Mono- and bis-nitrosylated propanediols for use in the treatment of thromboembolic diseases

Abstract

The present invention relates to a method for treating a pathological condition, which comprises administering a certain mono- and / or bis-nitrosylated propanediol (including its compositions and formulations), the administration of which is to a patient in need via intra-arterial infusion at a dose of approximately 0.01 to 3000 nmol kg. -1 min -1 The present invention relates to the use of mono- and / or bis-nitrosylated propanediols for the treatment of thromboembolic diseases.

Description

[Technical Field]

[0001] The present invention relates to a method for treating a pathological condition, which comprises administering a certain mono- and / or bis-nitrosylated propanediol (including its compositions and formulations), the administration of which is to a patient in need via intra-arterial infusion at a dose of approximately 0.01 to 3000 nmol kg. -1 min -1 It is the dosage.

[0002] The present invention also relates to the use of mono- and / or bis-nitrosylated propanediols for the treatment of thromboembolic diseases. [Background technology]

[0003] Any list or discussion of documents that are clearly previously published in this specification should not be taken as an endorsement that such documents are part of the latest technology or common general knowledge.

[0004] Organ ischemia, dysfunction, and failure after major surgery and in serious illness are serious complications that lead to significant comorbidities and death. There is a lack of specific medical therapies targeted at treating ischemia (e.g., increasing blood flow through vasodilation, enhancing collateral flow, avoiding local microthrombus / embolus formation through platelet inhibition, and reducing reperfusion injury through inhibition of oxygen radical production). Specifically, uncontrolled platelet activation is a significant pathological event in acute thromboembolism, vascular occlusion, and ischemic tissue injury, including mechanisms of thromboinflammation and immunothrombosis. Pulmonary embolism represents a disease state in which interactions between several key components, such as the cardiovascular and respiratory systems, inflammation, and coagulation, manifest.

[0005] One common and serious form of organ ischemia is a condition caused by acute arterial occlusion, a medical emergency where the damage caused depends on how long the affected organ can withstand ischemia, which can range from a few minutes for the brain to about 4-6 hours for the limbs. Symptoms of arterial occlusion include pain and loss of function. The longer these symptoms persist, the less likely the organ is to be saved. Dissolving or removing blood clots is crucial for revascularizing the tissue.

[0006] Nitric oxide (NO) is an important molecule in several biological systems. It has become widely recognized that endogenous NO plays a crucial role as a mediator of vasodilation in blood vessels.

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

[0008] NO donors have been used to treat heart disease since the mid-19th century, with nitroglycerin being the most well-known. However, currently available NO donors have problems, including the risk of side effects due to the development of tolerance and the release of NO throughout the circulatory system. Therefore, the most widely used existing organic nitrate NO donors require increasingly higher doses to maintain their effectiveness. For example, when increasing the dose of an existing NO donor in the treatment of tissues at risk of ischemic damage, the induced vasodilatory effect can cause a dangerous drop in blood pressure as blood vessels throughout the vascular system widen due to the released NO, thus limiting the usefulness of currently available NO donors.

[0009] Since the discovery that nitroglycerin and related compounds act through NO release, considerable resources have been invested in attempts to develop new and better NO donors. However, more than 30 years have passed since the groundbreaking discovery regarding NO, and progress in developing improved NO donors with high specificity and fewer side effects has been slow.

[0010] To further explain, nitrates most commonly used in clinical settings, such as nitroglycerin, are currently used to treat the symptoms of angina (chest pain). Organic nitrates work by relaxing blood vessels, thereby reducing the load on the heart while increasing the supply of blood and oxygen to it. Examples of organic nitrate drugs currently available include:

[0011] a) Nitroglycerin (glyceryl trinitrate) (1,2,3-propanetriol nitrate) is primarily administered sublingually today to suppress acute attacks of angina. However, severe headache and dizziness due to its rapid and common vasodilatory effect are frequent side effects. Nitroglycerin infusion concentrates are also available and are diluted with isotonic glucose or saline for intravenous administration. The development of tolerance (i.e., decreased effectiveness due to repeated or continuous administration) is a clinical problem in nitroglycerin (and other organic nitrates) therapy.

