 μm) were isolated for functional studies as described above. After the onset of resting basal tension, the vessels were subjected to treatment with PLC inhibitors and MLCK inhibitors, and the vasomotor response to physiological concentrations of ET-1 was examined.

[0229] As shown in Figure 4A, the role of phospholipase C (PLC) in mediating ET-1-induced coronary arteriole constriction was investigated in the presence of the selective PLC inhibitor U-73122 (5 μmol / L). In the absence of U-73122, control coronary arterioles constricted in response to ET-1 (0.1 nmol / L) by reducing their resting (R) diameter (46 ± 3 μm) by approximately 40%. In another group of vessels (resting diameter 46 ± 2 μm), pretreatment with U-73133 resulted in some loss of resting basal tone and reduced vasoconstriction in response to ET-1 (0.1 nmol / L). This result indicates the role of PLC in coronary arteriole constriction in response to ET-1. In coronary smooth muscle, PLC acts via non-selective cation channels to Ca 2+ This can activate the inflow. This concept involves inducing resting tension and causing contraction of ET-1 by extracellular Ca 2+ This is consistent with the observations presented in Figures 2A and 2C, which indicate that inflow is necessary. In Figure 4A, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of ET-1 addition (0 min).

[0230] As shown in Figure 4B, the role of MLCK in coronary arteriole constriction in response to ET-1 (0.1 nmol / L) was investigated using the selective MLCK inhibitor ML-9. In the absence of ML-9, ET-1 (0.1 nmol / L) resulted in constriction of control coronary arterioles by reducing their resting (R) diameter (43 ± 3 μm) by approximately 40%. In the presence of ML-9 (10 μmol / L), the vessels (resting diameter: 43 ± 2 μm) lost their basal tone, but the vasoconstriction induced by ET-1 (0.1 nmol / L) remained. This result indicates that MLCK activity is essential for the development of basal tone, but not essential for the initiation of vasoconstriction in response to ET-1. Therefore, inhibition of MLCK does not prevent coronary arteriole constriction in response to ET-1. In Figure 4B, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of ET-1 addition (0 min).

[0231] It is important to determine whether ET-1-induced contraction can be reversed by MLCK inhibition. Therefore, we conducted further studies to determine whether MLCK activation contributes to sustained ET-1 contraction. As shown in Figure 4C, ET-1-induced vasoconstriction was initially reversed by ML-9 (resting diameter 44±3 μm), but vasoconstriction gradually developed over time in the presence of ML-9. This indicates that the basal vasoconstriction portion is sensitive to MLCK inhibition, while vasoconstriction for ET-1 is insensitive to MLCK inhibition, as suggested by the data shown in Figure 4B. Therefore, ML-9 cannot reverse ET-1-induced vasoconstriction. In contrast, the ROCK inhibitor H-1152 effectively reverses vasoconstriction for ET-1 (resting diameter 44±3 μm) and maintains its dilated state. These microvascular data appear inconsistent with the established knowledge that molecular signaling related to smooth muscle contraction revolves around MLCK-mediated MLC phosphorylation.

[0232] The data presented in Figures 4B and 4C suggest that resting microvascular tone is controlled by MLCK activity, but vasoconstriction in response to ET-1 is likely mediated by a pathway unrelated to MLCK. Since ML-9 does not reverse vasoconstriction in response to ET-1, but H-1152 does, it is thought that ET-1 activates a ROCK-mediated signaling pathway that increases MLC phosphorylation by inhibiting MLCP activity, because the MLC phosphorylation level is determined by the balance between MLCK activity and MLC phosphatase (MLCP) activity. In Figure 4C, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of ET-1 addition (0 min).

[0233] In summary, ET-1-induced coronary arteriole constriction is mediated by PLC without the involvement of MLCK. This vasoconstriction mechanism differs from those reported in larger conduits. This MLCK-independent mechanism in coronary arterioles may be explained by the potential activation of ROCK, which can modulate vasoconstriction by inhibition of MLCP. To our knowledge, this is the first finding regarding MLCK-independent vasoconstriction for ET-1 in small coronary arterioles. The role of ROCK in mediating coronary arteriole constriction for ET-1 is further supported by the additional studies presented below.

[0234] The role of MLCK in coronary artery constriction in relation to PKC activation by PDBu. Since excessive activation of ET-1 and PKC contributes to many microvascular diseases, it is important to understand their similarities and differences in vasoconstriction mechanisms at the microcirculation level. As shown in Figures 4A-4C above, coronary arteries exhibit a unique MLCK-independent pathway for vasoconstriction in response to ET-1. However, both vasoconstrictors are extracellular Ca 2+Because it is dependent on inflow, it is unclear whether vasoconstriction in response to PKC activation is MLCK-independent (see Figures 3A-3D). To address this question, we tested whether the MLCK inhibitor ML-9 could prevent and / or reverse PDBu-induced vasoconstriction. We also compared the effects of ML-9 with the ROCK inhibitor H-1152.

[0235] As shown in Figure 5A, PDBu induced vasoconstriction by reducing the diameter to approximately 40% of the resting (R) diameter (50 ± 2 μm), which was approximately 60% vasoconstriction. Addition of ML-9 to this constricted vessel resulted in transient vasodilation (i.e., 178% of the resting diameter), and the diameter gradually returned to the control level (i.e., approximately 125% of the resting diameter) after 20 minutes. This change in diameter corresponds to approximately 70% of the relaxed diameter, which is approximately 30% vasoconstriction. The magnitude of this vasoconstriction in the presence of ML-9 was 50% lower than the initial vasoconstriction in the absence of ML-9 at time zero. This indicates that PDBu-induced vasoconstriction is partially (i.e., 50%) reversed by MLCK inhibition. Addition of the broad-spectrum PKC inhibitor Goe6983 to the vessel (resting diameter 38 ± 2 μm) completely reversed PDBu-induced vasoconstriction. In contrast, H-1152 did not affect vasoconstriction in response to PDBu (resting diameter 49±4 μm). These data indicate that PDBu activates PKC-dependent vasoconstriction and that MLCK plays a crucial role in the expression of resting tension. On the other hand, MLCK contributes approximately 50% to vasoconstriction in response to PKC activation. Furthermore, this vasoconstriction is independent of ROCK signaling. In Figure 5A, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of PDBu addition (at 0 minutes).

[0236] As shown in Figure 5B, prior exposure of blood vessels to Goe6983 did not alter resting (R) vascular tone (55±5 μm), but prevented the development of PDBu-responsive vasoconstriction. These results indicate that PKC does not contribute to the development of resting tone. However, PDBu does induce PKC-mediated vasoconstriction. These results are consistent with the findings presented in Figure 5A. In the presence of ML-9 (48±3 μm) or H-1152 (40±3 μm), coronary arterioles lost resting tone by doubling their diameter (i.e., by approximately 200%). These data indicate that MLCK and ROCK contribute to the development of resting vascular tone. Adding PDBu to a vascular bath containing either ML-9 or H-1152 resulted in progressive vasoconstriction over time, with the final diameter slightly below the resting level (R), i.e., approximately 75–90% of the resting diameter. The magnitude of these vasoconstrictions corresponded to approximately 55–63% vasoconstriction from dilated vessels (i.e., 200% of resting diameter). The magnitude of these vasoconstrictions falls within the range of initial PDBu-induced contraction without inhibitors (i.e., approximately 60% contraction, from 100% of resting diameter to 40% of resting diameter), as shown in Figure 5A. These data indicate that MLCK and ROCK signaling contributes only slightly (or not at all) to the initiation of PKC-mediated vasoconstriction. In Figure 5B, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of PDBu addition (0 min).

[0237] In summary, PDBu-induced coronary artery constriction is mediated by PKC activation, as this vasomotor response is blocked by the broad-spectrum PKC inhibitor Goe6983. Prior inhibition of MLCK by ML-9 does not prevent PDBu-induced vasoconstriction, therefore the initiation of PKC-mediated vasoconstriction is Ca 2+It appears to be independent of / calmodulin / MLCK signaling (Figure 5B). This is the first study to demonstrate MLCK-independent vasoconstriction induced by PKC activation in microvessels. It may involve direct phosphorylation of MLC or PKC-binding Ca 2+ Channel through Ca 2+ PKC activation, which triggers the influx, likely explains the vasoconstriction we observed. Meanwhile, MLCK activation contributes to approximately 50% of the sustained vasoconstriction induced by PKC (Figure 5A). The other 50% of vasoconstriction, which is insensitive to MLCK inhibition, is due to direct activation of MLC by PKC, or possibly PKC-binding Ca, as suggested in Figure 5B. 2+ It is highly likely that this is due to influx. Even if MLCK activity is impaired, it appears that coronary artery constriction in response to PKC activation can still be initiated. Furthermore, once vasoconstriction occurs, MLCK is involved in the persistence of this vasomotor activity. This appears to further complicate the use of MLCK inhibitors to effectively treat PKC-activated vascular diseases, as multiple pathways at the smooth muscle contraction element level are activated by PKC. However, importantly, ROCK signaling does not play a role in PKC-mediated vasoconstriction.

[0238] Mechanism of coronary artery constriction in response to ET-1: Roles of PKC and ROCK It has been suggested that ET-1-induced vasoconstriction is mediated by PKC activation. However, this hypothesis was based on data obtained from either large conduit arteries or microvessels that did not exhibit spontaneous basal tension. Therefore, the contribution of PKC signaling to ET-1-induced coronary arteriole constriction remains undetermined. Furthermore, since PKC activation is known to mediate many cardiovascular diseases through countless signaling mechanisms, determining whether PKC, as a broad kinase family, is involved in ET-1 vasoconstriction has clinical significance. To address this issue, porcine coronary arterioles were isolated and pressurized as described above. The vessels were subjected to pharmacological interventions to elucidate the involvement of PKC and ROCK in ET-1 vasoconstriction and to determine whether inhibition of these two signaling molecules can prevent and / or reverse vasoconstriction.

[0239] To investigate whether PKC activation is involved in ET-1-induced coronary arteriolar constriction, vasoconstriction in response to ET-1 and the PKC activator PDBu was compared in the absence or presence of the broad-spectrum cell-permeable PKC inhibitor bisindolylmaleimide XI (BIM XI, 1 μmol / L). As shown in Figure 6A, both ET-1 (0.1 nmol / L) and PDBu (0.1 μmol / L) constricted coronary arterioles to a similar extent from the resting (R) basal diameter (ET-1 group: 46 ± 6 μm, PDBu group: 43 ± 2 μm). Pretreatment of vessels with BIM XI induced vasodilation, and PDBu-induced vasoconstriction disappeared. However, in another group of vessels treated with BIM XI, ET-1-induced vasoconstriction remained. This result indicates that ET-1-induced vasoconstriction is not mediated by PKC activation, which differs from the findings in larger upstream coronary arteries. In Figure 6A, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of ET-1 or PDBu addition (0 min).

