Mono-nitrosolated and bis-nitrosolated propylene glycol for treatment of thromboembolic diseases

Abstract

The present invention relates to methods of treating conditions wherein such treatments comprise the administration of certain mono-and / or bis-nitrosolated propylene glycol, including compositions and formulations thereof, and wherein the administration is administered to a patient in need thereof via intraarterial infusion at a dose of about 0.01 nmol kg-1 min-1 to 3000 nmol kg-1 min-1. The invention also relates to the use of mono-nitrosolated propylene glycol and / or bis-nitrosolated propylene glycol for the treatment of thromboembolic diseases.

Description

Technical Field

[0001] This invention relates to methods of treating a condition, wherein such treatment includes administering certain mononitrosylpropanediol and / or dinitrosylpropanediol, including compositions and formulations thereof, and wherein said administration is via intra-arterial infusion at about 0.01 nmol / kg -1 min -1 Up to 3000 nmol kg -1 min -1 The appropriate dose should be administered to the patient who requires it.

[0002] This invention also relates to mononitrosyl propylene glycol and / or dinitrosyl propylene glycol for the treatment of thromboembolic diseases. Background Technology

[0003] The enumeration or discussion of obviously previously disclosed documents in this specification is not necessarily an admission that such documents are part of the prior art or common general knowledge.

[0004] Organ ischemia, dysfunction, and failure following major surgery and in critical illness are devastating complications that can lead to severe comorbidities and death. There is a lack of specific medical therapies to target ischemia (e.g., increasing blood flow through vasodilation, enhancing collateral blood flow, preventing local microthrombus / embolism by inhibiting platelets, and reducing reperfusion injury by inhibiting oxygen free radical production). Specifically, uncontrolled platelet activation is a key pathological event in acute thromboembolism, vascular occlusion, and ischemic tissue damage, encompassing mechanisms of thrombotic inflammation and immune thrombosis. Pulmonary embolism represents a disease condition in which interactions between several important components are involved: the cardiovascular and respiratory systems, inflammation, and coagulation.

[0005] A common and serious form of organ ischemia is caused by acute arterial occlusion, a medical emergency in which the damage will depend on how long the affected organ can tolerate ischemia—minutes in the brain and approximately 4 to 6 hours in the limbs. Symptoms of arterial occlusion include pain and loss of function. The longer these symptoms persist, the less likely the organ can be saved. Dissolving or removing the blood clot is crucial to restoring blood flow to the tissue.

[0006] Nitric oxide (NO) is an important molecule in several biological systems. It is widely recognized that endogenous NO is crucial as a vasodilator in blood vessels.

[0007] As we age, the production of endogenous NO in various organs ceases, leading to an increased risk of dysfunction and disease. Impaired NO production is associated with an increased risk of cardiovascular disease, including myocardial infarction, stroke, and peripheral ischemia (also known as limb ischemia).

[0008] Since the mid-19th century, NO donors have been used to treat heart disease, most notably nitroglycerin. However, currently available NO donors present several problems, including tolerance development and the risk of side effects due to the release of NO throughout the circulatory system. Therefore, increasingly higher doses are required to maintain the efficacy of the most commonly used organic nitrate NO donors. For example, the usefulness of currently available NO donors is limited in treating tissues at risk of damage due to local ischemia because increasing the dose of existing NO donors can lead to dangerously low blood pressure due to the vasodilation caused by the release of NO throughout the vascular system.

[0009] Since the discovery that nitroglycerin and related compounds exert their effects by releasing NO, significant resources have been invested in trying to develop new and better NO donors. However, more than 30 years have passed since the breakthrough discovery related to NO, and progress in developing improved NO donors with higher specificity and fewer side effects has been slow.

[0010] To further explain, the most commonly used nitrates in clinical settings, such as nitroglycerin, are currently used to treat the symptoms of angina (chest pain). Organic nitrates work by relaxing blood vessels, increasing the blood and oxygen supply to the heart, and simultaneously reducing its workload. Examples of currently available organic nitrate medications include: a) Nitroglycerin (trinitroglycerin ester) (1,2,3-propanetriol nitrate), which is now mostly administered sublingually to suppress acute attacks of angina. However, severe headache and dizziness due to its rapid and widespread vasodilation are frequently encountered side effects. Nitroglycerin infusion concentrates are also available and diluted in isotonic glucose or normal saline for intravenous infusion. Tolerance development (i.e., decreased efficacy with repeated or continuous administration) is a clinical problem with nitroglycerin (and other organic nitrates) therapy.

[0011] b) Isosorbide mononitrate (1,4:3,6-didehydro-D-glucol-5-nitrate), which is considered a preventative agent for angina. Tolerance development is an issue in long-term treatment regimens. Common side effects include headache and dizziness, as experienced with nitroglycerin.

[0012] c) Isosorbide dinitrate (1,4:3,6-didehydro-D-glucol-2,5-nitrate), which is used for acute and prophylactic treatment of angina and heart failure.

[0013] d) Pentaerythritol nitrate, a group of organic nitrates, is known to exert long-term antioxidant and anti-atherosclerotic effects through currently unknown mechanisms. Pentaerythritol tetranitrate has been investigated for nitrate tolerance (an undesirable development in nitrate therapy) and has been experimentally tested in pulmonary hypertension.

[0014] Already in vivo Tests on many of these organic nitrate compounds, as well as other nitrate and nitrite compounds, revealed that these compounds produce NO. For example, glyceryl trinitrate, ethyl nitrite, isobutyl nitrate, isobutyl nitrite, isoamyl nitrite, and butyl nitrite have been tested in rabbit models, and these compounds were found to produce NO. body Inside A significant correlation was found between NO production and its effect on blood pressure (Cederqvist). et al. , Biochem. Pharmacol. ,1994, 47 , 1047-53).

[0015] As our understanding of the importance of nitric oxide deepens, the importance of dietary components has also been recognized, as they may influence the availability of NO in the arginine-nitric oxide system and have been found to play a role in host defense (Larsen). et al. , N. Eng. J. Med ,2006, 355 (2792-3). Therefore, L-arginine and its esters, such as ethyl-L-arginine, methyl-L-arginine and butyl-L-arginine, have been used to increase the endogenous production of NO.

[0016] WO 2006 / 031191 describes compositions and methods for the therapeutic delivery of gaseous nitric oxide. Such compositions for delivering gaseous NO include compounds capable of forming reversible bonds or associating with NO, such as alcohols, carbohydrates, and proteins.

[0017] WO 2007 / 106034 describes a method for producing organic nitrites from a compound that is a monohydric alcohol / polyhydric alcohol or an aldehyde or ketone derivative thereof. The method involves degassing an aqueous solution of the compound and then purging it with gaseous nitric oxide (NO).

[0018] Nilsson, KF et al. , Biochem Pharmacol. ,82(3), 248-259 (2011) discussed the formation and identification of new bioactive organic nitrites.

[0019] WO 2020 / 109420 describes a method for preparing mononitrosyl propylene glycol and / or dinitrosyl propylene glycol, as well as the resulting compositions. The document explains that such compounds can treat conditions where NO has beneficial effects.

[0020] WO 2021 / 239906 describes how these compounds have been surprisingly shown to be effective in treating microbial infections.

[0021] The inventors have previously demonstrated that mixtures of mononitrosylpropanediol and / or dinitrosylpropanediol (PDNO) release NO with an extremely short half-life when infused into the lungs, thereby exerting a major effect in pulmonary circulation. However, the mechanism behind this rapid degradation of PDNO in pulmonary circulation remains unclear, as researchers have surprisingly found that PDNO can reach pulmonary circulation and exert its effects even when administered via other routes of administration (e.g., transdermal), whereas it was thought that the degradation of PDNO in other parts of the body should generally be less efficient and rapid compared to the lungs (WO 2021 / 239892).

[0022] Furthermore, despite over 50 years of research into the mechanisms of NO release from NO donors, including organic nitrites, the underlying mechanisms remain unclear. Most of the mechanistic studies have been conducted in vitro, making it nearly impossible to connect them to understanding what actually occurs in the extremely complex in vivo environment.

[0023] Besides involving different enzymatic and non-enzymatic channels in the metabolism of NO donors, the effectiveness of these channels can also be influenced by the local environment in the organ from which NO is released from the molecule, such as local pH, oxygen and carbon dioxide levels, and the state of inflammation in the tissue. Furthermore, the bioavailability of NO released from a specific NO donor can vary due to various factors, such as the concentration of oxygen free radicals in the tissue, which in turn can be affected by the metabolism of the NO donor itself; for example, NO released from nitroglycerin has been shown to increase oxygen free radical levels, potentially leading to tissue damage.

[0024] In summary, the complexity of the biotransformation of organic nitrates and NO itself makes it nearly impossible to predict what effect a given dose of NO donor will have in a particular organ, especially if that organ is affected by pathological processes such as ischemia or infection.

[0025] Numerous studies have focused on intravenous administration of NO donors. Intravenous infusion of low-dose PDNO has been shown to exert a vasodilatory effect primarily in the pulmonary circulation (Nilsson KF, Lundgren M, Agvald P, Adding LC, Linnarsson D, Gustafsson LE. Formation of new bioactive organic nitrites and their identification with gas chromatography-mass spectrometry and liquid chromatography coupled to nitrite reduction. Biochem Pharmacol. 2011;82(3):248-59; and Nilsson KF, Gustafsson LE. Treatment with new organic nitrites in pulmonary hypertension of acute experimental pulmonary embolism. PharmacolRes Perspect. 2019:e00462.). By studying this pharmacokinetic principle in experimental models of acute pulmonary hypertension from various causes, low-dose intravenous PDNO has been demonstrated to be an effective pulmonary vasodilator without causing systemic hypotension (Nilsson KF, Gustafsson LE. Treatment with new organicnitrites in pulmonary hypertension of acute experimental pulmonary embolism. Pharmacol Res Perspect. 2019:e00462; Nilsson KF, Gozdzik W, Frostell C, Zielinski S, Zielinska M, Ratajczak K, et al. Organic mononitrites of 1,2-propanediol act as an effective NO-releasing vasodilator in pulmonary hypertension and exhibit no cross-tolerance with nitroglycerin inanesthetized pigs).Drug Des Devel Ther. 2018;12:685-94; Nilsson KF, Gozdzik W, Zielinski S, Ratajczak K, Goranson SP, Rodziewicz S, et al. Pulmonary Vasodilation by Intravenous Infusion of Organic Mononitrites Of 1,2-Propanediol in Acute Pulmonary Hypertension Induced by Aortic Cross Clamping and Reperfusion: A Comparison With Nitroglycerin in Anesthetized Pigs. Shock. 2020;54(1):119-27, and Stene Hurtsen A, Zorikhin Nilsson I, Dogan EM, Nilsson KF. A Comparative Study of Inhaled Nitric Oxide and an Intravenously Administered Nitric Oxide Donor in Acute Pulmonary Hypertension. Drug Des Devel Ther. 2020;14:635-45).