[0012] b) Isosorbide mononitrate (1,4:3,6-dianhydro-D-glucitol-5-nitrate), which is taken as a preventive medication for angina. The problem with long-term treatment regimens is the development of tolerance. Frequent side effects include headaches and dizziness, similar to those caused by nitroglycerin.

[0013] c) Isosorbide dinitrate (1,4:3,6-dianhydro-D-glucitol-2,5-nitrate), which is taken both acutely and prophylactically for angina pectoris and heart failure.

[0014] d) Pentaerythrityl nitrate, a group of organic nitrates known to exert long-term antioxidant and anti-atherosclerotic effects through mechanisms not yet identified. Pentaerythrityl tetranitrate has been investigated in relation to nitrate resistance, an undesirable development in nitrate therapy, and has been experimentally tested in pulmonary hypertension.

[0015] Many of these organic nitrate compounds, as well as other nitrate and nitrite compounds, have been tested in vivo and found to produce NO. For example, in rabbit models, glyceryl trinitrate, ethyl nitrite, isobutyl nitrate, isobutyl nitrite, isoamyl nitrite, and butyl nitrite were tested and found to give a significant correlation between in vivo NO production and its effect on blood pressure (Cederqvist et al., Biochem. Pharmacol., 1994, 47, 1047-53).

[0016] With increasing knowledge of the importance of nitric oxide, the importance of dietary composition has also been recognized, as it may affect the availability of NO in the arginine-nitric oxide system, and its role in host defense has been discovered (Larsen et al., N.Eng.J.Med, 2006, 355, 2792--3). Therefore, L-arginine, as well as its esters such as ethyl-, methyl-, and butyl-L-arginine, have been used to increase endogenous NO production.

[0017] WO2006 / 031191 describes compositions and methods for use in the therapeutic delivery of gaseous nitric oxide. Such compositions for the delivery of gaseous NO include compounds capable of forming reversible binding or association with NO, such as alcohols, carbohydrates, and proteins.

[0018] WO2007 / 106034 describes a method for producing organic nitrites from compounds that are monohydric / polyhydric alcohols or their aldehyde or ketone derivatives. This method involves degassing an aqueous solution of the compound and subsequent purging with gaseous nitric oxide (NO).

[0019] Nilsson, KFet al., Biochem Pharmacol., 82(3), 248-259 (2011) discusses the formation and identification of novel bioactive organic nitrites.

[0020] WO2020 / 109420 describes a process for preparing mono- and / or bis-nitrosylated propanediols, along with the resulting compositions. This document explains that such compounds may be capable of treating conditions in which NO has beneficial effects.

[0021] WO2021 / 239906 describes how these compounds have been shown to be remarkably effective in treating microbial infections.

[0022] The inventors have previously shown that a mixture of mono- and / or bis-nitrosylated propanediol (PDNO), when injected into the lungs, releases NO with an ultrashort half-life, causing a dominant effect on the pulmonary blood circulation. However, the mechanism behind this rapid degradation of PDNO in the pulmonary blood circulation is unknown, and surprisingly, researchers have discovered that PDNO can reach the pulmonary blood circulation and exert its effects even when administered via other routes of administration, such as transdermal routes, suggesting that the degradation of PDNO in other parts of the body should generally be less effective and rapid compared to the lungs (WO2021 / 239892).

[0023] Furthermore, despite more than 50 years of research into the mechanisms of NO release from NO donors (including organic nitrites), the knowledge behind the mechanism of NO release from NO donors remains unclear. The majority of mechanism studies have been conducted in vitro, making it nearly impossible to bridge this to understanding what actually happens in the highly complex in vivo environment.

[0024] In addition to the involvement of different enzymatic and non-enzymatic pathways for the metabolism of NO donors, their effectiveness may be influenced by the local environment in the organ where NO release from the molecule occurs, such as local pH, oxygen and carbon dioxide levels, and the state of inflammation in the tissue. Furthermore, the bioavailability of NO released by a specific NO donor may be affected by various factors, such as the concentration of oxygen radicals in the tissue, which may be influenced by the metabolism of the NO donor itself. Specifically, the release of NO from nitroglycerin has been shown to increase the level of oxygen radicals, which may contribute to tissue damage.