[0240] To investigate whether ET-1-induced and PDBu-induced vasoconstriction can be prevented by inhibiting Rho kinase (ROCK) activation, vasoconstriction in response to ET-1 and PDBu was examined using the ROCK inhibitor H-1152. As shown in Figure 6B, coronary arterioles constricted to a similar extent in response to ET-1 (0.1 nmol / L, resting diameter 44±4 μm) and PDBu (0.1 μmol / L, resting diameter 47±4 μm). Pretreatment of coronary arterioles (resting diameter 40±3 μm) with H-1152 (3 μmol / L) eliminated resting (R) basal tone, but enhanced vasoconstriction in response to PDBu. However, in a different group of vessels (resting diameter 44±5 μm), vasoconstriction in response to ET-1 was prevented by H-1152. These results suggest that ROCK activation mediates ET-1-induced vasoconstriction, but not PDBu-induced vasoconstriction, likely due to inhibition of phosphatase activity (see Figures 7 and 8A-8D below). Therefore, the ROCK inhibitor H-1152 can be specifically used to prevent ET-1-induced vasomotor activity in coronary arteries. These data also suggest that H-1152 may be usable to prevent ET-1-induced microvasoconstriction during disease onset. In Figure 6B, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of ET-1 or PDBu addition (0 min).

[0241] To determine whether ET-1-induced vasoconstriction could be reversed by ROCK and / or PKC inhibition, vessels were first constricted with ET-1 (0.1 nmol / L) to a stable diameter (i.e., a reduction of approximately 40-50% from the resting (R) diameter). The vasoconstrictive state was then examined after the addition of either a ROCK inhibitor (H-1152, 3 μmol / L) or a broad-spectrum PKC inhibitor (BIM XI, 1 μmol / L). As shown in Figure 6C, H-1152, rather than BIM XI, effectively reversed ET-1-induced vasoconstriction within 30 seconds after drug administration, and the vessels remained dilated throughout the entire course of this study. The reversibility with BIM XI was less significant compared to that with H-1152. This study demonstrated that ET-1-induced vasoconstriction is not mediated by PKC activation. These results demonstrate that H-1152 is a potent agent that reverses ET-1-induced vasoconstriction, suggesting that H-1152 can be used to treat ET-1-induced microvasoconstriction. In Figure 6C, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of ET-1 addition (0 min).

[0242] Using the same approach, we investigated whether PDBu (0.1 μmol / L)-induced vasoconstriction could be maintained by H-1152 (3 μmol / L) and / or BIM XI (1 μmol / L). As shown in Figure 6D, PDBu-induced vasoconstriction was reversed by BIM XI, which is consistent with the results in Figure 5A when using a different PKC inhibitor, Goe6983. However, H-1152 only slightly attenuated vasoconstriction to PDBu within the first minute of drug administration, and vasoconstriction was observed throughout the entire course of this study. This study indicates that H-1152 is not an effective agent for reversing PKC activation-induced vasoconstriction. In Figure 6D, * For R, P < 0.05 relative to the resting diameter percentage, #The P<0.05 ratio was observed relative to the resting diameter percentage at the time of PDBu addition (0 min).

[0243] In summary, since PKC activation is known to cause aortic vasoconstriction in response to ET-1 stimulation, findings of PKC-independent vasoconstriction in response to ET-1 are specific to resistant arteries. Therefore, findings in upstream aortic vessels cannot be applied to downstream microvessels from the perspective of the mechanistic action of ET-1. The involvement of ROCK activation in microvasoconstriction in response to ET-1 is supported by this study. Most importantly, H-1152 not only selectively and effectively prevents but also reverses ET-1-induced vasoconstriction in coronary arterioles, supporting the clinical application of this inhibitor for the prevention and treatment of ET-1-induced coronary microvascular dysfunction.

[0244] Mechanism of coronary artery constriction in response to ET-1: The role of myosin light chain phosphatase (MLCP) in ROCK Our study demonstrates the involvement of ROCK activation in coronary arteriole constriction against ET-1 (Figure 6). Interpretation of these data depends on the specificity of H-1152 for ROCK inhibition. While inhibition of ROCK activity by H-1152 has been characterized using biochemical enzyme assays, cultured smooth muscle cell preparations, or vascular striae constriction studies, it should be noted that there is no data to support whether this also applies to vasomotor regulation of small arterioles. Most importantly, this concern needs to be addressed at the intact vascular level, exhibiting spontaneous basal tension as seen in vivo, without the involvement of confounding activation or inactivation of other signaling pathways by pharmacological agents. Therefore, if H-1152 is a candidate for treating microvascular dysfunction associated with ROCK activation, a mechanistic understanding of how H-1152 acts in intact microvessels is essential. Similarly, since ROCK activation leads to MLCP inhibition and consequently promotes vasoconstriction, we anticipate that H-1152 increases MLCP activity and induces vasodilation. In this case, the effect of H-1152 should be reversed by an MLCP inhibitor. In other words, the inhibitory effect of H-1152 on ET-1-induced vasoconstriction should disappear in the presence of an MLCP inhibitor. The following experiment supports this situation.

[0245] The role of MLCPs was investigated in isolated coronary arterioles using kallikrin A (CLA), a potent and selective cell-permeable MLCP inhibitor. CLA directly inhibits the protein phosphatase type 1 catalytic subunit (PP1cδ), one of the three subunits of MLCP. The other two components of MLCP are the regulatory complex of MYPT1 (also known as MBS, M110) as a downstream target of ROCK and PKC, as well as a 21kDa accessory subunit of unknown function. CLA promotes vasoconstriction by directly inhibiting phosphatase activity. It is unclear whether the inhibitory effect of H-1152 on ET-1-induced coronary arteriole constriction is mediated by direct activation of phosphatases or by inhibition of ROCK molecules upstream of phosphatases. CLA inhibits Ca in the plasma membrane. 2+ Because it affects channel activity, we used Ca in the coronary arteries.2+ These issues were addressed by exposing the cells to a free solution and H-1152 pretreatment, and then comparing the vasomotor response to ET-1 (0.1 nmol / L) in the absence and presence of CLA (0.1 μmol / L).

[0246] As shown in Figures 2A, 3A, and 3B, ET-1-induced vasoconstriction is Ca 2+ In the free solution, it was not present (resting diameter 45±5 μm, n=5, Figure 7), and vasoconstriction in response to ET-1 (0.1 nmol / L) was sensitive to H-1152 (resting diameter 44±5 μm, n=7, Figure 7, see also Figure 4C and Figures 6B and 6C). In contrast, as shown in Figure 7, vasoconstriction induced by CLA (0.1 μmol / L) was in response to extracellular Ca 2+ This was unrelated (resting diameter 50±5μm, n=6). Vasoconstriction relative to ET-1 was abolished by H-1152 (resting diameter 49±2μm, n=4), but this vasoconstriction was maintained by adding CLA. These data suggest, firstly, that, unlike ET-1, CLA is related to extracellular Ca 2+ It has been shown that it induces vasoconstrictive signaling without influx, and plasma membrane Ca 2+ This suggests the action of CLA downstream of the channel. This situation is related to extracellular Ca 2+ This is consistent with the results of phosphatase inhibition unrelated to influx. Secondly, H-1152 did not affect CLA-induced vasoconstriction, suggesting a downstream target of ROCK in CLA. These results also indicate that H-1152 inhibits ET-1-induced vasoconstriction not by directly acting on phosphatases, but by indirectly increasing phosphatase activity, for example, through ROCK inhibition. These studies not only demonstrate the specificity of H-1152 as a ROCK inhibitor but also demonstrate the downstream action of ROCK-mediated CLA as a phosphatase inhibitor. In Figure 7, * For R, P < 0.05 relative to the resting diameter percentage, # The P<0.05 ratio was observed relative to the resting diameter percentage at the time of CLA addition (0 min).

[0247] In summary, the possibility that H-1152 inhibits ET-1-induced vasoconstriction by directly acting on MLCP activity is ruled out in this study. Our research demonstrates the specificity of H-1152 as a potent ROCK inhibitor in intact microvessels. Furthermore, the inhibitory effect of H-1152 on ROCK is supported by a series of molecular studies described below.

[0248] Molecular evidence of ET-1-mediated phosphorylation of MYPT1 and MLC via ROCK activation Myosin light chain (MLC) activation via the phosphorylation process is crucial for smooth muscle contraction, and the level of MLC phosphorylation (pMLC), which is negatively regulated by phosphorylation of myosin phosphatase target subunit 1 (pMYPT1) in phosphatases, can be regulated by MLCP. Therefore, the level of pMLC at serine 19 (S19), corresponding to smooth muscle contractile activity, can be increased by inhibiting phosphatase activity via MYPT1 phosphorylation at threonine 850 (T850). However, it is unclear whether ET-1 directly activates MLC for coronary arteriole constriction or indirectly activates it through inhibition of phosphatase activity by MYPT1. It is also unclear whether ROCK is involved in the phosphorylation of MLC and MYPT1 for vasoconstriction. To address these questions, as described in the functional studies, porcine coronary arterioles (less than 100 μm in diameter) were isolated, pressurized, and basal tension was expressed. After pharmacological treatment (ET-1, PDBu, or H-1152), 2-3 blood vessels from each group of the same animal were flash-frozen in liquid nitrogen for biochemical analysis. The concentrations of the pharmacological agents used in molecular studies were the same as those used in functional studies in intact blood vessels. Coronary arteriole pMLC and pMYPT1 expression in response to ET-1 were analyzed, and the results were compared with those obtained from PKC activator PDBu.

[0249] Coronary arterioles were subjected to immunoblotting for pMYPT1 and pMLC using smooth muscle actin (SMA) expression as an equal protein loading standard. As shown in Figure 8A, pMYPT1 and pMLC were elevated in vessels treated with ET-1 (0.1 nmol / L) compared to vessels treated with the vehicle (control). In contrast, PDBu (0.1 μmol / L) caused only MLC phosphorylation (pMLC), and pMYPT1 was not elevated. These data indicate the involvement of MYPT1 phosphorylation in ET-1-induced signaling for vasoconstriction, which is a pathway distinct from the one triggered by PKC activation. Since MYPT1 phosphorylation is known to inhibit phosphatase activity, it can be concluded that ET-1-induced coronary arteriole constriction is mediated by phosphatase inhibition via MYPT1 phosphorylation. These molecular data are consistent with functional data showing that ET-1 and PDBu activate different pathways (Figures 3A-3D and 5A-5B) for coronary artery constriction, namely phosphatase-dependent and phosphatase-independent signaling, respectively. Representative data were analyzed from four independent experiments.

[0250] As shown in Figure 8B, when arteries were collected on the same day under three treatment conditions (control, ET-1, and PDBu), both ET-1 and PDBu treatment induced potent MLC activation (pMLC) at the S19 site, while only ET-1 treatment induced MYPT1 phosphorylation at the T850 site. pMYPT1 was elevated by ET-1 but not by PDBu, suggesting that ET-1 and PDBu induce different vasoconstrictive pathways. The data also suggest that PDBu induces Ca for vasoconstriction. 2+ It has been shown that it induces the calmodulin-MLCK pathway. On the other hand, ET-1 inhibits MLCP activity upon MYPT1 phosphorylation, thereby promoting Ca for vasoconstriction. 2+ It induces a sensitization mechanism. Representative data were analyzed from four independent experiments. Ctl is the control.