[0026] Previous animal studies have unexpectedly shown that low-dose intravenous infusion of PDNO (targeted pulmonary vasodilation) has renal protective and anti-inflammatory effects in a multi-organ failure model (aortic occlusion and reperfusion) (Nilsson KF, Gozdzik W, Zielinski S, Ratajczak K, Goranson SP, Rodziewicz S, et al. Pulmonary Vasodilation by Intravenous Infusion of Organic Mononitrites Of 1,2-Propanediol in Acute Pulmonary Hypertension Induced by Aortic CrossClamping and Reperfusion: A Comparison With Nitroglycerin in Anesthetized Pigs. Shock. 2020;54(1):119-27.). Studies have also shown that high-dose intravenous PDNO (for systemic effects) can improve renal function in a sheep model of renal ischemia-reperfusion (Nilsson KF, Sandin J, Gustafsson LE, Frithiof R. The novel nitric oxide donor PDNO attenuates ovine ischemia-reperfusion induced renal failure. Intensive Care Med Exp. 2017;5(1):29.). In these studies, local blood flow was not monitored (only general cardiac output was monitored), and therefore the knowledge of the dose required to affect blood flow, particularly blood flow in specific organs such as the brain (or even a specific side of the brain), limbs (e.g., a leg), kidneys (or a specific kidney), or parts of the intestine, has not been investigated. Nor has the relationship between such flow changes and their effects on systemic blood pressure and / or cardiac output been measured. Such studies were not conducted because they were not a natural continuation of development programs exploring the role of PDNO in pulmonary and systemic circulation. The findings of these studies presented in this paper are surprising because general development programs do not even provide any motivation to study this approach, and because the focus is completely different, it is impossible to infer the invention described in this paper from previous studies.

[0027] Therefore, clinically available NO donor drugs are usually administered intravenously, leading to systemic effects, including systemic hypotension, which is the most important dose-limiting side effect. Hypotension can be harmful, especially in critically ill patients, and may counteract the desired increase in blood flow by severely reducing perfusion pressure (Gresele P, Momi S, Guglielmini G. Nitric oxide-enhancing or-releasing agents as antithrombotic drugs. Biochem Pharmacol. 2019;166:300-12.).

[0028] It is well known that NO in organic nitrites can be released through both non-enzymatic (acid-catalyzed hydrolysis) and enzymatic pathways, but the importance of each pathway for NO release from organic nitrites (including PDNO) has not yet been determined. Since the lungs are generally considered metabolically active organs, it is expected that the degradation of PDNO in the lungs will be much more efficient than in other organs.

[0029] Given the above, there is a need for improved treatments using NO donors, particularly for conditions where NO has beneficial effects, overcoming one or more of the aforementioned drawbacks. This need is especially present in organs and body parts other than the lungs. Summary of the Invention

[0030] The inventors were surprised by the efficacy and effects of PDNO degradation observed during intra-arterial infusion testing. Specifically, they were surprised to find that the rapid release of nitric oxide induced beneficial effects without first allowing PDNO to pass through the pulmonary circulation. Even more surprisingly, the dosage of PDNO administered, and the intensity of its response in the blood vessels of the brain, extremities, kidneys, and intestines, were unexpected compared to the possible dosage when administered intravenously in local therapy, while avoiding or reducing systemic effects. The inventors were also surprised to find that intra-arterial administration required a lower dosage to achieve the desired effect compared to intravenous administration. Furthermore, the inventors were surprised to find that at higher doses administered intra-arterially, which do indeed affect systemic blood pressure, these drugs significantly influenced blood flow to specific target organs, where they had previously expected local effects to be diminished due to reduced driving pressure.

[0031] This invention also includes the surprising discovery that PDNO can induce an antithrombotic effect in the experiments conducted. For this to be achieved, NO must be delivered in sufficient quantity to enter the platelets. Given that platelets are surrounded by hemoglobin-filled red blood cells, which typically act as receivers for NO, and considering that NO has been shown to have a biological half-life in the millisecond range in the blood, it is quite unusual that PDNO appears to affect platelet function even at relatively low doses. Whether this means that PDNO is released from the platelets, or whether this is achieved through another unknown mechanism, remains unclear. However, the combined targeted local vasodilatory and antithrombotic effects of PDNO are surprising and have potentially enormous value in treating several forms of tissue ischemia.

[0032] Intra-arterial administration The inventors have unexpectedly discovered that administering certain mononitrosyl propylene glycol and / or dinitrosyl propylene glycol, including compositions and formulations thereof, to patients in need via intra-arterial infusion at certain doses can achieve local, site-specific treatment of diseases, particularly those in which NO is expected to have a beneficial effect, while avoiding unwanted systemic side effects.

[0033] As illustrated in the examples, when such compounds are administered intravenously, systemic effects are observed at doses much lower than those administered intra-arterially, before any systemic effects are observed.

[0034] Therefore, in a first aspect of the invention, a compound of formula (I) is provided: (I) Where R 1 R 2 and R 3 Each can be independently represented as H or -NO. Where n is 0 or 1; When n is 0, R 1 For H; and When n is 1, R 2 For H, The condition is R 1 R 2 and R 3 At least one of them represents -NO, This compound (I) is used to treat a condition, wherein the compound (I) is administered via intra-arterial infusion at a dose of approximately 0.01 nmol kg. -1 min -1 Up to 3000 nmol kg -1 min -1The dosage is administered to the patient who needs it, a method referred to herein as "the first method of the present invention".

[0035] According to the first method of the present invention, the following is further provided: ●A method using approximately 0.01 nmol kg -1 min -1 Up to 3000 nmol kg -1 min -1 A method of administering the dosage of compound (I) to a patient in need of treatment via intra-arterial infusion; and ●The use of the compound according to formula (I) in the manufacture of a pharmaceutical agent for treating a ailment, wherein the compound is present in an amount of about 0.01 nmol / kg. -1 min -1 Up to 3000 nmol kg -1 min -1 The compound is administered to the patient in need via intra-arterial infusion at a dose specified in the manual method.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] Without departing from the disclosure of this invention, all embodiments of the invention and specific features mentioned herein may be separated from or combined with any other embodiments and / or specific features mentioned herein (and thus more specific embodiments and specific features as disclosed herein are described).

[0038] As used herein, the term "comprising" will take its usual meaning in the art, indicating that the component includes, but is not limited to, the relevant features (i.e., including, etc.). Thus, the term "comprising" will include references to components that are substantially composed of the relevant substances.

[0039] As used herein, unless otherwise stated, the terms "consistses substantially of" and "consisting essentially of" will refer to the relevant component that, according to relevant measurements (e.g., by weight), comprises at least 80% (e.g., at least 85%, at least 90%, or at least 95%, such as at least 99%) of the specified substance. The terms "consistses substantially of" and "consisting essentially of" may be replaced by "consists of" and "consisting of," respectively.

[0040] To avoid any doubt, the term "comprising" will also include references to components that are "substantially composed of the relevant substance" (and in particular "composed of the relevant substance").

[0041] Those skilled in the art will understand that references to treatment of a specific symptom (or similarly, the treatment of the symptom) take on their normal meaning in the medical field. Specifically, the term can refer to the achievement of a reduction in the severity of one or more clinical symptoms and / or signs associated with the symptom. For example, in the case of pulmonary embolism, the term could refer to... via Vasodilation is used to reduce the severity of chest pain, shortness of breath, and / or pulmonary hypertension. Additionally, in the case of pulmonary embolism, the term can also refer to achieving pulmonary vasodilation or a reduction in pulmonary vascular resistance and right ventricular strain.

[0042] As used herein, references to "patient" refer to a living subject receiving treatment, including mammalian (e.g., human) patients. Specifically, the term "patient" can refer to a human subject. The term "patient" can also refer to an animal (e.g., a mammal), such as domestic pets (e.g., cats, and especially dogs), livestock, and horses.

[0043] The method according to the invention comprises administering an effective amount of a compound according to formula (I) to a patient in need. As used herein, the term "effective amount" will refer to the amount of compound that imparts a therapeutic effect to the treated patient. The effect may be objective (i.e., measurable by some test or biomarker) or subjective (i.e., the subject gives indication of the effect and / or feels the effect).

[0044] Compound (I) can be obtained at approximately 0.01 nmol kg -1 min -1 Approximately 300 nmol kg -1 min -1 For example, about 1 nmol kg -1 min -1 Approximately 300 nmol kg -1 min -1 Such as approximately 1 nmol kg -1 min -1 Approximately 100 nmol kg -1 min -1 or about 10 nmol kg -1 min -1 Approximately 3000 nmol kg -1 min -1 Such as 10 nmol kg -1 min -1 Approximately 300 nmolkg -1 min -1Dosage administration.

[0045] Preferably, the compound of formula (I) is expressed at about 1 nmol kg -1 min -1 Approximately 30 nmol kg -1 min -1 Administered at a dosage of approximately 1 nmol / kg. For example, compound (I) can be administered at a dosage of approximately 1 nmol / kg. -1 min -1 to less than about 30 nmol kg -1 min -1 Such as approximately 1 nmol kg -1 min -1 Approximately 25, 26, 27, 28 or 29 nmol kg -1 min -1 For example, about 1 nmol kg -1 min -1 Approximately 20 nmol kg - 1 min -1 Approximately 1 nmol kg -1 min -1 Approximately 15, 14, 13, 12 or 11 nmol kg -1 min -1 Such as approximately 1 nmol kg -1 min -1 Approximately 10 nmol kg -1 min -1 Dosage administration.

[0046] Compounds of formula (I) can be administered continuously, such as by continuous infusion, and this can occur over a period of up to 14 days, such as 7 days.

[0047] Compounds of formula (I) may be administered alternatively as a single dose or as repeated single injections (such as in bolus infusion).

[0048] Compound of formula (I) can be administered to any part of the arterial vascular system that is accessible to a medical professional for delivery of compound of formula (I) by infusion. Infusion can be achieved through surgical techniques, such as the use of catheter devices.

[0049] Intra-arterial administration can be used to deliver the compound according to formula (I) to the lower extremities, and this can be achieved by intra-arterial administration to the femoral artery.

[0050] Intra-arterial administration can also or alternatively be intended to deliver the compound according to formula (I) to the brain, and this can be achieved by intra-arterial administration to the carotid artery.

[0051] Intra-arterial administration may further or alternatively be intended to deliver the compound according to formula (I) into the intestine, and this can be achieved by intra-arterial administration to the superior mesenteric artery.

[0052] Intra-arterial administration may also or alternatively be intended to deliver the compound according to formula (I) to the kidney, adrenal gland and / or ureter, and this can be achieved by intra-arterial administration to the renal artery.

[0053] Therefore, the compound of formula (I) can be administered via intra-arterial infusion to the femoral artery, carotid artery, superior mesenteric artery, splenic artery, hepatic artery and / or renal artery.

[0054] When the compound of formula (I) is administered via intra-arterial infusion to the femoral artery and / or carotid artery, the compound can be administered at a dose of approximately 1 nmol / kg. -1 min -1 Approximately 300 nmol kg -1 min -1 Such as 1 nmol kg -1 min -1 Approximately 60 nmol kg -1 min -1 For example, 1 nmol kg -1 min -1 Approximately 50 nmol kg -1 min -1 Such as 1 nmol kg -1 min -1 Approximately 30 nmol kg -1 min -1 Dosage administration.