[0025] The complexity of bioconversion of organic nitrates and NO biology itself makes it virtually impossible to predict the effects of a given dose of NO donor on a specific organ, especially when this is affected by pathological processes such as ischemia or infection.

[0026] Many studies have focused on the intravenous administration of NO donors. Low doses of PDNO administered intravenously have been shown to exert vasodilation 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. Pharmacol Res Perspect. 2019:e00462). In investigations of this pharmacokinetic principle in several experimental models of acute pulmonary hypertension caused by various factors, it has been confirmed that low doses of intravenous PDNO are an efficient pulmonary vasodilator that does not cause systemic hypotension (Nilsson KF, Gustafsson LE. Treatment with new organic nitrites 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 in anesthetized 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、および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)。.

[0027] Previous animal studies have unexpectedly shown that low-dose intravenous infusion of PDNO (targeting pulmonary vasodilation) has renal protective and anti-inflammatory effects in a multi-organ failure model (aortic cross-clamping 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 Cross Clamping and Reperfusion: A Comparison With Nitroglycerin in Anesthetized Pigs. Shock. 2020;54(1):119-27.). Furthermore, high doses of intravenous PDNO (targeting systemic effects) have been shown to 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, knowledge of the doses required to affect blood flow, particularly in specific organs such as the brain (or more specifically parts of the brain), limbs (e.g., one leg), kidneys (or specific kidneys), or parts of the intestines, has not been studied. Nor has the relationship between such changes in flow and their effects on systemic blood pressure and / or cardiac output been measured. The reason such studies have not been conducted is that it is not a natural continuation of development programs to explore the role of PDNO in pulmonary and systemic circulation. The findings concerning these matters presented herein are astonishing.This is because previous research was unable to infer the inventions described herein, as the motivation for investigating this pathway was not provided at all in typical development programs and was conducted with a completely different focus.

[0028] Therefore, clinically available NO donor drugs are typically administered intravenously and result in systemic effects, including systemic hypotension, the most significant dose-limiting side effect. Low systemic blood pressure can be harmful, especially in critically ill patients, as it can offset the desired increase in blood flow by significantly reducing perfusion pressure (Gresele P, Momi S, Guglielmini G. Nitric oxide-enhancing or -releasing agents as antithrombotic drugs. Biochem Pharmacol. 2019;166:300-12.).

[0029] Currently, NO from organic nitrites can be released both non-enzymatically and enzymatically by acid-catalyzed hydrolysis, but the importance of each pathway for NO release from organic nitrites containing PDNO has not been established. Since the lungs are generally recognized as metabolically active organs, it could be expected that the degradation of PDNO in the lungs is far more potent than in other organs.

[0030] Considering the above, there is a need for improved treatment by NO donors that overcome one or more of the disadvantages described above, particularly for conditions in which NO has a beneficial effect. Such a need exists especially in organs and parts of the body other than the lungs. [Modes for carrying out the invention]

[0031] The inventors were surprised by the efficacy and effects of PDNO degradation discovered when testing intra-arterial infusion. Specifically, they were surprised by the rapid release of nitric oxide, which induces beneficial effects without first passing through the pulmonary circulation. Even more surprisingly, the doses that PDNO could deliver, as well as the magnitude of the response in the blood vessels of the brain, limbs, kidneys, and intestines, were unexpected compared to the doses possible when administered intravenously, while avoiding or reducing systemic effects in its local treatment. The inventors were also surprised that the dose required by intra-arterial administration to achieve the effect was lower compared to intravenous administration. The inventors were further surprised that when administered intra-arterial at higher doses that affected systemic blood pressure, these had a significant effect on blood flow across specific target organs where a reduction in local effect due to decreased driving pressure was expected.

[0032] The present invention also includes the surprising finding that PDNO was able to induce an antithrombotic effect in experiments conducted. For this to be achieved, NO must be delivered in an amount sufficient to enter the platelets. Given that platelets are surrounded by hemoglobin-filled red blood cells and act as a sink for NO, which is normally shown to have a biological half-life in the blood in the range of milliseconds, it is exceptional that PDNO appears to affect platelet function even at fairly low doses. It is unclear whether this means that NO from PDNO is released within the platelets, or whether this is achieved by another unknown mechanism. Nevertheless, the combination of PDNO's targeted local vasodilatory and antithrombotic effects is remarkable and has potentially great value in the treatment of several forms of tissue ischemia.