[0251] As shown in Figure 8C, we present the normalized quantitative analysis of pMYPT1 in smooth muscle actin (SMA) from four independent experiments. The data showed that ET-1 significantly increased pMYPT1 in the coronary arterioles. * indicates P<0.05 compared to the control.

[0252] As shown in Figure 8D, ET-1 consistently induces MYPT1 and MLC phosphorylation. It should be noted that the MLC phosphorylation induced by ET-1 was abolished by H-1152, which indicates Ca 2+ This indicates that activation of the calmodulin-MLCK axis is not responsible for pMLC-dependent vasoconstriction, but rather that ROCK activation is a key signaling pathway mediating vasoconstriction in response to ET-1. Although H-1152 did not affect basal levels of pMYPT1, it eliminated the elevation of pMYPT1, leading to the conclusion that ROCK activation by ET-1 is responsible for MYPT1 phosphorylation. Overall, these data demonstrate the involvement of upstream ROCK activation of myosin phosphatases in coronary artery constriction in response to ET-1. Quantitative analysis was obtained from normalized data in MLC from four independent experiments. In Figure 8D, * indicates P<0.05 compared to other groups.

[0253] In summary, ET-1 induces coronary artery constriction by increasing pMLC, which is a result of myosin phosphatase inhibition by phosphorylation (activation) of the MYPT1 subunit after ROCK activation. In contrast, PDBu / PKC induces vasoconstriction unrelated to ROCK / pMYPT1 signaling. 2+- It induces direct MLC phosphorylation and / or indirect activation of the calmodulin-MLCK axis. Our molecular data support the role of ROCK in ET-1-induced coronary arteriole constriction. The ROCK inhibitor H-1152 blocks this signaling pathway and inhibits ET-1-induced vasoconstriction. Furthermore, H-1152 is ineffective in inhibiting PKC activation-induced contractile signaling and, consequently, vasoconstriction. However, if PKC activation is clearly involved based on our data, calcium channel blockers can be utilized. This conclusion is consistent with and reflects the results of functional studies in intact arterioles presented in Figures 5A-5B and 6A-6D.

[0254] Phosphorylation of MLCs in isolated coronary artery smooth muscle cells by ET-1 and its reciprocal phosphorylation regulated by H-1152 ET-1 induces concentration-dependent contraction of intact coronary arterioles, but contractile signaling activation (pMLC) in smooth muscle cells responds to increased ET-1 levels, or to Ca in the contractile filaments within the cell. 2+ It is unclear whether this simply corresponds to an increase in sensitivity. This question is important when investigating the signaling mechanism of ET-1 activation. Furthermore, it is unclear whether the change in pMLC levels in smooth muscle is a function of ET-1 concentration, and whether H-1152 can inversely modulate ET-1-induced MLC phosphorylation levels in a manner consistent with that observed in functional studies of intact blood vessels. This information is important for interpreting molecular data corresponding to the functional behavior of blood vessels and for supporting the idea that ET-1 is antagonized by inhibiting MLC phosphorylation via the ROCK inhibitor H-1152.

[0255] As shown in Figure 9A, ET-1 concentration-dependently increased MLC phosphorylation (pMLC) in smooth muscle cells isolated from porcine coronary arterioles (less than 100 μm in diameter) under primary culture conditions. In the absence of ET-1 (C), the cells expressed basal levels of pMLC, which is consistent with the expression of spontaneous basal tension in intact vessels, indicating the viability and functional integrity of the MLCs in these cells. These data demonstrate that ET-1 can induce pMLC and support the role of pMLC in coronary arteriole constriction in relation to ET-1. Quantitative data were obtained from three independent experiments. The intensity of the immunoblotting bands was normalized to the level obtained from 10 nmol / L of ET-1 and expressed as relative intensity. In Figure 9A, M is mol / L.

[0256] To investigate the effect of H-1152 on ET-1-induced pMLCs, smooth muscle cells isolated from coronary arterioles (less than 100 μm in diameter) were treated with 0.1 nmol / L ET-1 (the same as used in functional studies) in primary culture under different concentrations of H-1152 (C) and in the presence of H-1152. As shown in Figure 9B, ET-1 activated MLCs (pMLCs) in the absence of H-1152, and the expression level of pMLCs was reduced in a concentration-dependent manner by H-1152. These data indicate that MLC phosphorylation levels are dependent on ROCK activity, supporting the concept that ET-1-induced coronary arteriole constriction is mediated by pMLCs via ROCK activation. Furthermore, ET-1 activated pMLCs almost completely disappear at micromolar concentrations of H-1152, in a manner consistent with the disappearance of basal tension and contraction in response to ET-1 (0.1 mmol / L) at the same levels as H-1152 in the coronary arterioles (Figures 5A-5B and 6A-6D). Quantitative data were obtained from three independent experiments. The intensity of the immunoblotting bands was normalized to the levels obtained under control (C) conditions without H-1152 and expressed as relative intensity. In Figure 9B, M is mol / L.

[0257] In summary, molecular data on ET-1-induced MLC phosphorylation elevation correlated fairly well with the degree of ET-1-induced vasoconstriction in a concentration-dependent manner (Figures 2A-2D). Data on concentration-dependent inhibition of MLC phosphorylation by H-1152 may help in selecting an effective H-1152 concentration to antagonize the vasoconstrictive activity of ET-1 or basal tension. Therefore, H-1152 may be used to treat ET-1-related microvasoconstriction and angina in ROCK activation-associated cardiovascular events.

[0258] Characteristics of ROCK isoform expression in cardiomyocytes, the left anterior descending artery, and coronary arteries. Two ROCK isoforms, ROCK1 and ROCK2, have been identified, but information on their distribution and relative expression in the coronary microvascular system is lacking. Here, we address these important issues as our data demonstrate a crucial role of ROCK in mediating coronary artery constriction relative to ET-1.

[0259] Coronary arteries and coronary arterioles were carefully isolated from porcine hearts, all vessels were pooled, homogenized, and lysed for immunoprecipitation (IP). The lysates were centrifuged to obtain supernatant (S) and precipitate (P) samples. These samples were loaded onto SDS-PAGE gels with input (I) and immunoblotted with antibodies against ROCK1 and ROCK2. As shown in Figure 10A, coronary vessels express both ROCK1 and ROCK2 isoforms, which can be precipitated and subsequently detected with their specific antibodies without cross-reactivity.

[0260] As shown in Figure 10B, ROCK isoforms were expressed in the aorta of different species, including different strains of mouse, pig, and rat (SD: Sprague Dawley, F: Fischer, LE: Long-Evans). Human samples were retinal tissue obtained from patients excised for ocular tumors (performed after informed consent with approval from the Baylor-Scott & White Health Review Board and in accordance with the principles of the Declaration of Helsinki). For this study, retinal samples were isolated from areas free of tumor tissue. As shown in Figure 10B, ROCK is ubiquitously expressed in the vascular system across different species. Except for the mouse and human samples, cleaved ROCK1 (130KD, arrow) was detected in aortic tissue. ROCK1 is a direct cleavage substrate of activated caspase-3, which is involved in myocardial apoptosis and cardiomyopathy. Cleavage of ROCK1 results in a 130KD variant that can activate caspase-3 via a positive feedforward loop, which may consequently promote apoptotic signaling in myocardial hypertrophy and / or heart failure. However, the physiological and pathophysiological roles of truncated ROCK1 within ductal arteries remain unclear. Interestingly, all rat strains tested primarily expressed the 130kD truncated ROCK1 isoform, with trace amounts of 160kD ROCK1 barely visible.

[0261] It is unclear whether ROCK isoforms are expressed in cardiomyocytes, and whether their expression levels differ from those in coronary arterioles. This question is important in considering whether the target of ROCK inhibitors is primarily in the cardiomyocytes or the coronary vascular system. As shown in Figure 10C, both cardiomyocytes and arteries express ROCK1 and ROCK2 at 4-fold and 2-fold higher expression levels in coronary arterioles, respectively. Cardiomyocyte samples contained tropomyosin but no α-actin, indicating that vascular tissue contamination was removed from these samples. Vascular samples contained α-actin but little to no tropomyosin, indicating high purity of the vascular samples. Immunoblot expression intensities were normalized by GAPDH and expressed as relative intensities (n=3 independent experiments). In Figure 10C, * indicates P<0.05 compared to cardiomyocytes.

[0262] Since there is no information available regarding the relative expression of ROCK1 to ROCK2 in the coronary vascular system, and it is unclear whether their expression levels differ between large conduital arteries and small resistance arterioles, we addressed these questions to aid in the future design of isoform-specific ROCK inhibitors. The left anterior descending (LAD) artery (the main coronary artery in the heart, approximately 3-4 mm in diameter) and its small downstream arterioles (less than 100 μm in diameter) were isolated for immunoblotting. As shown in Figure 10D, both ROCK isoforms were expressed in the coronary system, and after normalization with their own α-actin expression, there was no apparent difference in expression levels between large and small vessels (n=3 independent experiments). It is noteworthy that truncated ROCK1 (130KD) is readily detectable in the LAD artery but not in the small coronary arterioles.

[0263] Since ROCK2 appears to be the primary isoform expressed in coronary arterioles, we performed immunohistochemical studies of ROCK2 to localize its expression in cardiac tissue. As shown in Figure 10E, we identified arteries using smooth muscle actin expression (green) and co-localized them with vascular ROCK2 expression (integrated image). Both cardiomyocytes and arteries appear to express ROCK2, consistent with the Western blot data (Figure 10C). Immunohistochemical staining in arterioles was much stronger than that in cardiomyocytes, suggesting higher ROCK2 expression in arterioles. These immunohistochemical data are consistent with the Western blot of ROCK2 expression.

[0264] ROCK2 expression in smooth muscle compared to endothelial cells has not been determined in coronary arterioles. This is important because their differential expression may affect the arterial response to ET-1 and its vasomotor regulation. To address this question, coronary arterioles (less than 100 μm in diameter) were isolated and immunohistochemically stained with ROCK2 after cryo-excision. Arterial sections were stained with antibodies conjugated with red fluorescent dyes against eNOS and smooth muscle actin to localize endothelial cells and smooth muscle cells, respectively. ROCK2 was stained with antibodies conjugated with green fluorescent dyes. As shown in Figure 10F, ROCK2 expression in smooth muscle is evident. This is expected, as ET-1-induced vasoconstriction is endothelium-independent and mediated by ROCK. Endothelial cells also express ROCK2 to a visible degree.

[0265] In summary, coronary arterioles and cardiomyocytes express both ROCK1 and ROCK2 isoforms, with ROCK2 isoforms being primarily expressed in coronary arterioles. Coronary arteriole endothelial cells also express ROCK2 isoforms. However, their role in regulating vasomotor function remains undetermined. The expression of truncated ROCK1 (130KD) in vascular arteries, including coronary arteries, is intriguing. It is hypothesized that this expression may increase vascular susceptibility to disease onset. Nevertheless, this study also demonstrates another characteristic of coronary arterioles that distinguishes them from their upstream vascular vessels.