[0055] When compound of formula (I) is administered to the superior mesenteric artery via intra-arterial infusion, the compound can be delivered at approximately 3 nmol kg. -1 min -1 Approximately 3000 nmol kg -1 min -1 Such as approximately 30 nmol kg -1 min -1 Approximately 1000 nmol kg - 1 min -1 For example, about 100 nmol kg -1 min -1 Approximately 1000 nmol kg -1 min -1 Dosage administration.

[0056] When the compound of formula (I) is administered to the renal artery via intra-arterial infusion, the compound can be administered at a dose of approximately 0.01 nmol / kg. -1 min -1 Approximately 300 nmol kg -1 min -1 For example, about 1 nmol kg -1 min -1 Approximately 30 nmol kg -1 min -1 Dosage administration.

[0057] When applied to a specific artery, this also aims to include application to arterial branches originating from that artery. For example, when applied to the femoral artery, application could be to the superficial epigastric artery, superficial circumflex artery, external pudendal artery, femoral (or deep femoral) artery, or superficial femoral artery, all of which connect to the femoral artery. When applied to the carotid artery, this could be via a cerebral artery, namely the Circle of Willis.

[0058] According to the first method of the present invention, the condition to be treated can be a condition in which NO has a beneficial effect. That is, the compound according to formula (I) can be used to treat a condition in which NO (i.e., NO administration) has a beneficial effect.

[0059] As used herein, the term "beneficial effect" means an identifiable treatment and / or improvement of the condition of the patient being treated resulting from the use / application of the compounds / compositions of the present invention. Beneficial effects may be temporary or permanent and may be measured or determined by a physician or the patient themselves.

[0060] A specific result of this invention is that beneficial effects can be experienced locally, for example, only in a single organ of the patient, but without clinically significant systemic effects. These beneficial effects can be objective (i.e., measurable by some test or biomarker) or subjective (i.e., indicated and / or felt by the subject).

[0061] The condition to be treated can be selected from the following groups: Acute pulmonary vasoconstriction of various causes; pulmonary hypertension of various causes, including primary and secondary hypertension; preeclampsia; eclampsia; conditions requiring vasodilation of various causes; erectile dysfunction; systemic hypertension of various causes; regional vasoconstriction of various causes; local vasoconstriction of various causes; acute heart failure (with or without preserved ejection fraction [HFpEF]); coronary artery disease; myocardial infarction; ischemic heart disease; angina pectoris; unstable angina pectoris; arrhythmias; acute pulmonary hypertension in patients undergoing cardiac surgery; acidosis; airway inflammation; cystic fibrosis; COPD; immobile ciliary syndrome; pulmonary inflammation; pulmonary fibrosis; acute lung injury (ALI); adult respiratory distress syndrome; acute pulmonary... Edema; acute mountain sickness; asthma; bronchitis; hypoxia of various causes; ischemic diseases of various causes; stroke; cerebral vasoconstriction; gastrointestinal inflammation; gastrointestinal dysfunction; gastrointestinal complications; IBD; Crohn's disease; ulcerative colitis; liver disease; pancreatic disease; urethritis and cystitis; cystitis and ureteritis; skin inflammation; diabetic ulcers; diabetic neuropathy; psoriasis; inflammation of various causes; wound healing; organ protection under ischemia-reperfusion conditions; organ transplantation; tissue transplantation; cell transplantation; acute kidney disease; uterine relaxation; cervical relaxation; thromboembolic diseases, including diseases complicated by thromboembolism, such as various hematologic disorders; arterial occlusion, such as arterial embolism; and conditions requiring smooth muscle relaxation.

[0062] In particular, the condition to be treated can be pulmonary hypertension of different causes, including primary hypertension and secondary hypertension.

[0063] Possible specific conditions include those selected from the group consisting of: ischemic diseases of various causes; thromboembolic diseases, including diseases complicated by thromboembolism, such as various blood disorders; arterial thrombosis; peripheral ischemia (limb ischemia); thromboembolic stroke; pulmonary embolism; acute mesenteric ischemia (mesenteric artery occlusion); acute renal artery occlusion; arterial stenosis; arterial occlusion; splenic infarction; hepatic infarction; pulmonary infarction; Kawasaki disease; and arterial embolism.

[0064] Regarding the treatment of ischemic diseases, it should be understood that this includes treating / preventing conditions caused by arterial occlusion (partial or complete), which results in restricted blood flow to specific organs and ischemia or threatening ischemia.

[0065] Treatment can be manifested as a decrease in mean systemic arterial pressure (MAP) of no more than about 10% relative to the baseline MAP of the subject before treatment. For example, treatment can be manifested as a decrease in MAP of no more than about 9%, 8%, 7%, or 6% relative to the baseline MAP of the subject before treatment. In particular, treatment can be manifested as a decrease in MAP of no more than about 5% relative to the baseline MAP of the subject before treatment.

[0066] Treatment can be defined as a decrease in end-expiratory NO (ETNO) of no more than about 10% relative to the baseline ETNO in the subject before treatment. For example, treatment can be defined as a decrease in ETNO of no more than about 9%, 8%, 7%, or 6% relative to the baseline ETNO in the subject before treatment. In particular, treatment can be defined as a decrease in ETNO of no more than about 5% relative to the baseline ETNO in the subject before treatment.

[0067] Treatment can be defined as an increase in the methemoglobin fraction in the blood of a subject no greater than about 10% relative to baseline before treatment. For example, treatment can be defined as an increase in the methemoglobin fraction in the blood of a subject no greater than about 9%, 8%, 7%, or 6% relative to baseline before treatment. In particular, treatment can be defined as an increase in the methemoglobin fraction in the blood of a subject no greater than about 5% relative to baseline before treatment.

[0068] By “relative to baseline level,” we are referring to a comparison between the MAP level measured at the start of the study (i.e., before administration of compound (I)) and the MAP level after administration. The baseline level is the level before treatment begins and is used as a comparison for levels subsequently measured (e.g., immediately after the course of treatment or at a time point after the end of the course of treatment). Therefore, such comparisons are specific to the subjects or subject groups in question and are not absolute values.

[0069] Other specific conditions that can be treated include bacterial, fungal, viral, or parasitic infections.

[0070] Treatment of thromboembolic diseases The inventors have further and unexpectedly discovered that administration of certain mononitrosyl propylene glycol and / or dinitrosyl propylene glycol, including their compositions and formulations, to patients in need is particularly effective in inhibiting platelet aggregation and can be used to treat thromboembolic disorders.

[0071] Therefore, according to a second aspect of the invention, a compound of formula (I) is provided: (I) Where R 1 R 2 and R 3 Each can be independently represented as H or -NO. Where n is 0 or 1; When n is 0, R 1 For H; and When n is 1, R 2 For H, The condition is R 1 R2 and R 3 At least one of them represents -NO, The compound of formula (I) is used to treat thromboembolic diseases and is referred to herein as "the second method of the present invention".

[0072] According to a second method of the present invention, the following is further provided: ●A method for treating thromboembolic diseases by administering a compound of formula (I) to a patient in need; and ●The use of the compound according to formula (I) in the manufacture of a medicament for the treatment of thromboembolic diseases.

[0073] The inhibition of platelet aggregation appears to occur through a surprising mechanism different from other treatments, and is achieved by inhibiting the release of platelet particles and adenosine triphosphate (ATP).

[0074] Thromboembolic diseases are caused by the rupture of blood clots that form in blood vessels, are carried by the bloodstream, and cause at least partial blockage of the blood vessels.

[0075] Thromboembolic diseases awaiting treatment can be selected from the following groups: arterial thrombosis; myocardial infarction; cerebral venous thrombosis; portal venous thrombosis; peripheral ischemia (limb ischemia); thromboembolic stroke; pulmonary embolism; acute mesenteric ischemia (mesenteric artery occlusion); acute renal artery occlusion; arterial stenosis; arterial occlusion; venous thromboembolism, including deep vein thrombosis; and arterial embolism.

[0076] Thromboembolic diseases occur in both the venous and arterial systems, and to avoid any doubt, it is envisioned that the use of compounds according to formula (I) in the treatment of such diseases encompasses both intravenous and arterial treatment. Therefore, compounds according to formula (I) can be administered via intravenous infusion and / or intra-arterial infusion.

[0077] In addition, it is envisioned that treatment can be administered via other routes, including inhalation, nebulization, intramuscular administration, subcutaneous administration, transdermal administration, intranasal administration, sublingual administration, subconjunctival administration, rectal administration, tracheal administration, pulmonary administration, gastric administration, ureteral administration, enteral administration, urethral administration, bladder administration, oral administration, and enteral administration.

[0078] The compound according to formula (I) can be obtained at approximately 0.01 nmol kg -1 min -1 Up to 3000 nmol kg -1 min -1 Such as approximately 0.01 nmol kg -1 min -1 Approximately 300 nmol kg-1 min -1 For example, about 1 nmol kg -1 min -1 Approximately 300 nmol kg - 1 min -1 Such as approximately 1 nmol kg -1 min -1 Approximately 100 nmol kg -1 min -1 The dosage is as follows. Preferably, the compound of formula (I) is administered at about 1 nmol / kg. -1 min -1 Approximately 30 nmol kg -1 min -1 Administered at a dosage of approximately 1 nmol / kg. For example, compound (I) can be administered at a dosage of approximately 1 nmol / kg. -1 min -1 to less than about 30 nmol kg -1 min -1 Such as approximately 1 nmol kg -1 min -1 Approximately 25, 26, 27, 28, or 29 nmol kg -1 min -1 For example, about 1 nmol kg -1 min -1 Approximately 20 nmol kg -1 min -1 Approximately 1 nmol kg -1 min -1 Approximately 15, 14, 13, 12 or 11 nmol kg -1 min -1 Such as approximately 1 nmol kg -1 min -1 Approximately 10 nmol kg -1 min -1 Administered at a dose of approximately 1 nmol / kg. Furthermore, compound (I) can be administered at approximately 1 nmol / kg. -1 min -1 Approximately 5 nmol kg -1 min -1 Dosage administration.

[0079] The compound according to formula (I) can be administered to the vascular system in any manner, such as by intra-arterial infusion into any artery of any limb.

[0080] The compound according to formula (I) can be administered via intra-arterial infusion, such as into the femoral artery, carotid artery, renal artery or superior mesenteric artery.

[0081] Compounds according to formula (I) can be administered via intravenous infusion, such as into the central venous system, peripheral veins of the arms and legs, femoral veins, and scalp veins (especially scalp veins in newborns). Intravenous infusion can also be administered into deep or superficial veins, such as superficial varicose veins in the legs.

[0082] Treatment can be defined as an increase in the methemoglobin fraction in the blood of a subject no greater than about 10% relative to baseline before treatment. For example, treatment can be defined as an increase in the methemoglobin fraction in the blood of a subject no greater than about 9%, 8%, 7%, or 6% relative to baseline before treatment. In particular, treatment can be defined as an increase in the methemoglobin fraction in the blood of a subject no greater than about 5% relative to baseline before treatment.

[0083] By “relative to baseline level,” we are referring to a comparison between the MAP level measured at the start of the study (i.e., before administration of compound (I)) and the MAP level after administration. The baseline level is the level before treatment begins and is used as a comparison for levels subsequently measured (e.g., immediately after the course of treatment or at a time point after the end of the course of treatment). Therefore, such comparisons are specific to the subjects or subject groups in question and are not absolute values.