[0033] Intra-arterial administration The inventors have unexpectedly found that administering a particular mono- and / or bis-nitrosylated propanediol (including its compositions and formulations) to a patient in need thereof at a particular dose via intra-arterial infusion can achieve local and site-specific treatment of a pathological condition, particularly a pathological condition where NO is expected to have a beneficial effect, while avoiding undesirable systemic side effects.

[0034] As shown in the examples, when such a compound is administered intravenously, a systemic effect is seen at a dose much lower than the dose at which it can be administered intra-arterially before any systemic effect is seen.

[0035] Thus, in a first aspect of the invention, there is provided a compound of formula (I)

Chemical formula

[0036] According to the first method of the invention, · A compound of formula (I) is administered to a patient in need thereof via intra-arterial infusion at a dose of about 0.01 - 3000 nmol kg -1 min-1 A method of treating the disease by administering a certain dose, • Use of a compound according to formula (I) for the manufacture of a pharmaceutical product for the treatment of a disease, wherein the compound is administered to a patient in need via intra-arterial infusion at a dose of approximately 0.01 to 3000 nmol kg. -1 min -1 The use of the compound, administered in a dose, is further provided.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains.

[0038] All embodiments and specific features of the Invention referred to herein may be interpreted, either alone or in combination with any other embodiments and / or specific features referred to herein, without departing from the disclosure of the Invention (i.e., more specific embodiments and specific features as disclosed herein).

[0039] As used herein, the term “comprises” takes its ordinary meaning in the art, indicating that the components include, but are not limited to, the relevant features (i.e., include, in particular). Thus, the term “comprises” includes references to components that are essentially made up of the relevant substance(s).

[0040] As used herein, unless otherwise specified, the terms “consists essentially of” and “consisting essentially of” refer to the relevant component that makes up at least 80% (e.g., at least 85%, at least 90%, or at least 95%, e.g., at least 99%) of the specified substance(s) according to the relevant measurements (e.g., by weight). The terms “consists essentially of” and “consisting essentially of” may be replaced with “consists of” and “consisting of,” respectively.

[0041] To avoid ambiguity, the term “comprises” also includes references to the constituent substances (plural) “consisting essentially of” (and especially “consisting of”) related to the substance.

[0042] Those skilled in the art will understand that references to the treatment of a particular pathological condition (or similarly, the act of treating that condition) are in their usual sense in the medical field. In particular, the term may refer to achieving a reduction in the severity of one or more clinical symptoms and / or signs associated with that pathological condition. For example, in the case of pulmonary embolism, the term may refer to achieving a reduction in the severity of chest pain, shortness of breath, and / or pulmonary hypertension through vasodilation. Furthermore, in the case of pulmonary embolism, the term may refer to achieving pulmonary vasodilation, or a reduction in pulmonary vascular resistance and right ventricular load.

[0043] As used herein, a patient refers to a living subject being treated, including mammalian (e.g., human) patients. In particular, the term patient may refer to a human subject. The term patient may also refer to animals (e.g., mammals) such as domestic pets (e.g., cats, and especially dogs), livestock, and horses.

[0044] The method according to the present invention comprises administering an effective amount of a compound according to formula (I) to a patient in need thereof. As used herein, the term “effective amount” refers to the amount of a compound that confers a therapeutic effect to the patient being treated. The effect may be objective (i.e., measurable by several tests or markers) or subjective (i.e., the subject gives an indicator of the effect and / or feels the effect).

[0045] The compound of formula (I) is present in quantities of approximately 0.01 to 300 nmol kg. -1 min -1 For example, approximately 1 to approximately 300 nmol kg -1 min -1 For example, approximately 1 to approximately 100 nmol kg -1 min -1 , or approximately 10 to approximately 3000 nmol kg -1 min -1 For example, 10 to approximately 300 nmol kg -1 min -1 It may be administered in the following dose.