[0266] Differential expression of CPI-17 in the coronary network: vessel size-dependent CPI-17 expression The 17kDa PKC-enhanced myosin phosphatase inhibitor (CPI-17) is a phosphorylation-dependent inhibitory protein of MLCPs. Phosphorylation of CPI-17 at Thr38, reported in in vitro studies of large conduit arteries with PKC or ROCK, can enhance its inhibitory ability against MLCPs and promote vasoconstriction. It is unclear whether ET-1 can activate CPI-17 downstream of ROCK at physiological / pathophysiological concentrations to exert MLCP inhibition and induce coronary arteriolar constriction. Furthermore, no studies demonstrate whether CPI-17 activation is necessary for coronary arteriolar constriction in response to ET-1. To address these questions, aorta, cerebral arteries, coronary arteries (1 mm in diameter), and coronary arterioles (approximately 100 μm in diameter) were isolated from pigs for biochemical and immunohistochemical studies of phosphorylated CPI-17 (pCPI-17).

[0267] As shown in Figure 11A, both CPI-17 and pCPI-17 were easily detected in large conduit arteries (porcine cerebral arteries and aorta), but their expression was undetectable in porcine coronary arterioles (less than 100 μm in diameter). This unique CPI-17 expression leads to the hypothesis that its expression is dependent on blood vessel size.

[0268] To further investigate differential expression of CPI-17 in different vessel sizes within the same vascular bed, coronary arteries (approximately 1000 μm in diameter) and coronary arterioles (100 μm in diameter) were isolated and subjected to Western blot analysis for CPI-17. As shown in Figure 11B, CPI-17 was well expressed in larger coronary arteries but barely detectable in coronary arterioles with the same protein load (10 μg). Furthermore, CPI-17 expression remained very low even after a 5-fold increase in protein load (50 μg). It should be noted that these small vessel-sized protein samples may have been "contaminated" by some larger vessels (100–150 μm in diameter) during microvessel isolation. Nevertheless, these data suggest that CPI-17 may not be functionally important in regulating myosin phosphatase activity in coronary arterioles, given its sparse expression.

[0269] Further quantitative studies of CPI-17 and ROCK2 protein expression in coronary arteries of different sizes supported the concept of vessel size-dependent CPI-17 expression. As shown in Figure 11C, CPI-17 was barely detectable in narrow coronary arterioles (diameter 30–100 μm), but its expression level in arteries increased with increasing vessel size. Note that ROCK2 protein expression was independent of vessel size. 10 μg of protein was loaded into each lane. Expression levels after normalization with smooth muscle actin (SMA) are presented as relative ratios with reference to data obtained from 1000 μm vessel sizes from six independent experiments. In Figure 11C, * indicates P<0.05 compared to other groups.

[0270] To support the results of the Western blot analysis described above, immunostaining for CPI-17 was performed on frozen sections of large conduits and small coronary arterioles. Cell nuclei were stained using DAPI (4',6-diamidino-2-phenylindole). As shown in Figure 11D, CPI-17 was barely detectable in small coronary arterioles (approximately 20–50 μm in the left central panel), but its expression was evident in large coronary arteries (approximately 550 μm in the right panel). This result is consistent with differential CPI-17 protein expression from Western blot analysis (Figure 11C).

[0271] In summary, these findings indicate that CPI-17 is hardly expressed even in coronary arterioles (less than 100 μm in diameter). However, CPI-17 expression increases with increasing vessel size, suggesting that the regulation of myosin phosphatase activity by CPI-17 becomes more important in larger conduits. This finding suggests that CPI-17 plays a crucial role in smooth muscle contraction by inhibiting myosin phosphatase activity, and therefore contributes to the sustained vasoconstriction of contractile filaments. 2+ This supports biochemical and mechanical contractility studies of isolated vascular smooth muscle cells, striae, or vascular rings taken from large conduit vessels, which increase sensitivity. However, since CPI-17 is barely detectable or almost absent in these microvessels, this contractile signaling mechanism may not be important in narrow coronary arterioles. It is demonstrated here for the first time that CPI-17 is expressed along the same vascular tree based on arterial size, but is little to no expression at the arteriole level. In contrast, ROCK signaling for myosin phosphatase regulation is dominant in narrow coronary arterioles for vasoconstriction. Our study reveals unique biochemical features of coronary arterioles in vasomotor regulation. These findings may highlight the reasons for the failure of treatment of microvascular diseases based on large vessel data and emphasize the importance of targeting microvessels for effective treatment of microvascular diseases.

[0272] The effect of ROCK inhibitors on resting basal muscle tone, unrelated to endothelial nitric oxide (NO). As demonstrated in the series of studies above, ET-1-induced coronary artery constriction is ET A Receptor activation, extracellular Ca 2+ This process is primarily mediated by a series of events: inflow, ROCK activation, MYPT1 phosphorylation, myosin phosphatase inhibition, and the resulting increase in MLC phosphorylation. However, there is no available information on the effect of ROCK inhibition on the basal vasomotor tone of coronary arterioles. This is a significant concern because resting blood flow levels are primarily influenced by the basal arteriole tone levels of organ systems, including tissues and the heart. Furthermore, it is unclear whether ROCK inhibitor-induced vasodilation is mediated by the release of NO from the endothelium. These questions are also important because basal arteriole tone is generally elevated in patients with myocardial ischemia and / or hypertension associated with microvascular dysfunction. Therefore, determining the sensitivity and potency of ROCK inhibitors in antagonizing basal tone is essential for improving blood flow and managing hypertension by reducing resting arteriole resistance. Moreover, if ROCK inhibitor-induced vasodilation is mediated by the release of endothelial NO, the effectiveness of reversing vascular resistance would be impaired due to endothelial dysfunction, which is known to be associated with coronary artery disease. Therefore, it is necessary to consider the potential limitations of using ROCK inhibitors for medical purposes. Current clinical approaches also focus on improving endothelial function, such as treating patients with statins. To address these important questions, coronary arterioles (less than 100 μm in diameter) were isolated and pressurized for functional studies by constructing concentration-response curves of ROCK inhibitors. We compared the effectiveness of reversing basal tension with H-1152 and Y-27632, one of the most commonly used ROCK inhibitors for biomedical studies. NO synthase (NOS) inhibitors N1152 are specific NOS inhibitors for eliminating NO production. w ROCK-induced vasodilation was examined in the presence and absence of nitro-L-arginine methyl ester (L-NAME).

[0273] As shown in Figure 12A, isolated coronary arterioles exhibited resting basal tone (R) and dilated in a concentration-dependent manner with H-1152 (resting diameter 51±3 μm) and Y-27632 (resting diameter 50±5 μm). IC at 30 nmol / L of H-1152 during coronary arteriole dilation. 50 The value is the IC of Y-27632 at 3 μmol / L. 50 The effect was 100 times stronger than the stated value. The response to H-1152 showed a linear concentration-dependent relationship up to 300 nmol / L when the arteries approached their maximum dilation. This indicates the high specificity of H-1152 to its molecular target(s). Arterioles dilated maximally with 1 μmol / L of H-1152, and all vessels regained their resting tension after flushing out the H-1152. Repeated experiments showed no signs of decreased H-1152 responsiveness, and H-1152 did not immediately adversely affect vasomotor function.

[0274] As shown in Figure 12B, coronary artery vasodilation in response to H-1150 remained unchanged in the presence of L-NAME (10 μmol / L), indicating that the observed vasodilation was independent of endothelial NO release.

[0275] These results indicate that H-1152 is a more specific, sensitive, and potent ROCK inhibitor compared to Y-27632 in reversing basal tension without apparent toxicity. The action of this vasodilator is not mediated by endothelial NO release. Therefore, the clinical application of this ROCK inhibitor is not expected to depend on the functional state of the endothelium. This unique property is advantageous in clinical significance, as endothelial function related to NO bioavailability is generally impaired in many cardiovascular diseases. Thus, the endothelial NO-independent action of H-1152 is expected to have a wide range of applications in disease treatment, and H-1152 may be combined with statins as maintenance therapy. Overall, our data indicate that ROCK activation, without mediated by CPI-17 signaling, is the primary pathway mediating coronary artery constriction in response to ET-1. Our study also demonstrates that H-1152 is a selective and effective inhibitor of ROCK in both preventing and reversing ET-1-induced vasoconstriction in the coronary arteries. Our data support the present invention's technology, indicating that the ROCK inhibitor H-1152 is an excellent candidate for treating ET-1-related cardiovascular diseases, including microvascular dysfunction.

[0276] Tissue heterogeneity in the regulation and utilization of vasomotor activity by H-1152 Studies of the fundamental mechanisms of vasomotor regulation in the microcirculation provide a framework for characterizing the development of microvascular dysregulation in animal models of various diseases, namely hypertension, diabetes / hyperglycemia, ischemia / reperfusion injury, atherosclerosis, hyperlipidemia, lipopolysaccharide (LPS) shock, pathological angiogenesis, and C-reactive protein associated with inflammatory attacks. Microvessels from different vascular beds and related diseases exhibit unique features and mechanisms leading to microvascular dysfunction.

[0277] We investigated the microcirculation of healthy eyes / retina and compared its vasomotor regulation to that of the coronary microvascular system. Although both coronary and retinal circulation exhibit the same metabolism-dependent blood flow regulation phenomena, we found that they exhibit several different signaling pathways for vasomotor regulation and disease susceptibility. For example, we found that acetylcholine, when administered extraluminally, is a potent coronary artery vasoconstrictor in pigs, but a vasodilator in pig retinal arterioles. Furthermore, thrombin is a vasodilator in pig coronary arterioles but a potent vasoconstrictor in pig retinal arterioles. Serotonin is a potent endothelium-dependent vasodilator in coronary arterioles, but retinal arterioles cannot respond to serotonin.

[0278] Specifically, as shown in Figure 13A, isolated porcine coronary and retinal arterioles were pressurized to 60 cmH2O. The coronary arterioles exhibited basal tension at a bath temperature of 36-37°C (45±4 μm, n=5) and contracted in a concentration-dependent manner with acetylcholine. However, acetylcholine did not significantly dilate the retinal arterioles (resting diameter 43±3 μm, n=5). Similarly, as shown in Figure 13B, thrombin, using the same preparation as described in Figure 13A, induced dilation of the coronary arterioles (resting diameter 47±4 μm, n=5), but also induced constriction of the retinal arterioles (resting diameter 44±3 μm, n=5).

[0279] Coronary arterioles and retinal arterioles also exhibit different signaling pathways for vasomotor regulation. For example, PKC activation-induced coronary arteriole constriction is independent of Rho kinase signaling (i.e., insensitive to H-1152), while the latter plays a crucial role in mediating PKC-induced retinal arteriole constriction (i.e., sensitive to H-1152). In addition, while bradykinin-induced coronary arteriole dilation is partially mediated via endothelial hyperpolarizing factors, bradykinin-induced retinal arteriole dilation is entirely dependent on endothelial NO release. Porcine coronary arterioles express arginase-I for vasomotor regulation, while retinal arterioles primarily express arginase-II isoforms that contribute differently to the development of vascular dysfunction.