[0084] As mentioned above, the inhibition of platelet aggregation appears to occur through a surprising method different from other treatments, and is carried out by inhibiting the release of platelet particles and ATP release.

[0085] Therefore, the second method of the present invention also covers a method for inhibiting the release of platelet particles and / or ATP in a subject, said method comprising administering an effective amount of one or more compounds of formula (I) or pharmaceutical preparations thereof to a subject in need of such treatment. This aspect of the invention may include any of the features of the treatment of thromboembolic diseases outlined above.

[0086] Compounds and Compositions The specific compounds used in both the first and second methods of this invention are compounds according to formula (II). (II) Where R 2 and R 3 Each can be represented independently as H or -NO, provided that R is... 2 and R 3 At least one of them represents -NO.

[0087] There are two enantiomers of the compound according to formula (II), as shown below. R Form and S form: (II) R form (II) S form The compound of formula (I) may contain asymmetric carbon atoms as described above, and thus will exhibit optical isomerism.

[0088] All stereoisomers of the compounds according to formula (I) and mixtures thereof are included within the scope of this invention.

[0089] The other specific compounds used in both the first and second methods of this invention are compounds according to formula (III): (III) Where R 1 and R 3 Each can be represented independently as H or -NO, provided that R is... 1 and R 3 At least one of them represents -NO.

[0090] The additional specific compounds used in both the first and second methods of this invention are compounds according to formula (IV): (IV) Where R 4 and R 5 Each can be represented independently as H or -NO, provided that R is... 4 and R 5 At least one of them represents -NO.

[0091] The compounds used in both the first and second methods of the present invention may be present in the composition, said composition comprising: (a) one or more compounds of formula (I) as defined herein; and (b) One or more corresponding compounds of formula (I), but wherein R 1 R 2 and R 3 The H symbol represents 1,2-propanediol and / or 1,3-propanediol, and the composition may be referred to as the "composition" below.

[0092] The composition can be substantially non-aqueous. As used herein, with respect to both the first and second aspects of the invention, reference to "substantially non-aqueous" will mean containing less than 10% by weight, such as less than 9.9% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, or 1% by weight (such as less than 0.5% by weight or less than 0.1% by weight, for example less than 0.05% by weight, less than 0.01% by weight).

[0093] It should be understood that the composition may comprise a mixture of compounds falling into formula (I).

[0094] Specific compositions that may be mentioned include those compositions in which the composition comprises about 0.01% by weight to about 9% by weight (e.g., about 0.01% by weight to about 5% by weight, such as about 3% by weight to about 5% by weight, or about 5% by weight to about 7% by weight) one or a compound of formula (I).

[0095] Specific compositions that may be mentioned include those compositions in which the composition comprises one or more of a compound of formula (I) at a concentration of about 1 mM to about 1000 mM (e.g., about 5 mM to about 750 mM, such as about 5 mM to about 500 mM, or about 10 mM to about 203 mM).

[0096] To avoid any ambiguity, the unit mM refers to units of 10-1. -3 The concentration of compound (I) in the composition is expressed in mol / L, and in the case of a mixture of compounds (I) in the composition, it is based on the average molecular weight of compound (I) in the composition.

[0097] Specific compositions that may be mentioned include those compositions in which the composition comprises a compound according to formula (II). Preferably, the compound according to formula (II) is... S form.

[0098] The preferred compound is according to formula (II). S Form, because of its relationship with R Compared to other forms, it has a higher metabolic rate and a different metabolic pathway. Furthermore, S The forms have different metabolic degradation pathways, thus producing more than those R Metabolites that are less toxic than their metabolites.

[0099] Specific compositions that may be mentioned include those compositions in which the composition comprises a compound according to formula (III).

[0100] Preferably, the compound according to formula (II) is in the S form, but the product is envisioned to be of formula (II). S Form and RA mixture of the two, in which S The preferred form is an enantiomer in excess (ee).

[0101] In a specific embodiment, the compound according to formula (II) can be an enantiomerically excess of the compound in its S form. That is, the product greater than 50 ee% is in the S form, such as greater than or equal to 60 ee%, 70 ee%, 80 ee%, 90 ee%, 95 ee%, or 98 ee%. S form.

[0102] In examples where the product is a mononitrosylated compound according to formula (II), more than 50 wt.% of the product is nitrosylated at position 2 (i.e., R). 2 (Yes-NO), such as between about 55 wt.% and about 80 wt.%, nitrosylated at the 2 position, for example between about 55 wt.% and 75 wt.%.

[0103] Specific compositions that may be mentioned include those comprising essentially one or more compounds of formula I and corresponding compounds of formula I, but wherein R 1 R 2 and R 3 Compositions that represent the composition of H (i.e., 1,2-propanediol and / or 1,3-propanediol).

[0104] Other specific compositions may comprise one or more compounds of formula II and 1,2-propanediol (or particularly, substantially composed of one or more compounds of formula II and 1,2-propanediol, or more particularly, composed of one or more compounds of formula II and 1,2-propanediol).

[0105] Similarly, other compositions may include one or more compounds of formula III and 1,3-propanediol (or particularly, substantially composed of one or more compounds of formula III and 1,3-propanediol, or more particularly, composed of one or more compounds of formula III and 1,3-propanediol).

[0106] The term “consistently of…” means that there is at least 90% by weight of the defined feature, such as at least 95% by weight, 96% by weight, 97% by weight, 98% by weight, or 99% by weight of the defined feature.

[0107] In addition, specific compositions that may be mentioned include those compositions comprising one or more compounds of formula (II) and (III) and 1,2-propanediol and 1,3-propanediol (or particularly, substantially composed of one or more compounds of formula (II) and (III) and 1,2-propanediol and 1,3-propanediol, or more particularly, composed of one or more compounds of formula (II) and (III) and 1,2-propanediol and 1,3-propanediol).

[0108] Specific compositions that may be mentioned include those compositions in which the composition is substantially free of dissolved nitric oxide.

[0109] The term “substantially free” means that the composition contains less than 5 wt.%, 4 wt.%, 3 wt.%, 2 wt.% or 1 wt.% of dissolved nitric oxide, such as less than 0.5 wt.% or 0.1 wt.%.

[0110] In addition, the specific composition may include: (a) One or more compounds of formula IV (IV) Where R 4 and R 5 Each can be represented independently as H or -NO, provided that R is... 4 and R 5 At least one of them represents -NO; and (b) 1,2-Propanediol.

[0111] The composition can be administered alone or via a known pharmaceutical composition / formulation.

[0112] Therefore, the composition may be included in a pharmaceutical preparation, wherein the pharmaceutical preparation contains one or more pharmaceutically acceptable excipients.

[0113] Those skilled in the art will understand that references to pharmaceutical formulations herein refer to compositions in pharmaceutical formulation form, and will include references to all embodiments thereof and to particular forms thereof.

[0114] As used herein, pharmaceutically acceptable excipients include excipients, adjuvants, carriers, diluents, pH adjusters and buffers, tension modifiers, stabilizers, permeability enhancers, wetting agents, etc. In particular, such excipients may contain adjuvants, diluents, or carriers.

[0115] The specific drug formulations that may be mentioned include those in which the drug formulation includes at least one pharmaceutically acceptable excipient.

[0116] The specific drug formulations that may be mentioned contain one or more pharmaceutically acceptable excipients that are substantially non-aqueous.

[0117] The compound according to formula (I) can be administered to the patient (i.e. the subject) in combination with a suitable aqueous buffer (such as a non-nucleophilic and weakly basic buffer).

[0118] More specific examples that may be mentioned include those in which the buffer solution has a pH of about 7.1 to about 10 (e.g., about 8 or about 9.2), such as carbonate (e.g., NaHCO3) buffer or phosphate buffer or mixtures thereof.

[0119] Specifically, the buffer solution can be a carbonate buffer at pH 9.2, a phosphate buffer at pH 8.0 (e.g., 0.154 mol buffer), or a NaHCO3 buffer at pH 8.0.

[0120] To avoid any doubt, the specific uses of the compounds of formula (I) mentioned herein can also be applied to compositions and pharmaceutical preparations containing compounds of formula (I) as described herein.

[0121] Wherever the word “about” is used in the context of quantities (e.g., absolute amounts of individual components or components of a composition, such as weight, volume, size, diameter, etc., or relative amounts (e.g., percentages) (including concentrations and ratios), time ranges, and parameters (e.g., temperature), it should be understood that such variables are approximate and therefore can vary from the actual figures specified herein by ±10%, for example ±5%, and preferably ±2% (e.g., ±1%). This is true even if such figures are first presented as percentages (e.g., 'about 10%' can mean ±10% of the number 10, i.e., any value between 9% and 11%). Attached Figure Description

[0122] Figure 1: Study protocol for dose-response experiments of intravenous and organ-directing arterial infusion of 1,2-propylene glycol organic mononitrite (PDNO) in the carotid and femoral arteries (Figure A) and the renal and superior mesenteric arteries (Figure B).

[0123] Figure 2: Effects of organ-directed intra-arterial infusion of 1,2-propylene glycol organic mononitrite (PDNO) and intravenous administration of PDNO on regional blood flow, mean systemic arterial pressure (MAP), and end-tidal nitric oxide concentration (ETNO) in the left carotid artery (Figures A and B) and left common femoral artery (Figures C and D). Data are mean with standard error of mean (SEM). * indicates statistical significance relative to baseline (BL) in the femoral and carotid artery experiments (P < 0.05). #This indicates statistical significance relative to BL during intravenous administration (P < 0.05).

[0124] Figure 3: Effects of organ-directed infusion of 1,2-propylene glycol organic mononitrite (PDNO) and intravenous administration of PDNO on regional blood flow, mean systemic arterial pressure (MAP), and end-tidal nitric oxide (ETNO) in the superior mesenteric artery (SMA, Figures A and B) and left renal artery (Figures C and D). Data are mean with standard error of mean (SEM). * indicates statistical significance relative to baseline (BL) in the renal artery test and vasopressin (VP) in the SMA test (p < 0.05). # This indicates statistical significance relative to BL during intravenous administration (p < 0.05).

[0125] Figure 4: Calculation of end-tidal nitric oxide (ETNO) dose ratio between intravenous and intra-arterial administration of 1,2-propylene glycol organic mononitrite (PDNO) in the left common femoral artery, left common carotid artery, left renal artery, and superior mesenteric artery. Using logarithmic PDNO, ETNO = B0e B1剂量 (Figure A) The dose relationship linearized to the natural logarithmic ETNO, ln ETNO = B1 dose + ln B0 (Figure B). Estimated coefficients and standard errors (Figure C). Significance is shown for comparisons with intravenous (iv) reference (ref) 1 and with iv ref 2, respectively. Unless otherwise stated, iv ref 1 applies to intracarotid and femoral artery infusions, and iv ref 2 applies to intrarenal and superior mesenteric artery infusions. a : The dose multiple required to increase log(ETNO) to a similar level compared to the IV dose. #: Intercept for femoral artery infusion compared to carotid artery infusion. #: Intercept for both renal artery infusion and IV (ref 2). P All values ​​below 0.001 are statistically significant. *: P <0.05, **: P <0.01, ***: P <0.001.

[0126] Figure 5 Arterial fraction of methemoglobin during organ-directed intra-arterial infusion of 1,2-propylene glycol organic mononitrite (PDNO) and intravenous administration of PDNO. Data are mean with standard error of mean (SEM).