[0046] Preferably, the compound of formula (I) is present in a quantity of about 1 to about 30 nmol kg. -1 min -1 It is administered in the following doses. For example, the compound of formula (I) is approximately 1 to approximately 30 nmol kg. -1 min -1 Less than, for example, about 1 to about 25, 26, 27, 28, or 29 nmol kg -1 min -1 For example, approximately 1 to 20 nmol kg -1 min -1 Approximately 1 to 15, 14, 13, 12, or 11 nmol kg -1 min -1 For example, approximately 1 to 10 nmol kg -1 min -1 It may be administered in the following dose.

[0047] The compound of formula (I) may be administered continuously, such as by continuous infusion, over a period of up to 14 days, for example, 7 days.

[0048] The compound of formula (I) may be administered as a single dose or as repeated single shots, such as a bolus injection.

[0049] The compound of formula (I) can be administered by a medical professional to any part of the arterial vascular system accessible for delivery of the compound of formula (I) by infusion. Infusion can be achieved by surgical techniques, such as through the use of a catheter device.

[0050] Intra-arterial administration may be intended to deliver the compound according to formula (I) to the lower extremities, which can be achieved by intra-arterial administration into the femoral artery.

[0051] Intra-arterial administration may also, or alternatively, be intended to deliver the compound according to formula (I) to the brain, which can be achieved by intra-arterial administration into the carotid artery.

[0052] Intra-arterial administration may further, or alternatively, be intended to deliver the compound according to formula (I) to the intestines, which can be achieved by intra-arterial administration into the superior mesenteric artery.

[0053] Intra-arterial administration may further, or alternatively, be intended to deliver the compound according to formula (I) to the kidney, adrenal gland, and / or ureter, which can be achieved by intra-arterial administration into the renal artery.

[0054] Therefore, the compound of formula (I) can be administered by intra-arterial injection into the femoral artery, carotid artery, superior mesenteric artery, splenic artery, hepatic artery, and / or renal artery.

[0055] When the compound of formula (I) is administered to the femoral artery and / or carotid artery via intra-arterial infusion, this amounts to approximately 1 to approximately 300 nmol kg. -1 min -1 For example, 1 to approximately 60 nmol kg -1 min -1 For example, 1 to approximately 50 nmol kg -1 min -1 For example, 1 to approximately 30 nmol kg -1min -1 It may be administered in the following dose.

[0056] When the compound of formula (I) is administered to the superior mesenteric artery via intra-arterial infusion, this amounts to approximately 3 to 3000 nmol kg. -1 min -1 For example, approximately 30 to 1000 nmol kg -1 min -1 For example, approximately 100 to 1000 nmol kg -1 min -1 It may be administered in the following dose.

[0057] When the compound of formula (I) is administered to the renal artery via intra-arterial infusion, this amounts to approximately 0.01 to 300 nmol kg. -1 min -1 For example, approximately 1 to approximately 30 nmol kg -1 min -1 It may be administered in the following dose.

[0058] When administering to a specific artery, this is also intended to include administration to the arterial branches derived from that artery. For example, when administering to the femoral artery, administration can be to the superficial epigastric artery, superficial circumflex artery, external pudendal artery, deep femoral (or profunda femoris) artery, or superficial femoral artery, all of which are connected to the femoral artery. When administering to the carotid artery, this can be done via the cerebral artery, i.e., the Circle of Willis.

[0059] The conditions treated according to the first method of the present invention may be conditions in which NO has a beneficial effect. That is, the compound according to formula (I) is useful in the treatment of NO, i.e., conditions in which NO administration has a beneficial effect.

[0060] As used herein, the term “beneficial effect” means that the use / administration of the compound / composition of the present invention results in an identifiable treatment and / or improvement of the patient’s condition. Beneficial effects may be temporary or permanent and may be measured or determined by a healthcare professional or the patient themselves.

[0061] A particular outcome of the present invention is that beneficial effects may be experienced locally, for example, in only one organ of the patient, but without clinically significant systemic effects. Beneficial effects may be objective (i.e., measurable by several tests or markers) or subjective (i.e., the subject provides an indicator of the effect and / or feels the effect).