[0280] Under experimental pathological conditions, we found that 90 minutes of ischemia impairs retinal arteriole dilation in response to endothelium-dependent agonists; however, this duration of ischemia is not sufficient to induce coronary arteriole dysfunction without subsequent reperfusion. We found that activation of pro-inflammatory c-Jun N-terminal kinase (JNK) signaling may cause vasoconstrictive abnormalities in the porcine retinal microvascular system. However, in porcine coronary arterioles, JNK signaling does not contribute to vascular damage. Despite the fact that the primary functions of blood flow regulation in the microcirculation are the same across different tissues, the retinal and coronary microvascular systems appear to exhibit different sensitivities and signaling pathways to vasomotor regulation and vasomotor dysfunction. Since multiple virulence factors in addition to ET-1 can activate ROCK, the application of the ROCK inhibitor H-1152 would likely be beneficial to blood flow and tissue survival overall.

[0281] It should be noted that our findings do not rule out the possibility of activation of the same pathway in different tissues / organs for several diseases. On the other hand, our findings suggest that systemic interventions are not always beneficial or effective in treating tissue or organ-specific microvascular diseases. Instead, target (or tissue)-selective approaches should be considered due to the heterogeneity of vasomotor dysregulation / dysregulation and the possibility of involvement of different signaling mechanisms in specific organs / tissues. Here, we demonstrate a novel signaling mechanism for coronary artery constriction to ET-1 and the key role of ROCK activation in mediating this vasoconstriction (or spasm). Our data suggest a unique advantage of using H-1152 in neutralizing adverse effects, specifically in microvascular disease-related ROCK activation in general. Since ET-1 is one of the major virulence factors and its biological action is mediated by ROCK, the use of H-1152 is desirable in this respect. This concept may be applicable to other tissues / organs such as the retina and other microvascular beds.

[0282] Mechanism of coronary artery constriction induced by ET-1 and PKC activator PDBu Studies of conduit arteries demonstrate that ET-1-induced vasoconstriction is mediated by the activation of PKC signaling. Since ET-1 and PKC activation may contribute to the pathogenesis of many coronary artery diseases, elucidating their molecular signaling underlying the vasoconstriction mechanism in coronary arterioles, where myocardial blood flow is predominantly regulated, is beneficial. Based on the data presented in this example, a model diagram illustrating the intracellular mechanism of ET-1 and PKC activator PDBu-induced coronary arteriole constriction can be generated. This model diagram is shown in Figure 14.

[0283] ET-1 (0.1 nmol / L) is ET A It binds to the receptor and is activated, then binds to phospholipase C(PLC), and presumably T-type voltage-gated Ca 2+ Channel (T-VGCC) and / or Ca 2+ Extracellular calcium (Ca) is released via permeable nonselective cation channels (NSCCs). 2+ ) induces influx. Cytoplasmic Ca 2+ The increase in RhoA stimulates the active GTP-bound form, activating ROCK. 37 Activated ROCK phosphorylates the regulatory subunit of myosin phosphatase target subunit 1 (MYPT1) of myosin light chain phosphatase (MLCP), resulting in inactivation of phosphatase activity. Vasoconstriction occurs after inactivation of the 20 kDa regulatory myosin light chain (MLC 20 This occurs when phosphorylation (P) of ) increases. The expression / maintenance of either resting basal tension or vasoconstriction induced by ET-1 initiates ROCK-dependent regulation of extracellular Ca to initiate MLC phosphorylation. 2+Influx is required. While the direct involvement of ROCK in MLC phosphorylation cannot be ruled out, our study demonstrates an essential role of ROCK in mediating coronary artery constriction in response to ET-1. Under physiological and / or pathophysiological conditions, hyperpharmacological concentrations of ET-1 (10 nmol / L), not observed in vivo, lead to internal storage, i.e., IP3 and / or store-operated Ca 2+ Ca from the sarcoplasmic reticulum (SR) via channels 2+ It is noteworthy that this induces release. 2+ The increase is likely to trigger other biological events in addition to vasomotor activation. Resting microvascular tone is likely to trigger extracellular Ca 2+ Influx and myosin light chain kinase (MLCK) 20 Ca 2+ It should be noted that ROCK activity is maintained by calmodulin (CaM)-dependent phosphorylation. 20 By regulating phosphorylation, it contributes to maintaining resting vascular tone. Therefore, both MLCK inhibitors (ML-9) and ROCK inhibitors (H-1152) eliminate resting vascular tone. Activation of PKC signaling by PDBu (0.1 μmol / L) leads to L-type voltage-gated Ca 2+ By opening the channel (L-VGCC), extracellular Ca 2+ It induces inflow, and subsequent CaM / MLCK axis activation contributes to 50% of sustained vasoconstriction. However, ML-9 cannot prevent the contractile response to PDBu, so Ca 2+ The / calmodulin / MLCK axis does not appear to be involved in the initiation of PKC-mediated vasoconstriction. This is because MLC is involved in a PKC-induced chain reaction. 20This suggests the possibility of direct phosphorylation / activation. While activated PKC has been shown to phosphorylate MYPT1, promoting vasoconstriction in ductile arteries (dashed line in the figure), pMYPT1 levels are not increased by PDBu in coronary arterioles, indicating that vasoconstriction in response to PKC activation is independent of MLCP inhibition. Phosphorylation of CPI-17 by either PKC or ROCK (brown octagon in the figure) also causes vasoconstriction by inactivating MLCPs. However, despite its abundance in ductile arteries, CPI-17 is barely detectable in coronary arterioles, suggesting a limited role in regulating microvasoconstriction by PKC or ROCK activation. Since coronary arteriole constriction in response to PKC activation is insensitive to H-1152, this vasomotor activity appears independent of ROCK. Therefore, ET-1 and PKC-induced coronary arteriole constriction are mediated by different signaling pathways. The series of events that cause ET-1-induced vasoconstriction are highlighted with thick lines in the schematic diagram, and the inhibitors used to probe the pathways involved are also shown.

[0284] Example 2: Rho kinase (ROCK) inhibition in combination with cancer therapy, immunosuppressants, and substance abuse (e.g., drug or alcohol abuse) In this embodiment, we extend the above findings to cancer therapy, as solid evidence has been gathered for several chemotherapy drugs that cause coronary microvascular complications. New evidence is emerging that many anticancer therapies (e.g., antibodies, kinase inhibitors) cause systemic hypertension and contribute to adverse coronary microvascular dysfunction. Such conditions lead to a new subspecialty called cardio-oncology or cardiovascular oncology. However, due to insufficient research in the study of microvasculars, which are the origin of this clinical problem, there are no specific drugs to address these issues. Hereinafter, we propose anticancer drugs (doxorubicin, 5-FU, bevacizumab, and tyrosine kinase inhibitors, e.g., dasatinib, nilotinib) that have well-established side effects including hypertension, microvascular disease, and / or cardiotoxicity.

[0285] In this embodiment, the findings above are extended to immunosuppressive therapies requiring tacrolimus, cyclosporine A, or other drugs in the same category, as clinical evidence suggests that tacrolimus, cyclosporine A, or other drugs in the same category contribute to coronary microvascular dysfunction and vascular disease. A challenge for heart transplant recipients is that they cannot perceive chest pain because the nerve supply from the donated heart is not connected to the recipient's nervous system.

[0286] There is evidence suggesting that coronary adverse events from drugs such as doxorubicin or tacrolimus are associated with elevated ET-1 levels. In addition, drug-induced arteriosclerosis with underlying causes of ET-1 expression abnormalities has been reported, although these drugs have not been systemically investigated in experiments using coronary arteries. We propose that several causative drugs directly or indirectly activate ROCK signaling (e.g., by increasing ET-1 expression), and that H-1152 can effectively reverse the adverse effects of anticancer drugs. Therefore, H-1152 may be the first drug that can not only treat hypertension and tissue ischemia but also improve quality of life and reduce the likelihood of death from cardiovascular side effects caused by anticancer therapy. When co-administered with anticancer therapy, H-1152 may also enhance the effectiveness of chemotherapy and radiotherapy, which are less effective under hypoxic conditions.

[0287] In this embodiment, the above findings are extended to substance abuse associated with coronary artery spasm and / or microvascular dysfunction (e.g., cocaine, nicotine, and alcohol).

[0288] Background technology Microvascular dysfunction contributes to countless cardiovascular diseases associated with diabetes, hypertension, hyperlipidemia, obesity, hyperglycemia, and many other medical complications. Despite its high prevalence and known association with adverse clinical outcomes, effective medical care remains unavailable due to a limited understanding of its pathophysiology. Microvascular diseases are associated with impaired blood flow regulation and can ultimately lead to irreversible tissue damage and organ failure. Due to the technical challenges in studying microvessels—e.g., the complexity of the network for visualization / imaging / isolation, and the limited availability of tissue for molecular characterization—advancements in microvascular research have been either linear or stagnant over the past decade. Consequently, knowledge regarding the mechanisms of microvascular dysfunction has been primarily inferred from studies in large conduit vessels or cultured vessels / non-vascular cells.

[0289] Using methods we developed to study microvascular function, we identified that overproduction of endothelin-1 (ET-1), the most potent vasoconstrictor known in the human cardiovascular system with pro-inflammatory activity, contributes to microvascular dysfunction in affected hearts and retinas due to Rho kinase (ROCK) activation. Furthermore, the ROCK inhibitor H-1152 not only prevents but reverses ET-1 vasoconstriction, consequently restoring microvascular function. In our experience, H-1152 exhibits the best efficacy / specificity for reversing the action of ET-1, and we have completed a proof-of-concept study at the translational level in an animal model of cardiovascular disease.

[0290] In this embodiment, we propose expanding the use of H-1152 to treat cancer therapy-related CVD, immunosuppressive therapy-related CVD, and substance abuse-related CVD that have not been investigated in the microcirculation using isolated and pressurized arteries. Certain anticancer drugs with known cardiovascular side effects exhibit vasoconstrictive activity, which is thought to be induced directly by ROCK activation or indirectly by ET-1 overproduction. There is also evidence suggesting that cocaine induces ET-1 expression from vascular endothelial cells, which may lead to underlying smooth muscle cell contraction. Similarly, impaired microvascular endothelial function among heart transplant recipients has been associated with elevated coronary ET-1 concentrations during follow-up. We propose that these adverse effects can be reversed by H-1152. For example, H-1152 can be applied in conjunction with cancer therapy to enhance the responsiveness of chemotherapy and radiotherapy, which are less effective under tissue hypoxia / ischemia, as well as mitigating hypertension and cardiovascular complications, and thus improving flow / oxygen delivery. When co-administered with narcotics, H-1152 may alleviate tissue ischemia. When co-administered with immunosuppressants, H-1152 may reduce or delay the progression of coronary microvascular dysfunction / disease.

[0291] Summary of proposed treatment methods Microvascular dysfunction presents with countless clinical manifestations, ranging from ischemic heart disease to diabetic complications, nephropathy, systemic / pulmonary hypertension, stroke, blindness, pre-eclampsia, and dementia with cognitive impairment. However, there are no approved drugs specifically for the treatment of microvascular disease.