[0127] Figure 6Platelet aggregation in porcine whole blood (with heparin as an anticoagulant) was analyzed in vitro using a multiplate agglutinator. A coagulated blood clot was administered intravenously to anesthetized pigs (n=5) to induce pulmonary embolism (PE), followed by intravenous administration of PDNO at 160 nmol / kg / min. Arterial blood was collected at specific time points: pre-embolization (baseline), post-embolization, during PDNO infusion, and immediately after PDNO infusion cessation, and transferred to cuvettes for multiplate aggregation analysis. Blood was stimulated with the PAR-1 agonist peptide SFLLRN, and platelet aggregation (manifested as an increase in electrical resistance) was tracked over 10 minutes. Data are presented as individual values ​​and medians (line values). P-values ​​are derived from analyses using the Dunn test.

[0128] Figure 7 Anti-aggregation efficacy of PDNO. Suspensions of human platelets separated by activation with the PAR-1 agonist SFLLRN were analyzed in the absence of PDNO (hollow bars) and in the presence of different concentrations of PDNO (solid bars). Platelet aggregation was assessed by the increase in transmittance in a Chronolog aggregometer. PDNO was introduced into platelets 2 min before SFLLRN. Bars represent the mean and standard deviation (n=5).

[0129] Figure 8 Platelet aggregation in whole blood. Human blood suspensions were stimulated with the PAR-1 agonist SFLLRRN in the absence of PDNO (hollow bars) and in the presence of PDNO (black bars) or nitroglycerin (gray bars). NO donor drug was introduced into the bloodstream 2 min before PDNO (hollow bars). Platelet aggregation was analyzed as an increase in impedance over 10 min and expressed as the area under the curve. Bars represent the mean and standard deviation (n=4).

[0130] Figure 9 Increased Ser 239-specific VASP phosphorylation in platelets. Human platelet suspensions isolated by stimulation with PDNO or nitroglycerin. Platelets were exposed to NO donors for 5 min, followed by Western blotting to detect phosphorylated VASP. These experiments were performed on unstimulated and SFLLRN-stimulated platelets. PAR-1 agonists were added 2 min after PDNO / nitroglycerin. Bars represent mean and standard deviation (n=3).

[0131] Figure 10 A study protocol involving the intra-arterial infusion of 1,2-propylene glycol mono-organic nitrite (PDNO, n=12) into the organ-targeting artery during 6 hours after left common femoral artery (CFA) embolization, either in the CFA or in a control state (no infusion, n=12).

[0132] Figure 11: Blood flow (Figure A), femoral vascular resistance (FVR, Figure B), femoral venous partial pressure of oxygen (FV pO2, Figure C), femoral venous oxygen consumption (FV VpO2, Figure D), femoral venous lactate production (FV lactate production, Figure E), and the increase in lactate levels between central arterial blood and femoral venous blood (lactic acid increment, Figure F) during the 6-hour period following left common femoral artery (CFA) embolization, in either the CFA local arterial infusion of 1,2-propylene glycol mono-organic nitrite (PDNO, n=12) or the control group (no infusion, n=12). P-values ​​represent differences between groups. Data are presented as mean ± standard error of mean.

[0133] Figure 12: Mean systemic arterial blood pressure (MAP, Figure A) and end-tidal nitric oxide level (ETNO, Figure B) during the 6-hour period following left common femoral artery (CFA) embolization, either during local arterial infusion of 1,2-propylene glycol mono-organic nitrite (PDNO, n=12) at the CFA site or in the control group (no infusion, n=12). Data are presented as mean ± standard error of mean.

[0134] Example The invention is illustrated by the following examples, which are not intended to limit the general scope of the invention.

[0135] Example 1: Composition For the studies reported below, a composition was used comprising a mixture of 1-(nitrosooxy)prop-2-ol and 2-(nitrosooxy)prop-1-ol (referred to herein as PDNO) and 1,2-propanediol (PD; or propylene glycol). The composition contained 6.5 w / w% to 7.1 w / w% PDNO, with the balance being propylene glycol.

[0136] Processes for preparing compounds according to formula (I) as defined above, and compositions comprising these compounds, particularly substantially non-aqueous compositions comprising the compound and compositions for the following studies, can be found in WO 2020 / 109420, which is incorporated herein by reference.

[0137] To avoid ambiguity, compounds of formula (I) may also be referred to herein as the acronym PDNO, which indicates that such compounds (including all examples and their specific characteristics) are used in the methods and uses described with respect to the invention. Furthermore, when describing compositions containing PDNO that also contain PD, PD refers to propylene glycol corresponding to a compound of formula (I), that is, PD is according to formula (I) but wherein R 1 R 2 and R 3 The same compound representing H.

[0138] Example 2: Intra-arterial administration study PDNO exhibits ultra-rapid release and a very short half-life, the latter of which has never been definitively determined. The aim of this study was to compare intra-arterial versus intravenous (iv) infusion of PDNO in terms of local organ blood flow and systemic effects, and to estimate the effects of PDNO on... in vivo Half-life. A summary of the study is provided below, detailing the methods described.

[0139] In anesthetized, instrumented pigs (n=14), PDNO (0.01 nmol / kg) was infused intra-arterially into the left common carotid artery (CCA), left common femoral artery (CFA), left renal artery (RA), and superior mesenteric artery (SMA). -1 min -1 Up to 3000 nmol kg - 1 min -1 ) and intravenous administration of PDNO (3 nmol kg) -1 min -1 Up to 100 nmol kg -1 min -1 The results were compared. Intra-arterial and central venous injections (PDNO 0.5 μmol to 4 μmol) in the left CCA and left CFA were used to estimate the PDNO levels in anesthetized, instrumented pigs (n=2). in vivo Half-life. Measurement area blood flow, mean systemic arterial pressure (MAP), end-tidal nitric oxide (ETNO), and arterial blood gas.

[0140] From 1 nmol kg -1 min -1 Initially, during their respective intra-arterial infusions, CFA and CCA blood flow rates increased significantly from baseline, while SMA blood flow rate increased from 30 nmol / kg. -1 min -1 It begins to increase significantly, while RA infusion and IV infusion do not affect blood flow. During IV infusion, MAP is at 10 nmol / kg. -1 min -1 The decrease was significant, equivalent to 30 nmol kg during CCA and CFA infusions, respectively. -1 min -1 and 100 nmol kg -1 min -1 And 3000 nmol kg in the SMA experiment -1 min -1The ETNO dose equivalents between intravenous administration (reference) and CFA, CCA, RA, and SMA infusions were 2.7, 2.9, 2.6, and 47, respectively. This was determined by measuring the cycle time between the intra-arterial and intravenous infusion / infusion sites and using dose equivalents from infusion and injection experiments. in vivo The half-life is estimated to be 5.5 s to 6.1 s (infusion) and 3.0 s to 3.9 s (injection).

[0141] Compared to intra-arterial (RA) and intravenous (IV) infusions, organ-directed intra-arterial (CCA, CFA, and SMA) infusions of PDNO dose-dependently increase organ-specific blood flow with no or only mild systemic effects at effective doses. A significant right shift in the dose-response effect on PDNO concentration was observed when comparing intra-arterial and IV infusions, likely due to the rapid degradation of PDNO in the blood. in vivo The half-life is estimated to be in the range of 3.0 s to 6.1 s.

[0142] research group Sixteen 3-month-old domestic pigs (a crossbreed of Swedish Country Breed, Hampshire, and Yorkshire; average weight 30 kg [range 27 kg to 34 kg], sex ratio 1:1) were included in the study. These experiments were conducted at the animal laboratory of Örebro University Hospital (Örebro, Sweden) from March 7 to 18, 2022, May 9 to 13, 2022, and September 15, 2023. Ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden; approval number 00259-2022). The study was conducted in accordance with Directive 2010 / 63 / EU on the protection of animals used for scientific purposes and, where possible, followed the Aspirin Reduced Risk of Initial Vascular Events (ARRIVE) guidelines.

[0143] Surgical preparation and measurement According to previous reports (Stene Hurtsen et al.), A Comparative Study of Inhaled Nitric Oxide and an Intravenously Administered Nitric Oxide Donor in Acute Pulmonary Hypertension (Drug Des Devel Ther. 2020;14:635-645), anesthetize and ventilate the animals. Basic equipment setup (intravenous access for liquids and anesthetics, pulmonary artery catheter, urinary catheter insertion) is as previously described (Stene Hurtsen et al., , Drug Des Devel Ther. 2020;14:635-645). A Comparative Study of Inhaled Nitric Oxide and an Intravenously Administered Nitric Oxide Donor in Acute Pulmonary HypertensionThe procedure was performed using a catheter placed in both brachial arteries under ultrasound guidance. A 5-Fr catheter (Cordis Corporation, Florida, USA) was placed in the right brachial artery for catheter access, and a 4-Fr catheter (Cordis Corporation, USA) was placed in the left brachial artery for arterial blood pressure measurement and arterial blood gas analysis.

[0144] Preparation of the femoral and carotid arteries The femoral and common carotid arteries were equipped with flow probes (3 mm, Transonic System Inc., New York, USA). Using a guidewire (Terumo Radiofocus® STIFF type, 0.035 angle, Terumo Corporation, Tokyo, Japan), the angiography catheter (Soft-VuBerenstein 5Fr, Queensbury, NY, USA or SOFT-VU Sos Omni® selective 5 Fr, Queensbury, NY, USA) was positioned in the left carotid or left femoral artery via a 5 Fr guide (Cordis Corportation, USA). Position was confirmed by angiography. A microcatheter (TerumoProgreat®, 2.7 Fr. Terumo cooperation, Tokyo, Japan) was inserted into the angiography catheter and positioned proximal to the flow probe. The angiography catheter was then withdrawn to the brachiocephalic trunk in the carotid artery test and to the external iliac artery in the femoral artery test. In the femoral artery experiment, peripheral venous access was obtained in the superficial veins of the distal lower extremity (BD venflon pro safety, 0.9 mm, Switzerland).

[0145] In both animals, PDNO was repeatedly injected via microcatheters in the left common femoral artery (1 μmol to 4 μmol), left carotid artery (0.5 μmol to 2 μmol), and CVC in the superior vena cava (0.5 μmol to 4 μmol). Additionally, 10 ml of 5% sodium bicarbonate solution was injected at these sites.

[0146] Renal artery preparation and SMA preparation A 3 mm flow probe (Transonic System, USA) was delivered to the renal artery via a retroperitoneal incision, and the SMA was closed via laparotomy using a 6 mm flow probe (Transonic System, USA). A microcatheter (Rebar) was delivered to the target artery in a similar manner to that described previously. TM-18 Microcatheter, Microtherapeutics, Inc., California, USA), used to withdraw the angiography catheter to the aorta in both the renal artery test and the SMA test.

[0147] After the equipment was installed, 5000 E heparin (Leo Pharma A / S, Denmark) was administered, followed by a no-intervention period. The animals were covered with heating blankets to maintain normal body temperature. At the end of the experiment, the animals were given an overdose of propofol, followed by 20 mL of potassium chloride (2 mM, B. Braun Medical Inc., Pennsylvania, USA), and euthanasia was confirmed by the absence of systolic blood pressure and ETCO2.