[0062] The conditions being treated are: Acute pulmonary vasoconstriction of different causes, pulmonary hypertension of different causes including primary and secondary hypertension, preeclampsia, eclampsia, conditions of different causes requiring vasodilation, erectile dysfunction, systemic hypertension of different causes, localized vasoconstriction of different causes, regional vasoconstriction of different causes, acute heart failure (with or without ejection fraction preservation (HFpEF)), coronary heart disease, myocardial infarction, ischemic heart disease, angina pectoris, unstable angina, cardiac arrhythmias, acute pulmonary hypertension in cardiac surgery patients, acidosis, airway inflammation, cystic fibrosis, COPD, cilia syndrome, lung inflammation, pulmonary fibrosis, acute lung injury (ALI), adult respiratory distress syndrome, acute pulmonary edema, acute altitude sickness, The group may be selected from asthma, bronchitis, hypoxia of different causes, ischemic disease of different causes, stroke, cerebral vasoconstriction, inflammation of the gastrointestinal tract, gastrointestinal dysfunction, gastrointestinal complications, IBD (inflammatory bowel disease), Crohn's disease, ulcerative colitis, liver disease, pancreatic disease, inflammation of the bladder of the urinary tract, inflammation of the urinary tract, bladder and ureters, skin inflammation, diabetic ulcers, diabetic neuropathy, psoriasis, inflammation of different causes, wound healing, organ protection in ischemia-reperfusion conditions, organ transplantation, tissue transplantation, cell transplantation, acute kidney disease, uterine laxity, cervical laxity, thromboembolic diseases including thromboembolic diseases such as various hematological disorders, arterial occlusion such as arterial embolism, and conditions requiring smooth muscle relaxation.

[0063] In particular, the conditions being treated may include pulmonary hypertension of different causes, including primary and secondary hypertension.

[0064] Specific pathological conditions may include those selected from the group consisting of ischemic diseases of different causes, thromboembolic diseases including various blood disorders, arterial thrombosis, peripheral ischemia (ischemia of the limbs), thromboembolic stroke, pulmonary embolism, acute mesenteric ischemia (occlusion of the mesenteric artery), acute renal artery occlusion, arterial stenosis, arterial occlusion, splenic infarction, hepatic infarction, pulmonary infarction, Kawasaki disease, and arterial embolism.

[0065] Regarding the treatment of ischemic diseases, it should be understood that this includes the treatment / prevention of conditions resulting from (partial or complete) arterial occlusion, where blood flow to a specific organ is restricted and ischemic or threatens to be ischemic.

[0066] Treatment can be indicated by a reduction in mean systemic arterial pressure (MAP) of approximately 10% or less compared to baseline MAP in the pre-treatment subject. For example, treatment can be indicated by a reduction in MAP of approximately 9%, 8%, 7%, or 6% or less compared to baseline MAP in the pre-treatment subject. In particular, treatment can be indicated by a reduction in MAP of approximately 5% or less compared to baseline MAP in the pre-treatment subject.

[0067] Treatment may be indicated by a decrease of approximately 10% or less in end-tidal nitric oxide (ETNO) compared to baseline ETNO in the pre-treatment subject. For example, treatment may be indicated by a decrease of approximately 9%, 8%, 7%, or 6% or less in ETNO compared to baseline ETNO in the pre-treatment subject. In particular, treatment may be indicated by a decrease of approximately 5% or less in ETNO compared to baseline ETNO in the pre-treatment subject.

[0068] Treatment may be indicated by an increase of approximately 10% or less in the blood methemoglobin fraction compared to baseline in the subject before treatment. For example, treatment may be indicated by an increase of approximately 9%, 8%, 7%, or 6% or less in the blood methemoglobin fraction compared to baseline in the subject before treatment. In particular, treatment may be indicated by an increase of approximately 5% or less in the blood methemoglobin fraction compared to baseline in the subject before treatment.

[0069] "Compared to baseline levels" refers to a comparison between the MAP levels measured at the start of the study (i.e., before administration of the compound of formula (I)) and the MAP levels after administration. Baseline levels are the levels immediately before the start of treatment and are used as a comparison point for subsequent measured levels (e.g., immediately after the course of treatment or after the completion of the course of treatment). Therefore, such comparisons are specific to the subject or group of subjects in question and are not absolute values.