[0292] A key factor hindering the identification of effective drugs is the lack of a mechanistic approach to understanding microvascular dysfunction and targeting the underlying pathophysiology. The isolation and pressurized microvascular techniques described above have enabled us to address microvascular diseases mechanistically. We demonstrated that microvascular arterioles exhibit distinct physiological and pathophysiological characteristics compared to upstream conduits, and that ET-1 overproduction contributes to microvascular dysfunction in porcine models with cardiac and retinal diseases. ET-1-induced pathophysiology (i.e., excessive arterial constriction) is mediated by ROCK activation in the microcirculation, which can be functionally restored by its specific inhibitor, H-1152. Therefore, H-1152 may be the first effective agent to address the unmet need in the treatment of microvascular diseases.

[0293] A recent review of a comprehensive meta-analysis of international clinical trials in chemotherapy involving 6,241 patients showed that cancer treatment-induced hypertension is the most frequent case of cardiovascular toxicity, particularly in combinations of anticancer drug therapies. Hypertension is recognized as the most common comorbidity among various types of cancer, and this is one of the high-risk factors for cancer survivors with comorbid cardiovascular disease. Furthermore, anticancer drugs including doxorubicin, 5-FU, bevacizumab, dasatinib, and nilotinib, and immunosuppressants including tacrolimus, may promote coronary artery disease with microvascular dysfunction. Potential mechanisms such as vascular dilution, endothelial dysfunction, impaired vasodilation, oxidative stress, and enhanced vasoconstrictive effects have been suggested for anticancer therapy-associated hypertension and microvascular dysfunction. Animal data specifically suggest that angiogenesis inhibitors, such as tyrosine kinase inhibitors, which block access to vascular endothelial growth factor 2 (VEGFR2) signaling, and antibody drugs, which directly bind to vascular endothelial growth factor, play a role in mitigating vasodilator nitric oxide and vascular dilution in hypertension. However, our understanding of the mechanisms of hypertension associated with anticancer therapy in humans is very limited, and treatments are largely empirical rather than based on solid scientific evidence.

[0294] Currently, due to the lack of available or sufficient clinical trial data to define specific guidelines for antihypertensive therapy in cancer patients, a pragmatic approach is generally employed. Drugs used to treat chemotherapy-associated hypertension include angiotensin-converting enzyme and angiotensin II receptor blockers, calcium channel blockers, and other antihypertensives not directly related to the pathogenesis of anticancer drug-associated hypertension and microangiopathy. Furthermore, these antihypertensives are intended for the management of chronic hypertension, and the evidence supporting their use for the management of cancer-induced hypertension remains insufficient. It should be noted that a variety of complications are well recognized, including organ damage, hypotension, angioedema, hyperkalemia, diarrhea, hyperglycemia, renal injury, dizziness, urinary urgency, fatigue, weight gain, bradycardia, and several others associated with the use of antihypertensive drugs. Therefore, the undesirable side effects, inconsistent efficacy, and empirical use of these conventional antihypertensives in combination with anticancer therapy remain a concern. It is noteworthy that we have reported the adverse effects of ET-1 on endothelial function, vasodilation, oxidative stress, and vasoconstriction, all of which may contribute to the development of hypertension and tissue ischemia. Interestingly, tissue ischemia / hypoxia, a common phenomenon in most malignancies, can lead to resistance to chemotherapy and radiotherapy by inducing angiogenesis, impaired vascularization, increased cell migration / metastasis, and cell quiescence, as well as reduced chemosensibility. Therefore, we propose that hypertension and vascular complications induced by several common anticancer drugs (e.g., doxorubicin, 5-FU, bevacizumab, nilotinib), immunosuppressants (e.g., tacrolimus, cyclosporine A), and substance abuse (e.g., cocaine) are mediated by ET-1 via microvascular ROCK activation or directly activate ROCK signaling.

[0295] We observed the vasoconstrictive effects of dasatinib in three pilot studies (two in porcine retinal arterioles and one in mouse ophthalmic arteries). Dasatinib, along with nilotinib and bosutinib, is one of the second-generation tyrosine kinase inhibitors (TKIs) used as a frontline therapy for chronic myeloid leukemia. While these newer TKIs show excellent clinical outcomes, they also present increased vascular events and toxicity. For example, growing clinical evidence suggests that dasatinib can cause drug-induced PAH. At its effective clinical concentrations, we found that dasatinib induced a 15–20% reduction in arterial constriction, estimated based on the relationship between diameter and flow, resulting in a more than 50% reduction in blood flow. This is consistent with the hypothesis that observed cardiovascular complications of some anticancer drugs may be due to arterial constriction.

[0296] In this specification, we also propose that the ROCK inhibitor H-1152 can effectively reverse the vasoconstrictive effects of anticancer drugs, thereby improving the effectiveness of anticancer therapy. This H-1152 technology can expand the therapeutic range of microvascular diseases to include anticancer therapy.

[0297] For initial studies, four different anticancer agents were selected—doxorubicin as an anthracycline antibiotic / topoisomerase inhibitor, 5-FU as a thymidylate synthase inhibitor, bevacizumab as a VEGF / angiogenesis inhibitor, and dasatinib as a TKI—to represent four different categories of anticancer mechanisms. These examples cover a broad range of anticancer therapeutics. One immunosuppressant, tacrolimus, and the addictive substance cocaine or its metabolites will also be studied in vitro for their vasoconstrictive effects.

[0298] We investigate the vasomotor activity of coronary and retinal arterioles isolated from pigs because this large animal model is similar to humans in terms of cardiovascular physiology / pathophysiology and is applicable to translational medicine. It is noteworthy that the heart is a major organ affected by hypertension accompanied by the development of coronary microvascular dysfunction due to anticancer drugs. Furthermore, changes in the retinal microvascular system in healthy individuals are independently associated with a future risk of type 2 diabetes, congestive heart failure, and cardiovascular mortality. Notably, the development of retinal microvascular dysfunction precedes the clinical development of diabetic retinopathy in type 1 and type 2 diabetes. Therefore, studying the vasomotor activity of arterioles in these two organ systems has important clinical value not only for understanding CVD development but also for understanding treatment.

[0299] Coronary and retinal arterioles with diameters of 40–80 μm were isolated, pressurized in a dual reservoir cacanula insertion system, and studied using video microscopy techniques as described above. It should be noted that the microvessels studied in our laboratory exhibited spontaneous basal tension and presented vasomotor activity similar to that seen in vivo. Therefore, the collected data reflect microvascular behavior in intact microcirculation. We evaluated both the effects of these chemotherapeutic agents on the microvascular system and the ability of ROCK inhibitors such as H-1152 to reverse any negative effects on the microvascular system.

[0300] Figure 15 is a plot showing that dasatinib, an anti-cancer tyrosine kinase inhibitor, induces constriction of small ocular resistance arteries, and this response can be reversed by H-1152. Briefly, small ocular resistance arteries were isolated from mice for vasomotor studies using video microscopy techniques. The vessels were pressurized to 75 cmH2O (55 mmHg) using a dual reservoir system to induce basal tension (resting diameter <100 μm). Dasatinib, an anti-cancer tyrosine kinase inhibitor, was added to the saline surrounding the vessels. Dasatinib (5 μM) induced significant vasoconstriction (i.e., a decrease in resting diameter), which was reversed by subsequent administration of the Rho kinase inhibitor H-1152 (3 μM). 2+ The maximum diameter was obtained by replacing the solution with free physiological saline.

[0301] The compounds, compositions, and methods of the appended claims are not limited to the specific compounds, compositions, and methods described herein, but are intended to illustrate some aspects of the claims. Any functionally equivalent compounds, compositions, and methods are intended to be included within the claims. Various modifications of compounds, compositions, and methods, in addition to those shown and described herein, are intended to be included within the appended claims. Furthermore, while only certain representative compounds, components, compositions, and method steps are specifically described herein, other combinations of those compounds, components, compositions, and method steps, even if not specifically listed, are intended to be included within the appended claims. Thus, combinations of steps, elements, components, or constituents are included, whether explicitly mentioned or less explicitly mentioned herein, and even if not explicitly described.

[0302] As used herein, the term “comprising” and its variations are used synonymously with the term “including” and its variations, and are open and non-restrictive terms. While the terms “comprising” and “including” are used herein to describe various embodiments, the terms “essentially consisting of” and “consisting of” may be used instead of “comprising” and “including,” and are also disclosed, to provide more specific embodiments of the invention. Unless otherwise noted, all figures used herein and in the claims to represent geometry, dimensions, etc., should be understood as at least and not as an attempt to limit the application of the doctrine of equivalents to the claims, and should be interpreted taking into account the number of significant figures and common rounding methods.

[0303] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field to which the disclosed invention pertains. Publications cited herein and materials from which they are cited are incorporated herein by reference.

Claims

1. A method for treating or preventing microvascular dysfunction in a subject in need thereof, comprising administering to the subject an effective amount of a Rho kinase (ROCK) inhibitor.

2. The method according to claim 1, wherein the microvascular dysfunction is associated with elevated endothelin-1 (ET-1) levels.

3. A method for inducing microvasodilation in a subject, comprising administering an effective amount of a Rho kinase (ROCK) inhibitor to the subject.

4. A method for reversing endothelin-1-induced vasoconstriction in a subject requiring such reversal, comprising administering an effective amount of a Rho kinase (ROCK) inhibitor to the subject.

5. A method for treating or preventing a disease, disorder, or condition associated with microvascular dysfunction of a subject, comprising administering an effective amount of a Rho kinase (ROCK) inhibitor to the subject.

6. The method according to claim 5, wherein the disease, disorder, or condition is associated with elevated endothelin-1 (ET-1) levels.

7. The method according to claim 5 or 6, wherein the disease, disorder, or condition is unable to respond to a calcium channel blocker.

8. The method according to any one of claims 5 to 7, wherein the disease, disorder, or condition is unable to respond to vasodilators such as calcium channel blockers or nitrates.

9. The method according to any one of claims 5 to 8, wherein the disease, disorder, or condition is unable to respond to statins.

10. The method according to any one of claims 5 to 9, wherein the disease, disorder, or condition is not responsive to an anti-anginal drug such as lanolazine.

11. The method according to any one of claims 5 to 10, wherein the disease, disorder, or condition includes microvascular angina (also known as cardiac syndrome X, CSX).

12. The method according to any one of claims 5 to 11, wherein the disease, disorder, or condition includes coronary microvascular dysfunction / disease (CMD) as a standalone diagnosis or from other conditions such as systemic lupus erythematosus (SLE) or substance abuse.

13. The method according to any one of claims 5 to 12, wherein the disease, disorder, or condition includes drug-resistant hypertension.

14. The method according to any one of claims 5 to 13, wherein the disease, disorder, or condition includes drug-induced hypertension such as hypertension induced by the administration of an anticancer drug.

15. The method according to any one of claims 5 to 14, wherein the disease, disorder, or condition includes coronary artery spasm, percutaneous coronary intervention (PCI)-related refractory myocardial ischemia, cerebral vasospasm after subarachnoid hemorrhage, drug-resistant systemic hypertension, drug-induced hypertension (e.g., bevacizumab-induced hypertension), drug-induced cardiotoxicity (e.g., cardiotoxicity induced by chemotherapeutic agents such as 5-fluorouracil or immunosuppressants such as cyclosporine A or tacrolimus), pulmonary arterial hypertension, substance abuse (e.g., cocaine)-related myocardial infarction, diabetes-induced microangiopathy, microangiopathy, asymptomatic stroke, or any combination thereof.