[0148] Femoral artery test and carotid artery test PDNO was infused using an infusion pump and carrier solution (1.4% bicarbonate) via a microcatheter at eight doses (0.01 nmol kg). -1 min -1 0.1 nmol kg -1 min -1 1 nmol kg -1 min -1 3 nmol kg -1 min -1 10 nmol kg - 1 min -1 30 nmol kg -1 min -1 100 nmol kg -1 min -1 Administered at 300 nmol kg⁻¹ min⁻¹ over 20 minutes in the left common femoral artery and left carotid artery, in block randomization (Figure 1A (carotid and femoral arteries) and Figure 1B (renal and superior mesenteric arteries)). Between local intra-arterial infusion routes, four doses (3 nmol kg⁻¹ min⁻¹, 20 minutes each) were administered via a central venous catheter. -1 min -1 10 nmol kg -1 min -1 30 nmol kg -1 min -1 and 100 nmol kg -1 min -1 (Each dose is administered intravenously over 10 minutes) as a reference. There is a 60-minute washout period between PDNO administrations.

[0149] Renal artery test and superior mesenteric artery (SMA) test Eight doses of PDNO (0.01 nmol kg) were administered via microcatheter into the left renal artery. -1 min -1 0.1 nmolkg -1 min -1 1 nmol kg -1 min -1 3 nmol kg -1 min -1 10 nmol kg -1 min -1 30 nmol kg -1 min -1 100 nmol kg -1 min -1 and 300 nmol kg -1 min -1 Each dose is administered over 20 minutes. In SMA, 0.06 E kg of vasopressin (Empressin®, OrPha Swiss) is simultaneously infused. -1 min -1 (40 IU 2 ml -1 During this period, there were 7 doses (3 nmol kg). -1 min -1 10 nmol kg -1 min -1 30 nmol kg -1 min -1 100 nmol kg -1 min -1 300 nmol kg - 1 min -1 1000 nmol kg -1 min -1 and 3000 nmol kg -1 min -1 Each dose is administered over 20 minutes. Intravenous infusion is given between local arterial infusions, with a 60-minute washout period in between.

[0150] Data collection Hemodynamic and ventilation parameters, including local blood flow to the target organ and exhaled nitric oxide fraction (FENO; CLD 77, ECO PHYSICS, Switzerland) (Acqknowledge Software, BIOPAC® Systems Inc, California, USA), were continuously recorded. Arterial blood gas analysis was collected at the end of all doses (GEM5000, Werfen, Massachusetts, USA).

[0151] Data analysis and statistics The primary outcomes were effects on regional blood flow, systemic blood pressure, and end-tidal nitric oxide (ETNO). These data are presented as means with standard error of mean (SEM). Normality was determined using the Shapiro-Wilk normality test, and linear mixed models were performed using SPSS (version 27, IBM Corp., Armonk, NY, USA) to evaluate changes relative to baseline, with graphs created in GraphPad Prism 9.4.1 (GraphPad Software, Inc., San Diego, CA, USA). Secondary outcomes were hemodynamic and blood gas measurements. Intra-arterial and intravenous dose comparisons were performed. Linear mixed models were fitted where the fixed effects were route of administration (classification), dose (continuous), and the interaction between dose and route, and the random effect was subject ID (from...). nlme v.3.1-16 lme (Using R4.2.2(16)). To compare dosages, use ANOVA Function comparison of estimated coefficients (using emmeans v.1.8.4-1 lstrends (Estimated) and adjusted using Tukey's HSD test. The circulation time from the intra-arterial injection / infusion site (carotid and femoral arteries) to the intravenous injection / infusion site was calculated by subtracting the time from the injection of PDNO or sodium bicarbonate into the intra-arterial catheter and the first expiration with the increase of ETNO and end-tidal CO2, respectively. Then, it was estimated by linear regression analysis. in vivo Half-life (ln N(t) = kxt + ln (N(0)) and T) 1 / 2 = ln 2 / k). A p-value < 0.05 is considered statistically significant.

[0152] Hemodynamic results During intra-arterial administration of PDNO at each infusion site, left femoral artery blood flow and left carotid artery blood flow were within 1 nmol / kg. -1 min -1 The flow rate of SMA began to increase from 30 nmol / kg.-1 min -1 The rate of increase began to rise (P < 0.05 compared to baseline, Figures 2A and 2B (left carotid artery), Figures 2C and 2D (left femoral artery), and Figures 3A and 3B (SMA)). The contralateral (i.e., right) artery was unaffected in the femoral artery test, but increased at levels above 30 nmol / kg in the carotid artery test. -1 min -1 The mean systemic arterial pressure (MAP) increased significantly (P < 0.05, Figure 2A) in both the carotid and femoral artery tests. - 1 min -1 and 100 nmol kg -1 min -1 The pressure began to decrease (P < 0.05, as shown in Figures 2B and 2D, respectively); and in the SMA experiment, the mean systemic arterial pressure (MAP) was at 3000 nmol / kg. -1 min -1 The effect began to decrease (P < 0.05, Figure 3B). Intravenous administration of PDNO 10 nmol / kg was observed. -1 min -1 The MAP was affected accordingly (P > 0.05, Figures 2B, 2D, 3B, and 3D). Except for the last three doses in the carotid artery experiment, cardiac output remained unchanged during the experiment (P < 0.05). These results surprisingly suggest that intra-arterial administration of PDNO can be performed at higher doses than intravenous administration to achieve increased local therapeutic efficacy without overall systemic effects. Furthermore, unexpectedly, intravenous PDNO infusion did not affect organ blood flow compared to intra-arterial infusion, indicating the need for an intra-arterial route to effectively treat systemic organs.

[0153] ETNO In the femoral artery test, carotid artery test, and renal artery test, end-tidal NO (ETNO) at 30 nmol / kg - 1 min -1 The rate of increase began to rise (P < 0.05 compared to baseline) in SMA experiments at 3000 nmol / kg. -1 min -1 (Compared to baseline, P < 0.05, Figures 2B, 2D, 3B, and 3D). The corresponding increase in intravenous experiments was at 10 nmol / kg. -1 min -1 (P < 0.05 compared to baseline, Figures 2 and 3).

[0154] For femoral artery infusion, carotid artery infusion, renal artery infusion, and SMA infusion, using logETNO, the estimated coefficients between the administration routes and therefore the estimated dose ratios of the equivalent effects compared to intravenous infusion were 2.7, 2.9, 2.6, and 47, respectively, with intravenous PDNO infusion as a reference (Figure 4).

[0155] These results are consistent with those observed in hemodynamics.

[0156] Arterial blood gas Compared with baseline, the methemoglobin fraction was significantly higher in the SMA experiment at the two highest doses (P < 0.05). Figure 5 Other routes of administration did not alter the methemoglobin fraction, which was particularly surprising and unexpected. In the carotid and femoral artery tests, SaO2 at 30 nmol / kg... -1 min -1 and 100 nmol kg -1 min -1 The value decreased significantly (P < 0.05 compared to baseline).

[0157] Estimation of circulation time and half-life from artery to vein The median (25%–75%) cycle times from the intracarotid and femoral artery injection sites to the first expiration of increased ETNO were 13.5 s (12.3–15.4, n = 13) and 13.7 s (12.9–15.2, n = 9), respectively, and the median cycle times from the central venous injection site to the first expiration of increased ETNO were 5.0 s (4.3–5.9, n = 29) and 5.3 s (3.3–5.8, n = 9), respectively. As depicted in Table 1, the cycle times between the intracarotid and femoral artery injection sites and the central venous injection site were calculated to be 8.5 s and 8.4 s, respectively. Using the increase in ETNO integral caused by intra-arterial and central venous PDNO injection and the cycle times at each injection site in linear regression analysis, assuming first-order kinetics, in vivo The summative half-life of PDNO was estimated to be 3.6 s (3.0–4.4, 95% CI; half-life estimates for each dose are shown in Table 2 below). The dose-equivalent ratios of PDNO were estimated using the same equation from the carotid artery infusion, femoral artery infusion, and central venous infusion experiments (Figures 4A and 4B). in vivo The average half-life is 5.8 s.

[0158] Table 1: Half-life estimates based on injection and infusion experiments of PDNO in two anesthetized pigs. Circulation time between intra-arterial and intravenous injection sites was also estimated by injecting 10 ml of 5% sodium bicarbonate solution. Dose ratios (DRs) are derived from intra-arterial and intravenous infusion experiments (Figures 1 to 10 ml). Figure 5 ).

[0159]

[0160] in conclusion Compared to renal artery infusion and intravenous infusion, organ-directed intra-arterial (carotid, femoral, and supramesenteric) infusion of PDNO dose-dependently increases organ-specific blood flow. The effective doses required to increase carotid, supramesenteric, and femoral artery blood flow have no or only a slight effect on systemic blood pressure. A significant right shift in the dose-response effect on end-tidal NO concentration was observed when comparing intra-arterial and intravenous injection, likely due to the rapid breakdown of PDNO in the blood. Intra-arterial organ-directed infusion doses can reach up to 300 nmol / kg. -1 min -1 During this period, methemoglobin levels were below 3%. PDNO in vivo The half-life varies slightly depending on the experimental setup, but is estimated to be in the range of 3.0 s to 6.1 s.

[0161] Example 3: Antiplatelet aggregation effect of intravenous PDNO administration method research group Five 3-month-old domestic pigs (a crossbreed of Swedish country breed, Hampshire, and Yorkshire) were used. They had free access to food and water before the morning of the experiments. Ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden; Approval No. 00259-2022). These experiments were conducted in the animal laboratory at Örebro University Hospital (Örebro, Sweden) and in accordance with Directive 2010 / 63 / EU on the protection of animals used for scientific purposes.

[0162] Animal preparation and monitoring According to Stene Hurtsén et al. (Stene Hurtsen et al.), A Comparative Study of Inhaled Nitric Oxide and an Intravenously Administered Nitric Oxide Donor in Acute Pulmonary Hypertension (Drug Des Devel Ther. 2020;14:635-645) Establishing and maintaining anesthesia and ventilation, the difference being that the preoperative medication was changed to 0.05 ml / kg. -1 Zoletil forte (50 mg / ml each)-1 ) and Medetomidin (1 mg ml) -1 A mixture of Domitor Vet. Basic instruments (arterial catheter, pulmonary artery catheter, urinary catheter insertion) are as previously described (Stene Hurtsen et al.). A Comparative Study of Inhaled Nitric Oxide and an Intravenously Administered Nitric Oxide Donor in Acute Pulmonary Hypertension (Drug Des Devel Ther. 2020;14:635-645). 5 ml kg was administered via arterial catheter. -1 Blood was drawn into a 50 ml syringe, and a moderate dose of thrombin (0.1 U / ml) was administered. -1 To promote blood clotting in the syringe for subsequent pulmonary embolization, a 20 Fr catheter was inserted into the right jugular vein to inject the clotting blood. Intravenous access for fluids and anesthetics was established via a 7 Fr guide (Cordis Corportation, USA) in the left external jugular vein. Exhaled nitric oxide (NO) levels were measured via an endotracheal tube (Ecophysics, Diernten, Switzerland). The animals were covered with heating blankets to maintain normal body temperature. After the experiment, the animals were given an overdose of propofol, followed by 20 mL of potassium chloride (B. Braun Medical Inc., Pennsylvania, USA), and euthanasia was confirmed by ECG and ETCO2.