[0070] Further specific conditions that can be treated include bacterial, fungal, viral, or parasitic infections.

[0071] Treatment of thromboembolic diseases The inventors have further unexpectedly discovered that administering certain mono- and / or bis-nitrosylated propanediols (including their compositions and formulations) to patients in need is particularly effective in inhibiting platelet aggregation, and that this is useful in the treatment of thromboembolic disorders.

[0072] Therefore, according to a second aspect of the present invention, a compound of formula (I) is, [ka] In the formula, R 1 , R 2 , and R 3 However, each independently represents either H or -NO. n is 0 or 1, If n is 0, R 1 However, H is, If n is 1, R 2 However, H is, However, R 1 , R 2 , and R 3 The condition is that at least one of them represents -NO, A compound is provided for use in the treatment of thromboembolic diseases, which is referred to herein as "the second method of the present invention."

[0073] According to the second method of the present invention, A method for treating thromboembolic disease by administering the compound of formula (I) to a patient in need thereof, The use of compounds according to formula (I) for the manufacture of pharmaceuticals for the treatment of thromboembolic diseases is further provided.

[0074] Inhibition of platelet aggregation occurs in a remarkable way, distinct from other treatments, and appears to proceed through the inhibition of platelet particle release and ATP release.

[0075] Thromboembolic diseases are conditions that occur when blood clots formed inside blood vessels loosen and are carried by the bloodstream, resulting in at least partial blockage of the vessels.

[0076] The thromboembolic diseases treated may be selected from a group consisting of arterial thrombosis, myocardial infarction, cerebral venous thrombosis, portal vein thrombosis, peripheral ischemia (ischemia of the limbs), thromboembolic stroke, pulmonary embolism, acute mesenteric ischemia (occlusion of the mesenteric artery), acute renal artery occlusion, arterial stenosis, arterial occlusion, venous thromboembolism including deep vein thrombosis, and arterial embolism.

[0077] Thromboembolic diseases can occur in both the venous and arterial systems, and to avoid any doubt, the use of compounds according to formula (I) in the treatment of such diseases is assumed to encompass both venous and arterial treatment. Therefore, compounds according to formula (I) may be administered via intravenous and / or intra-arterial infusion.

[0078] Furthermore, it is anticipated that treatment may be administered through other routes, including inhalation, spray, intramuscular, subcutaneous, transdermal, intranasal, sublingual, subconjunctival, rectal, tracheal, pulmonary, gastric, ureteral, enteral, urethral, ​​bladder, oral, and enteral administration.

[0079] The compounds represented by formula (I) are approximately 0.01 to 3000 nmol kg. -1 min -1 For example, approximately 0.01 to approximately 300 nmol kg -1 min -1 For example, approximately 1 to approximately 300 nmol kg -1 min -1 For example, approximately 1 to approximately 100 nmol kg -1 min -1 It may be administered in doses of approximately 1 to approximately 30 nmol kg. Preferably, the compound of formula (I) is administered in doses of approximately 1 to approximately 30 nmol kg. -1 min -1 It is administered in the following doses. For example, the compound of formula (I) is approximately 1 to approximately 30 nmol kg. -1 min -1 Less than, for example, about 1 to about 25, 26, 27, 28, or 29 nmol kg -1 min -1 For example, approximately 1 to 20 nmol kg -1 min -1 Approximately 1 to 15, 14, 13, 12, or 11 nmol kg -1 min -1 For example, approximately 1 to 10 nmol kg -1 min -1 It can be administered in the following doses. Furthermore, the compound of formula (I) is approximately 1 to approximately 5 nmol kg. -1 min -1 It may be administered in the following dose.

[0080] Compounds according to formula (I) can be administered into the vascular system by any means, for example, to any artery in any limb via intra-arterial injection.

[0081] Compounds according to formula (I) can be administered via intra-arterial injection into the femoral artery, carotid artery, renal artery, or superior mesenteric artery, among others.