16. The method according to any one claim, wherein the disease, disorder, or condition includes coronary microvascular dysfunction or vascular disease caused by an immunosuppressant such as tacrolimus or cyclosporine A.

17. The method according to claim 16, wherein the subject includes an organ transplant recipient.

18. The method according to claim 17, wherein the subject includes a heart transplant recipient.

19. The method according to claim 17, wherein the subject includes recipients of organ transplants other than heart transplants.

20. The method according to any one of claims 1 to 19, wherein the ROCK inhibitor selectively inhibits ROCK2 activity rather than ROCK1 activity.

21. The method according to any one of claims 1 to 19, wherein the ROCK inhibitor selectively inhibits ROCK1 activity rather than ROCK2 activity.

22. The ROCK inhibitor is of formula I, 【Chemistry 1】 During the ceremony, L is O or SO ２ And, R １ However, H, hydroxy, NH ２ , NHR', or NR'R', R ２ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, or (C １ -C ６ ) is an alkoxy, R ３ is H, halogen, hydroxy, (C １ -C ６ -C １ -C ６ ), alkoxy, NH ２ 2, NHR', NR'R', or CN, and R ４ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ５ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ６ However, H, (C １ -C ８ ) alkyl, (C １ -C ６ ) Alkylene-R'', (C １ -C ６ ) Alkylene-C(O)-R', (C １ -C ６ ) Alkylene-C(O)O-R', (C １ -C ６ ) Alkylene-C(O)NH ２ , (C １ -C ６ ) Alkylene-C(O)NHR', (C １ -C ６ ) Alkylene-C(O)NR'R', C(O)OR', C(O)R', C(O)NH ２ , C(O)NHR', C(O)NR'R', C(O)-(C １ -C ６ ) Alkilen-NH ２ , C(O)-(C １ -C ６ ) Alkylene-NHR', C(O)-(C １ -C ６ ) Alkylene-NR'R', C(O)O-(C １ -C ６ ) Alkilen-NH ２ , C(O)O-(C １ -C ６ ) alkylene-NHR', or C(O)O-(C １ -C ６ ) Alkilen-NR'R', R ７ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ８ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, n is 1, 2, 3, or 4, m is independently 1, 2, 3, or 4 each time it appears. R' is (C １ -C ６ ) alkyl, (C ３ -C ８ ) Cycloalkyl, (C ５ -C １０ ) Heterocyclyl, or (C ６ -C １０ ) is an aryl, R'' is (C ３ -C ８ ) Cycloalkyl, (C ５ -C １０ ) Heterocyclyl, or (C ６ -C １０ ) It is an aryl, defined by formula I, The method according to any one of claims 1 to 21, or the stereoisomers thereof and / or the tautomers thereof and / or pharmaceutically acceptable salts thereof.

23. The method according to any one of claims 1 to 22, wherein the ROCK inhibitor comprises isoquinoline sulfonamide.

24. The ROCK inhibitor is of formula IA, 【Chemistry 2】 During the ceremony, R １ However, H, hydroxy, NH ２ , NHR', or NR'R', R ２ is H, halogen, hydroxy, (C １ -C ６ -alkyl, or (C １ -C ６ -alkoxy, and R ４ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ６ is H, (C １ -C ８ ), alkyl, (C １ -C ６ ), alkylene-R'', (C １ -C ６ ), alkylene-C(O)-R', (C １ -C ６ ), alkylene-C(O)O-R', (C １ -C ６ ), alkylene-C(O)NH ２ , (C １ -C ６ ), alkylene-C(O)NHR', (C １ -C ６ ), alkylene-C(O)NR'R', C(O)OR', C(O)R', C(O)NH ２ , C(O)NHR', C(O)NR'R', C(O)-(C １ -C ６ ), alkylene-NH ２ , C(O)-(C １ -C ６ ), alkylene-NHR', C(O)-(C １ -C ６ ), alkylene-NR'R', C(O)O-(C １ -C ６ ), alkylene-NH ２ , C(O)O-(C １ -C ６ ), alkylene-NHR', or C(O)O-(C １ -C ６ ), alkylene-NR'R', and R' is (C １ -C ６ ) alkyl, (C ３ -C ８ ) Cycloalkyl, (C ５ -C １０ ) Heterocyclyl, or (C ６ -C １０ ) is an aryl, defined by formula IA, The method according to any one of claims 1 to 23, or the stereoisomers thereof and / or the tautomers thereof and / or pharmaceutically acceptable salts thereof.

25. The method according to any one of claims 1 to 24, wherein the ROCK inhibitor comprises fasudil (HA-1077).

26. The method according to any one of claims 1 to 24, wherein the ROCK inhibitor comprises glycyl-H 1152.

27. The method according to any one of claims 1 to 24, wherein the ROCK inhibitor comprises H-1152.

28. The method according to any one of claims 1 to 24, wherein the ROCK inhibitor comprises lipasudil.

29. The method according to any one of claims 1 to 24, wherein the ROCK inhibitor is selected from the group consisting of fasudil, hydroxyfasudil, dimethylfasudil, H-1152, H-1152P, H-1152 dihydrochloride, ripasudil, netalusdil, belmosdil, SAR407899, their prodrugs, their salts, or combinations thereof.

30. The method according to any one of claims 1 to 21, wherein the ROCK inhibitor is selected from the group consisting of AR-12286, AT-13148, BA-1049(R), FCN-016, GSK429286A, PHP-201, RKI-1447, Y-27632, Y-30141, Y-32885, Y-39983, their prodrugs, their salts, or combinations thereof.

31. The method according to any one of claims 1 to 30, further comprising measuring the circulating endothelin-1 (ET-1) level of the subject to determine an appropriate drug administration plan for the ROCK inhibitor.

32. The method according to any one of claims 1 to 31, wherein the subject exhibits an increase in circulating endothelin-1 (ET-1) levels.

33. A method for treating cancer in a person in need of such treatment, comprising administering an effective dose of cancer therapy to the person in combination with an effective dose of a ROCK inhibitor.

34. A method for improving the effectiveness of cancer therapy, comprising co-administering an effective amount of a ROCK inhibitor in combination with the cancer therapy.

35. The method according to claim 33 or 34, wherein administering the effective amount of the ROCK inhibitor in combination with the cancer therapy includes administering the ROCK inhibitor simultaneously with the cancer therapy.

36. The method according to claim 33 or 34, wherein administering the effective amount of the ROCK inhibitor in combination with the cancer therapy includes administering the ROCK inhibitor after the administration of the cancer therapy.

37. The method according to claim 33 or 34, wherein administering the effective amount of the ROCK inhibitor in combination with the cancer therapy includes administering the ROCK inhibitor before administering the cancer therapy.

38. The method according to any one of claims 33 to 37, wherein the effective amount of ROCK inhibitor comprises an effective amount for treating or preventing a cardiovascular disease caused by the cancer therapy, such as drug-induced hypertension or drug-induced cardiotoxicity.

39. The method according to any one of claims 33 to 38, wherein the cancer therapy includes the administration of a chemotherapeutic agent.

40. The method according to claim 39, wherein the chemotherapeutic agent comprises a DNA alkylating agent, an antitumor antibiotic, an antimetabolite, a tubulin stabilizer, a tubulin destabilizer, a hormone antagonist, a topoisomerase inhibitor, a protein kinase inhibitor, an HMG-CoA inhibitor, a CDK inhibitor, a cyclin inhibitor, a caspase inhibitor, a proteasome inhibitor, a metalloproteinase inhibitor, an antisense nucleic acid, a triple helix DNA, a nucleic acid aptamer, a molecularly modified viral agent, a bacterial agent, or an exotoxic agent, or any combination thereof.

41. The aforementioned chemotherapeutic agents include cytidine arabinoside, cytarabine, methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin), cisplatin (CDDP), dexamethasone, algrabine, cyclophosphamide, sarcoridine, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, and topote. The method according to claim 39 or 40, comprising can, leucovorin, carmustine, streptozosin, taxol and its derivatives, tamoxifen, dacarbazine, rituximab, daunorubicin, 1-β-D-arabinofuranosilcytosine, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, fludarabine, docetaxel, bevacizumab, trastuzumab, FOLFOX4, bortezomib, carfilzomib, ixazomib, or any combination thereof.

42. The method according to any one of claims 33 to 41, wherein the cancer therapy includes the implementation of radiotherapy.

43. A method for treating or preventing cancer treatment-induced hypertension in a subject undergoing cancer treatment, comprising administering an effective amount of a Rho kinase (ROCK) inhibitor to the subject.

44. A method for treating or preventing a substance abuse-related cardiovascular disease, comprising administering an effective amount of a ROCK inhibitor to the subject.

45. The method according to claim 44, wherein the substance abuse-related cardiovascular disease includes cocaine-induced cardiotoxicity.

46. The method according to claim 44 or 45, wherein the substance abuse-related cardiovascular disease includes cocaine-induced myocardial infarction.

47. The method according to any one of claims 33 to 46, wherein the ROCK inhibitor selectively inhibits ROCK2 activity rather than ROCK1 activity.

48. The method according to any one of claims 33 to 46, wherein the ROCK inhibitor selectively inhibits ROCK1 activity rather than ROCK2 activity.

49. The ROCK inhibitor is of formula I, 【Transformation 3】 During the ceremony, L is O or SO ２ And, R １ However, H, hydroxy, NH ２ , NHR', or NR'R', R ２ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, or (C １ -C ６ ) is an alkoxy, R ３ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ４ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ５ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ６ However, H, (C １ -C ８ ) alkyl, (C １ -C ６ ) Alkylene-R'', (C １ -C ６ ) Alkylene-C(O)-R', (C １ -C ６ ) Alkylene-C(O)O-R', (C １ -C ６ ) Alkylene-C(O)NH ２ , (C １ -C ６ ) Alkylene-C(O)NHR', (C １ -C ６ ) Alkylene-C(O)NR'R', C(O)OR', C(O)R', C(O)NH ２ , C(O)NHR', C(O)NR'R', C(O)-(C １ -C ６ ) Alkilen-NH ２ , C(O)-(C １ -C ６ ) Alkylene-NHR', C(O)-(C １ -C ６ ) Alkylene-NR'R', C(O)O-(C １ -C ６ ) Alkilen-NH ２ , C(O)O-(C １ -C ６ ) alkylene-NHR', or C(O)O-(C １ -C ６ ) Alkilen-NR'R', R ７ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ８ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, n is 1, 2, 3, or 4, m is independently 1, 2, 3, or 4 each time it appears. R' is (C １ -C ６ ) alkyl, (C ３ -C ８ ) Cycloalkyl, (C ５ -C １０ ) Heterocyclyl, or (C ６ -C １０ ) is an aryl, R'' is (C ３ -C ８ ) Cycloalkyl, (C ５ -C １０ ) Heterocyclyl, or (C ６ -C １０ ) It is an aryl, defined by formula I, The method according to any one of claims 33 to 48, or the stereoisomer forms thereof and / or the tautomer forms thereof and / or pharmaceutically acceptable salts thereof.