[0163] Experimental protocol After the intervention period, pulmonary embolism was induced by injecting coagulated blood into the right external jugular vein to achieve a target mean pulmonary artery pressure of 45 mmHg to 55 mmHg. Simultaneously, intravenous norepinephrine infusion was initiated and titrated to maintain a mean systemic arterial pressure above 60 mmHg. The fractional oxygen inhaled (FiO2) was increased to maintain normal arterial oxygenation, and the respiratory rate was increased to be adjusted for partial hypercapnia. Approximately 15 minutes later, 160 nmol / kg was started. -1 min -1 PDNO was administered intravenously for approximately 15 minutes, and then the infusion was stopped. After the experiment, the animals were euthanized by rapid intravenous injection of 40 mM potassium chloride following a bolus injection of 200 mg propofol. Cardiac arrest was confirmed using hemodynamic and respiratory measurements.

[0164] Hemodynamic and ventilatory parameters, including end-tidal NO concentration, were continuously recorded (AcqknowledgeSoftware, BIOPAC® Systems Inc., California, USA). Recordings were taken at baseline (pre-pulmonary embolization), post-pulmonary embolization, and at PDNO 160 nmol / kg. -1min -1 Arterial and mixed venous blood samples were collected during infusion and 15 min after PDNO infusion was stopped. Arterial and mixed venous blood samples were analyzed using a blood gas analyzer (GEM5000), and arterial blood samples collected from the heparin syringe were immediately transferred to cuvettes for multiplate aggregation analysis. Blood was stimulated with the PAR-1 agonist peptide SFLLRN, and platelet aggregation (manifested as an increase in electrical resistance) was tracked over a 10-minute period and calculated as the area under the curve (see the method in Example 4 for details).

[0165] Statistical analysis The primary outcome was the change in platelet aggregation induced by the PAR-1 agonist peptide SFLLRN between pulmonary embolization and intravenous PDNO infusion (P < 0.05 was observed by paired comparisons [Dunn's test], with Friedman's test showing P < 0.05 if all four time points were included). Secondary outcomes were the comparison of changes in hemodynamic variables, end-tidal NO, and blood gas values ​​between pulmonary embolization infusion and no PDNO infusion. No statistical analysis was performed on secondary outcomes.

[0166] result Prior to pulmonary embolization, the animals had normal hemodynamic and respiratory variables. Pulmonary embolization induced acute pulmonary hypertension, accompanied by systemic hypotension (reversible by norepinephrine infusion), arterial deoxygenation (reversible by increasing FiO2), hypercapnia (partially reversible by increasing respiratory rate), and increased end-tidal NO. Intravenous PDNO infusion resulted in decreased pulmonary and systemic vascular resistance and increased end-tidal NO; these effects were reversed upon discontinuation of PDNO infusion. Compared with pre-PDNO infusion, intravenous PDNO infusion during pulmonary embolization statistically significantly reduced PAR-1 agonist peptide SFLLRN-induced platelet aggregation (P=0.0143). Figure 6 ).

[0167] in conclusion Data showed that intravenous PDNO during pulmonary embolization surprisingly inhibited platelet aggregation. In vitro (Measurement), and is an effective treatment for thromboembolic diseases. Combined with the local effects (such as vasodilation) achieved by the application of PDNO, the present invention can further provide an improved treatment for thromboembolic diseases.

[0168] Example 4: Other antithrombotic studies Platelet aggregation in whole blood The efficacy of PDNO was evaluated by analyzing the aggregation reaction of thrombin-mimicking hexapeptide SFLLRN. in vitroPotential antithrombotic effect. This peptide is an agonist of the major thrombin receptor (called protease-activated receptor-1 (PAR-1)). The inhibitory effect of PDNO was elucidated in isolated human platelet suspensions and whole blood.

[0169] Heparinized human or porcine blood was diluted 1:1 (0.5 ml) with Krebs-Ringer glucose (KRG) buffer (isoketone saline, pH 7.4). The diluted blood was transferred to analytical cuvettes and placed in a multiplate aggregator. In vitro (human blood) and [other blood samples] were measured by the increase in impedance between two platinum electrodes. In vitro Platelet aggregation in (pig blood). Measurements were taken under stirring conditions at 37°C.

[0170] exist in vitro In the experiment, PDNO or nitroglycerin (used as a control drug) was introduced 2 min before the hexapeptide SFLLRN. This hexapeptide acts as an agonist of the major thrombin receptor (called protease-activated receptor-1 (PAR-1)). In vitro In the experiment, anesthetized pigs were administered PDNO intravenously. After blood collection, the diluted blood was transferred to a Multiplate instrument and stimulated with SFFLRN. Aggregation responses were recorded over 10 minutes and expressed as the area under the curve.

[0171] The potential anti-aggregation activity of PDNO was investigated by measuring the increase in transmittance in aliquots of platelets separated after the addition of SFLLRN (Born aggregation assay). Figure 7 As shown, PDNO concentration-dependently inhibits SFLLRN-induced platelet aggregation, indicating that it has antithrombotic activity.

[0172] Aggregation of separated platelets The aggregation of platelets in whole blood (human blood containing heparin as an anticoagulant) was analyzed as an increase in resistance (the impedance change between two platinum electrodes) using a multiplate aggregator.

[0173] Heparinized blood was obtained from healthy volunteers and immediately mixed with acidic citrate glucose (ACD) solution, with a recommended volume of 5 parts blood to 1 part ACD. Platelets were centrifuged at 220 x g for 20 min to obtain platelet-rich plasma. Subsequently, platelets were precipitated by a second centrifugation (480 x g for 20 min) and gently resuspended in KRG buffer. 0.3 ml of the separated platelet suspension (2.5 x 10⁸ platelets / ml) was placed in a Chrono-log aggregator and analyzed under stirring at 37°C. Platelets were stimulated with PDNO or nitroglycerin for 2 min, followed by activation with SFLLRN. Platelet aggregation was assessed as an increase in transmittance (%) through a cuvette (transmittance in platelet-free KRG represents 100%).

[0174] Figure 8 The results showed that under these experimental conditions, PDNO, rather than nitroglycerin, significantly inhibited SFLLRN-induced platelet aggregation. This finding indicates that PDNO exerts an antithrombotic effect even in the presence of whole blood and hemoglobin.

[0175] Detection of protein phosphorylation by Western blotting In platelets and vascular smooth muscle cells, the cytoskeleton-binding protein vasodilator-stimulated phosphoprotein (VASP) is a major molecular target of the NO / cyclic guanosine monophosphate (GMP) signaling pathway. Ser239-specific phosphorylation of VASP after platelet exposure to NO is considered a key molecular mechanism of NO's anti-aggregation / anti-thrombotic effects. This study analyzed the effect of PDNO on Ser239-specific VASP phosphorylation using Western blotting.

[0176] Aliquots of the separated platelet suspension were exposed to PDNO or nitroglycerin for 5 min. This series of experiments was performed on both resting platelets and SFLLRN-stimulated platelets.

[0177] Specifically, NO / ring GMP-induced Ser239 phosphorylation on vasodilator-stimulated phosphoprotein (VASP) was detected by Western blotting. Platelet suspensions (0.2 ml) were stimulated with PDNO or nitroglycerin for 5 min in the absence or presence of SFLLRN. A hexapeptide was added 2 min after NO donation. Platelets were lysed with 50 µl of sodium dodecyl sulfate sample buffer and the proteins were further denatured at 95 °C for 5 min. Platelet proteins were separated by electrophoresis and then blotted onto a polyvinylidene fluoride membrane. To determine VASP phosphorylation, the membrane was incubated with a Ser239-specific VASP antibody, followed by a horseradish peroxidase antibody, and visualized using a Fuji LAS chemiluminescence meter.

[0178] like Figure 9 As shown, PDNO, rather than nitroglycerin, induced a significant increase in Ser239-specific VASP phosphorylation. These data indicate that PDNO induces significant NO / cyclic GMP signaling in human platelets, and this is associated with significant anti-aggregation effects. Figure 7 and Figure 8 ).

[0179] Example 5: Intra-arterial infusion of PDNO for the treatment of acute lower extremity embolism research group Twenty-four 3-month-old domestic pigs (a crossbreed of Swedish country breed, Hampshire, and Yorkshire (sex ratio 1:1, weight 32 ± 4 kg)) were used. They had free access to food and water before the morning of the experiments. Ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden; approval number 00259-2022). These experiments were conducted from September 12 to 30, 2022, and January 24 to 31, 2023, in the animal laboratory of Örebro University Hospital, following Directive 2010 / 63 / EU on the protection of animals used for scientific purposes.

[0180] Animal preparation and monitoring According to Stene Hurtsén et al. (Stene Hurtsen et al.), A Comparative Study of Inhaled Nitric Oxide and an Intravenously Administered Nitric Oxide Donor in Acute Pulmonary Hypertension (Drug Des Devel Ther. 2020;14:635-645) For the first 14 animals, and for the last 10 animals, the preoperative medication was changed to 0.05 ml / kg. -1 Zoletil forte (250 mg + 250 mg) and Medetomidin (1 mg ml -1 A mixture of Domitor Vet and other instruments. Basic instruments (arterial catheter, pulmonary artery catheter, urinary catheter insertion) are as previously described (Stene Hurtsen et al.). A Comparative Study of Inhaled Nitric Oxide and an Intravenously Administered Nitric Oxide Donor in Acute Pulmonary Hypertension (Drug Des Devel Ther. 2020;14:635-645). 1 ml kg was drawn via arterial catheter using a 5 ml syringe (Cordis Medical ApS, Denmark). -1Blood was used for subsequent embolization. Intravenous access for fluids and anesthetics was established via a 7 Fr guide (Cordis Corportation, USA) in the left external jugular vein. A 5 Fr catheter (Soft-Vu Berenstein 5Fr, Queensbury, NY, USA) was placed through the left venous access and advanced to the common femoral vein at the deep vein bifurcation. Similarly, a 5 Fr catheter (Soft-Vu Berenstein, USA) was inserted into the brachial artery via a 5 Fr (Cordis Corportation, USA) guide and advanced to the common femoral artery (CFA). Position was confirmed by fluoroscopy. Blood flow probes (Transonic Systems Inc., NY, USA) were placed on either side of the CFA, 3 mm on the left and 6 mm on the right. A laser Doppler probe (Perimed, Sweden) was placed in the peroneus muscle of the hind limb, and a microdialysis catheter (MDialysis AB, Sweden) was placed in the tibialis anterior muscle. Exhaled nitric oxide was measured via an endotracheal tube (Eco Physical, Diernten, Switzerland). The animals were covered with heating blankets to maintain normal body temperature. After the experiment, the animals were given an overdose of propofol, followed by 20 mL of potassium chloride (B. Braun Medical Inc., Pennsylvania, USA), and euthanasia was confirmed by ECG and ETCO2.

[0181] Experimental protocol After the intervention period, 1 ml / kg of autologous blood clot was administered via a 5 Fr catheter (Berenstein, USA). -1 (Clothing time at least 2 hours) 5 ml min -1 Selective embolization of the left common femoral artery. One hour post-embolization, participants were randomly assigned to receive PDNO3 nmol kg. -1 min -1 Treatment group or control group (no infusion) Figure 10 NO donors were administered via a microcatheter (Terumo Progreat®, 2.7Fr., Terumo cooperation, Tokyo, Japan) using an infusion pump with a carrier solution (1.4% bicarbonate). This microcatheter was inserted into the right brachial artery via a 5Fr catheter (Berenstein, USA) and guided to the common femoral artery. In both groups, the 5Fr catheter (Berenstein, USA) was withdrawn to the aortic bifurcation. Reperfusion time was 6 hours. At the end of the protocol, muscle biopsies were performed on both tibialis anterior muscles and formalin (Solveco AB, Russäberg, Sweden) for blinded histopathological evaluation.

[0182] Continuous recording of hemodynamic and ventilation parameters, including local blood flow in CFA (AcqknowledgeSoftware, BIOPAC® Systems Inc, California, USA). Laser Doppler signals, exhaled nitric oxide fraction (FENO, instrument), arterial blood samples, local venous blood samples, and blood gas analysis were collected according to protocol. Figure 10 ).

[0183] The primary outcome was left femoral artery blood flow. Secondary outcomes were ischemic index scores at 6 hours (concentrations of lactate, potassium, aspartate aminotransferase, lactate dehydrogenase, and creatine kinase (Ref. 19667877)), lower extremity microcirculation (via laser Doppler), and histopathology of lower extremity muscle biopsies. Side effects (methemoglobinemia, systolic blood pressure) were measured. Figure 10 Collect measurement values.

[0184] calculate Femoral vein resistance (FVR): Femoral artery blood flow / MAP.

[0185] Lactate increment (Δlactic acid): central arterial lactate level - femoral vein lactate level.

[0186] Lactic acid production: femoral artery blood flow × Δlactic acid × -1.

[0187] Central arterial saturation and femoral vein saturation: (partial pressure of oxygen [pO2] 2,94) / (pO2 2,94 + (4,76 × (10 (-0,441 × (pH-7,4))))^2,94).

[0188] O2 content in the central artery and femoral vein: ((saturation × Hb) / 10 × 1.31 + 0.025 × pO2) × 10.

[0189] Femoral vein O2 consumption: (arterial O2 content - femoral vein O2 content) × (femoral artery blood flow / 1000).

[0190] Statistical analysis Normality was analyzed using the Shapiro-Wilk test. A linear mixed model was used to analyze the normally distributed data, with group and time as the primary factors, followed by interactions, and then multiple interactions. Normally distributed data are presented as mean ± standard error of mean. Data deviating from the normal distribution were analyzed using the Mann-Whitney U test and presented as median (interquartile range [IQR] from the 25th to the 75th percentile). All statistics were performed in SPSS (version 27, IBM Corp., Armonk, NY, USA) and graphs were generated in GraphPad Prism 9.4.1 (GraphPad Software, Inc., San Diego, CA, USA).

[0191] result The PDNO group and the control group were similar at baseline, but the control group had higher cardiac output (CO) and femoral venous oxygen consumption (FV O2) at baseline. Blood flow in the common femoral artery (CFA) disappeared in both groups 15 minutes after embolization, and the blood flow in the PDNO group was significantly higher after the start of infusion (Figure 11). Similarly, the femoral venous resistance (FVR) in the PDNO group was significantly lower 1 hour after infusion (Figure 12). Heart rate, CO, and MAP were similar among the groups.

[0192] Compared with the control group, the PDNO group had higher, but not significantly higher, pO2, FV VO2, and FV lactate production in the femoral vein (FV) (Figure 11). Three hours after infusion, the femoral vein CO2 level was significantly higher in the control group, while the FV O2 level was generally higher in the PDNO group, but not significantly. Similarly, the PDNO group had higher ETNO and MHb levels and generally lower, but not significantly, pO2 (Figure 12).

[0193] in conclusion Data showed that, compared with the control group, intra-femoral artery infusion in experimental acute limb embolism in pigs increased femoral blood flow without systemic side effects. Further analysis will indicate whether this vasodilatory effect also translates into improved metabolism and reduced lower limb injury.

Claims

1. A compound of formula (I): (I) Where R 1 R 2 and R 3 Each can be independently represented as H or -NO. Where n is 0 or 1; When n is 0, R 1 For H; and When n is 1, R 2 For H, The condition is R 1 R 2 and R 3 At least one of them represents -NO, The compound of formula (I) is used to treat a condition, wherein the compound of formula (I) is administered via intra-arterial infusion at a dose of approximately 0.01 nmol kg. -1 min -1 Up to 3000 nmol kg -1 min -1 The appropriate dose should be administered to the patient who requires it.

2. The compound used according to claim 1, wherein the compound of formula (I) is present at about 0.01 nmol kg -1 min -1 Approximately 300 nmol kg -1 min -1 For example, about 1 nmol kg -1 min -1 Approximately 300 nmol kg -1 min -1 Such as approximately 1 nmol kg -1 min -1 Approximately 100 nmol kg -1 min -1 or about 10 nmol kg -1 min -1 Approximately 3000 nmol kg -1 min -1 Dosage administration.

3. The compound used according to any of the preceding claims, wherein the compound of formula (I) is present at a concentration of about 1 nmol / kg. -1 min -1 Approximately 30 nmol kg -1 min -1 Dosage administration.

4. The compound used according to any of the preceding claims, wherein the compound of formula (I) is applied continuously, optionally for a period of up to 14 days, such as 7 days.

5. The compound used according to any of the preceding claims, wherein the compound of formula (I) is administered to the femoral artery and / or carotid artery via intra-arterial infusion.

6. The compound used according to any one of claims 5, wherein the compound of formula (I) is administered via intra-arterial infusion at a dose of about 1 nmol / kg. -1 min -1 Approximately 300 nmol kg -1 min -1 Such as 1 nmol kg -1 min -1 Approximately 60 nmolkg -1 min -1 For example, 1 nmol kg -1 min -1 Approximately 50 nmol kg -1 min -1 Such as 1 nmol kg -1 min -1 Approximately 30 nmol kg -1 min -1 The dose is administered to the femoral artery and / or the carotid artery.

7. The compound used according to any one of claims 1 to 4, wherein the compound of formula (I) is administered to the superior mesenteric artery via intra-arterial infusion.

8. The compound used according to claim 7, wherein the compound of formula (I) is administered via intra-arterial infusion at a dose of about 3 nmol kg. -1 min -1 Approximately 3000 nmol kg -1 min -1 Such as approximately 30 nmol kg -1 min -1 Approximately 1000 nmol kg -1 min -1 For example, about 100 nmol kg -1 min -1 Approximately 1000 nmol kg -1 min -1 The dose was administered to the superior mesenteric artery.

9. The compound used according to any one of claims 1 to 4, wherein the compound of formula (I) is administered to the renal artery via intra-arterial infusion.

10. The compound used according to claim 9, wherein the compound of formula (I) is administered via intra-arterial infusion at a dose of about 0.01 nmol / kg. -1 min -1 Approximately 300 nmol kg -1 min -1 For example, about 1 nmol kg -1 min -1 Approximately 30 nmol kg -1 min -1 The dose was administered to the renal artery.

11. The compound used according to any of the preceding claims, wherein the compound is used to treat a condition in which NO has a beneficial effect.

12. The compound used according to any of the preceding claims, wherein the symptom is selected from the group consisting of: Acute pulmonary vasoconstriction of various causes; pulmonary hypertension of various causes, including primary and secondary hypertension; preeclampsia; eclampsia; conditions requiring vasodilation of various causes; erectile dysfunction; systemic hypertension of various causes; regional vasoconstriction of various causes; local vasoconstriction of various causes; acute heart failure (with or without preserved ejection fraction [HFpEF]); coronary artery disease; myocardial infarction; ischemic heart disease; angina pectoris; unstable angina pectoris; arrhythmias; acute pulmonary hypertension in patients undergoing cardiac surgery; acidosis; airway inflammation; cystic fibrosis; COPD; immobile ciliary syndrome; pulmonary inflammation; pulmonary fibrosis; acute lung injury (ALI); adult respiratory distress syndrome; acute pulmonary... Edema; acute mountain sickness; asthma; bronchitis; hypoxia of various causes; ischemic diseases of various causes; stroke; cerebral vasoconstriction; gastrointestinal inflammation; gastrointestinal dysfunction; gastrointestinal complications; IBD; Crohn's disease; ulcerative colitis; liver disease; pancreatic disease; urethritis and cystitis; cystitis and ureteritis; skin inflammation; diabetic ulcers; diabetic neuropathy; psoriasis; inflammation of various causes; wound healing; organ protection under ischemia-reperfusion conditions; organ transplantation; tissue transplantation; cell transplantation; acute kidney disease; uterine relaxation; cervical relaxation; thromboembolic diseases, including diseases complicated by thromboembolism, such as various hematologic disorders; arterial occlusion, such as arterial embolism; and conditions requiring smooth muscle relaxation.

13. The compound used according to claim 12, wherein the condition to be treated is selected from the group consisting of: ischemic diseases of various causes; thromboembolic diseases, including diseases complicated by thromboembolism, such as various hematologic disorders; arterial thrombosis; peripheral ischemia (limb ischemia); thromboembolic stroke; pulmonary embolism; acute mesenteric ischemia (mesenteric artery occlusion); acute renal artery occlusion; arterial stenosis; arterial occlusion; splenic infarction; hepatic infarction; pulmonary infarction; Kawasaki disease; and arterial embolism.

14. A compound of formula (I): (I) Where R 1 R 2 and R 3 Each can be independently represented as H or -NO. Where n is 0 or 1; When n is 0, R 1 For H; and When n is 1, R 2 For H, The condition is R 1 R 2 and R 3 At least one of them represents -NO, The compound of formula (I) is used to treat thromboembolic diseases.

15. The compound used according to claim 14, wherein the compound is administered via intravenous infusion and / or intra-arterial infusion.

16. The compound used according to claim 14 or claim 15, wherein the compound is present at about 0.01 nmol / kg. -1 min -1 Up to 3000 nmol kg -1 min -1 Dosage administration.

17. The compound used according to any one of claims 14 to 16, wherein the compound is present at about 0.01 nmol / kg. -1 min -1 Approximately 300 nmol kg -1 min -1 For example, about 1 nmol kg -1 min -1 Approximately 300 nmol kg -1 min -1 Dosage administration.

18. The compound used according to claim 17, wherein the compound is present in a concentration of about 1 nmol / kg. -1 min -1 Approximately 300 nmol kg -1 min -1 Dosage administration.

19. The compound used according to claim 17, wherein the compound is present in a concentration of about 1 nmol / kg. -1 min -1 approximately 10 nmol kg -1 min -1 Dosage administration.

20. The compound used according to any one of claims 14 to 19, wherein the compound is administered to the femoral artery, carotid artery, renal artery or superior mesenteric artery via intra-arterial infusion.

21. The compound used according to any one of claims 14 to 20, wherein the thromboembolic disease to be treated is selected from the group consisting of: arterial thrombosis; peripheral ischemia (limb ischemia); thromboembolic stroke; pulmonary embolism; acute mesenteric ischemia (mesenteric artery occlusion); acute renal artery occlusion; arterial stenosis; arterial occlusion; and arterial embolism.

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