50. The method according to any one of claims 33 to 49, wherein the ROCK inhibitor comprises isoquinoline sulfonamide.

51. The ROCK inhibitor is of formula IA, 【Chemistry 4】 During the ceremony, R １ However, H, hydroxy, NH ２ , NHR', or NR'R', R ２ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, or (C １ -C ６ ) is an alkoxy, R ４ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ６ However, H, (C １ -C ８ ) alkyl, (C １ -C ６ ) Alkylene-R'', (C １ -C ６ ) Alkylene-C(O)-R', (C １ -C ６ ) Alkylene-C(O)O-R', (C １ -C ６ ) Alkylene-C(O)NH ２ , (C １ -C ６ ) Alkylene-C(O)NHR', (C １ -C ６ ) Alkylene-C(O)NR'R', C(O)OR', C(O)R', C(O)NH ２ , C(O)NHR', C(O)NR'R', C(O)-(C １ -C ６ ) Alkilen-NH ２ , C(O)-(C １ -C ６ ) Alkylene-NHR', C(O)-(C １ -C ６ ) Alkylene-NR'R', C(O)O-(C １ -C ６ ) Alkilen-NH ２ , C(O)O-(C １ -C ６ ) alkylene-NHR', or C(O)O-(C １ -C ６ ) Alkilen-NR'R', R' is (C １ -C ６ ) alkyl, (C ３ -C ８ ) Cycloalkyl, (C ５ -C １０ ) Heterocyclyl, or (C ６ -C １０ ) is an aryl, defined by formula IA, The method according to any one of claims 33 to 50, or the stereoisomer forms thereof and / or the tautomer forms thereof and / or pharmaceutically acceptable salts thereof.

52. The method according to any one of claims 33 to 51, wherein the ROCK inhibitor comprises fasudil (HA-1077).

53. The method according to any one of claims 33 to 51, wherein the ROCK inhibitor comprises glycyl-H 1152.

54. The method according to any one of claims 33 to 51, wherein the ROCK inhibitor comprises H-1152.

55. The method according to any one of claims 33 to 51, wherein the ROCK inhibitor comprises lipasudil.

56. The method according to any one of claims 33 to 51, wherein the ROCK inhibitor is selected from the group consisting of fasudil, hydroxyfasudil, dimethylfasudil, H-1152, H-1152P, H-1152 dihydrochloride, ripasudil, netalusdil, belmosdil, SAR407899, their prodrugs, their salts, or combinations thereof.

57. The method according to any one of claims 33 to 48, wherein the ROCK inhibitor is selected from the group consisting of AR-12286, AT-13148, BA-1049(R), FCN-016, GSK429286A, PHP-201, RKI-1447, Y-27632, Y-30141, Y-32885, Y-39983, their prodrugs, their salts, or combinations thereof.

58. A method for evaluating the microvascular function of a target, To measure the coronary blood flow velocity of the subject before and after administration of ROCK inhibitors, This includes determining the coronary blood flow reserve of the subject before and after administration of the ROCK inhibitor, and calculating the change in the coronary blood flow reserve of the subject as evaluated by the administration of the ROCK inhibitor. A method in which a change in the coronary blood flow reserve of the subject, as evaluated by the administration of the ROCK inhibitor, that falls below expectations indicates that the subject exhibits coronary microvascular dysfunction.

59. The method according to claim 58, wherein the coronary blood flow velocity of the subject is invasively measured, for example, during coronary angiography or angioplasty.

60. The method according to claim 58, wherein the coronary blood flow velocity of the subject is measured non-invasively, for example, using MRI or PET.

61. The method according to any one of claims 58 to 60, wherein the coronary blood flow reserve of the subject is the ratio of the peak diastolic blood flow velocity to the resting peak diastolic blood flow velocity.

62. The method according to any one of claims 58 to 61, wherein a change in CFR at a fixed intracoronal dose of less than approximately 2.5, such as a change in CFR at a fixed intracoronal dose of less than approximately 2.3 or a change in CFR at a fixed intracoronal dose of less than approximately 2, indicates that the subject exhibits coronary microvascular dysfunction.

63. The method according to any one of claims 58 to 62, further comprising administering an effective amount of the ROCK inhibitor to the subject to achieve better blood flow if the subject exhibits a change in coronary blood flow reserve induced by the administration of the ROCK inhibitor that falls below expectations.

64. The method according to claim 63, further comprising selecting the patient for prescription of a ROCK inhibitor to treat underlying coronary microvascular dysfunction.

65. The method according to claim 63, further comprising selecting an alternative therapy to treat the subject if the effective amount of ROCK inhibitor fails to improve the CFR or results in only slight improvement to the CFR.

66. The method according to any one of claims 58 to 61, wherein a change in CFR at a fixed intracoronal dose greater than approximately 2.5, such as a change in CFR at a fixed intracoronal dose greater than approximately 2.7 or a change in CFR at a fixed intracoronal dose greater than approximately 3, indicates that the subject does not exhibit coronary microvascular dysfunction.

67. The method according to any one of claims 58 to 66, wherein the method is also applicable to cerebral or peripheral angiography when a ROCK inhibitor is used to calculate blood flow reserve or microcirculatory resistance index (IMR).

68. The method according to any one of claims 58 to 66, wherein the method is also applicable to a non-invasive diagnostic method (e.g., CT, ultrasound, MRI, PET, etc.) based on changes in blood supply before and after administration of a ROCK inhibitor.

69. The method according to any one of claims 58 to 68, wherein the ROCK inhibitor selectively inhibits ROCK2 activity rather than ROCK1 activity.

70. The method according to any one of claims 58 to 68, wherein the ROCK inhibitor selectively inhibits ROCK1 activity rather than ROCK2 activity.

71. The ROCK inhibitor is of formula I, 【Transformation 5】 During the ceremony, L is O or SO ２ And, R １ However, H, hydroxy, NH ２ , NHR', or NR'R', R ２ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, or (C １ -C ６ ) is an alkoxy, R ３ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ４ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ５ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ６ However, H, (C １ -C ８ ) alkyl, (C １ -C ６ ) Alkylene-R'', (C １ -C ６ ) Alkylene-C(O)-R', (C １ -C ６ ) Alkylene-C(O)O-R', (C １ -C ６ ) Alkylene-C(O)NH ２ , (C １ -C ６ ) Alkylene-C(O)NHR', (C １ -C ６ ) Alkylene-C(O)NR'R', C(O)OR', C(O)R', C(O)NH ２ , C(O)NHR', C(O)NR'R', C(O)-(C １ -C ６ ) Alkilen-NH ２ , C(O)-(C １ -C ６ ) Alkylene-NHR', C(O)-(C １ -C ６ ) Alkylene-NR'R', C(O)O-(C １ -C ６ ) Alkilen-NH ２ , C(O)O-(C １ -C ６ ) alkylene-NHR', or C(O)O-(C １ -C ６ ) Alkilen-NR'R', R ７ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ８ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, n is 1, 2, 3, or 4, m is independently 1, 2, 3, or 4 each time it appears. R' is (C １ -C ６ ) alkyl, (C ３ -C ８ ) Cycloalkyl, (C ５ -C １０ ) Heterocyclyl, or (C ６ -C １０ ) is an aryl, R'' is (C ３ -C ８ ) Cycloalkyl, (C ５ -C １０ ) Heterocyclyl, or (C ６ -C １０ ) It is an aryl, defined by formula I, The method according to any one of claims 58 to 70, or the stereoisomer forms thereof and / or the tautomer forms thereof and / or pharmaceutically acceptable salts thereof.

72. The method according to any one of claims 58 to 71, wherein the ROCK inhibitor comprises isoquinoline sulfonamide.

73. The ROCK inhibitor is of formula IA, 【Transformation 6】 During the ceremony, R １ However, H, hydroxy, NH ２ , NHR', or NR'R', R ２ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, or (C １ -C ６ ) is an alkoxy, R ４ However, H, halogen, hydroxy, (C １ -C ６ ) alkyl, (C １ -C ６ ) Alkoxy, NH ２ , NHR', NR'R', or CN, R ６ However, H, (C １ -C ８ ) alkyl, (C １ -C ６ ) Alkylene-R'', (C １ -C ６ ) Alkylene-C(O)-R', (C １ -C ６ ) Alkylene-C(O)O-R', (C １ -C ６ ) Alkylene-C(O)NH ２ , (C １ -C ６ ) Alkylene-C(O)NHR', (C １ -C ６ ) Alkylene-C(O)NR'R', C(O)OR', C(O)R', C(O)NH ２ , C(O)NHR', C(O)NR'R', C(O)-(C １ -C ６ ) Alkilen-NH ２ , C(O)-(C １ -C ６ ) Alkylene-NHR', C(O)-(C １ -C ６ ) Alkylene-NR'R', C(O)O-(C １ -C ６ ) Alkilen-NH ２ , C(O)O-(C １ -C ６ ) alkylene-NHR', or C(O)O-(C １ -C ６ ) Alkilen-NR'R', R' is (C １ -C ６ ) alkyl, (C ３ -C ８ ) Cycloalkyl, (C ５ -C １０ ) Heterocyclyl, or (C ６ -C １０ ) is an aryl, defined by formula IA, The method according to any one of claims 58 to 72, or the stereoisomers thereof and / or the tautomers thereof and / or pharmaceutically acceptable salts thereof.

74. The method according to any one of claims 58 to 73, wherein the ROCK inhibitor comprises fasudil (HA-1077).

75. The method according to any one of claims 58 to 73, wherein the ROCK inhibitor comprises glycyl-H 1152.

76. The method according to any one of claims 58 to 73, wherein the ROCK inhibitor comprises H-1152.

77. The method according to any one of claims 58 to 73, wherein the ROCK inhibitor comprises lipasudil.

78. The method according to any one of claims 58 to 73, wherein the ROCK inhibitor is selected from the group consisting of fasudil, hydroxyfasudil, dimethylfasudil, H-1152, H-1152P, H-1152 dihydrochloride, ripasudil, netalusdil, belmosdil, SAR407899, their prodrugs, their salts, or combinations thereof.

79. The method according to any one of claims 58 to 70, wherein the ROCK inhibitor is selected from the group consisting of AR-12286, AT-13148, BA-1049(R), FCN-016, GSK429286A, PHP-201, RKI-1447, Y-27632, Y-30141, Y-32885, Y-39983, their prodrugs, their salts, or combinations thereof.

80. A pharmaceutical composition or kit comprising: an effective amount of a ROCK inhibitor for treating or preventing microvascular dysfunction in a subject in need thereof; an effective amount of a ROCK inhibitor for inducing microvasodilation in a subject; an effective amount of a ROCK inhibitor for reversing ET-1-induced vasoconstriction in a subject; an effective amount of a ROCK inhibitor for treating or preventing a disease, disorder, or condition associated with microvascular dysfunction in a subject; and / or an effective amount of a ROCK inhibitor for treating or preventing a substance abuse-related cardiovascular disease in a subject.

81. A pharmaceutical composition or kit comprising an effective amount of a ROCK inhibitor and an effective amount of a chemotherapeutic agent.

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