[0082] Compounds according to formula (I) can be administered intravenously to the central venous system, peripheral veins of the arms and legs, femoral veins, and scalp veins (especially scalp veins in neonates). Intravenous infusion may also be performed to deep or superficial veins, such as superficial veins of varicose veins in the legs.

[0083] Treatment may be indicated by an increase of approximately 10% or less in the blood methemoglobin fraction compared to baseline in the subject before treatment. For example, treatment may be indicated by an increase of approximately 9%, 8%, 7%, or 6% or less in the blood methemoglobin fraction compared to baseline in the subject before treatment. In particular, treatment may be indicated by an increase of approximately 5% or less in the blood methemoglobin fraction compared to baseline in the subject before treatment.

[0084] "Compared to baseline levels" refers to a comparison between the MAP levels measured at the start of the study (i.e., before administration of the compound of formula (I)) and the MAP levels after administration. Baseline levels are the levels immediately before the start of treatment and are used as a comparison point for subsequent measured levels (e.g., immediately after the course of treatment or after the completion of the course of treatment). Therefore, such comparisons are specific to the subject or group of subjects in question and are not absolute values.

[0085] As mentioned above, inhibition of platelet aggregation is achieved in a remarkable way that differs from other treatments, and appears to proceed by inhibiting the release of platelet particles and ATP release.

[0086] Therefore, the second method of the present invention also encompasses a method of inhibiting platelet particle release and / or ATP release in a subject, the method comprising administering to a subject in need thereof an effective amount of one or more compounds of formula (I) or a pharmaceutical formulation thereof. This aspect of the present invention may include any of the features outlined above with respect to the treatment of thromboembolic diseases.

[0087] Compounds and Compositions Specific compounds for use in both the first and second methods of the present invention are compounds according to formula (II),

Chemical Formula

[0088] There are two enantiomers of the compound according to formula (II), which are the R-form and the S-form shown below.

Chemical Formula

Chemical Formula

[0092] Further specific compounds for use in both the first and second methods of the present invention are compounds according to formula (IV),

Chemical formula

[0093] Compounds for use in both the first and second methods of the present invention are (a) one or more compounds of formula (I) as defined herein, and (b) one or more corresponding compounds of formula (I), but where R 1 , R 2 , and R 3 represent H (e.g., 1,2-propanediol and / or 1,3-propanediol), and may be present in a composition, which composition may be referred to hereinafter as the "composition".

[0094] The composition may be substantially non-aqueous. As used herein in connection with both the first and second aspects of the present invention, the reference to "substantially non-aqueous" refers to a component containing less than 10% by weight, for example, 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 less than 1% by weight (e.g., less than 0.5% by weight or less than 0.1% by weight, e.g., less than 0.05% by weight, less than 0.01% by weight) of water.

[0095] It should be understood that the composition may contain a mixture of compounds corresponding to formula (I).

[0096] Specific compositions that may be mentioned include those in which the composition contains one or more compounds of formula (I) in about 0.01% to about 9% by weight (e.g., about 0.01% to about 5% by weight, e.g., about 3% to about 5% by weight, or about 5% to about 7% by weight).

[0097] Specific compositions that may be mentioned include those in which the composition contains one or more compounds of formula (I) in concentrations of about 1 to about 1000 mM (e.g., about 5 to about 750 mM, e.g., about 5 to about 500 mM, or about 10 to about 203 mM).

[0098] To avoid any ambiguity, the unit mM is 10 -3 This refers to the concentration of the compound of formula (I) in a composition in mol / L, and if the composition contains a mixture of compounds of formula (I), it is based on the average molecular weight of the compounds of formula (I) in the composition.

[0099] Specific compositions that may be mentioned include those in which the composition contains a compound according to formula (II). Preferably, the compound according to formula (II) is the S-isomer.

[0100] The S-isomer of the compound according to formula (II) is preferred because it has a higher metabolic rate and a different metabolic pathway than the R-isomer. Furthermore, the S-isomer has a different metabolic degradation pathway and produces metabolites that are less toxic than those of the R-isomer.

[0101] Specific compositions that may be mentioned include those in which the composition contains a compound according to formula (III).

[0102] Preferably, the compound according to formula (II) is the S-isomer, but the product is a mixture of both the S-isomer and the R-isomer of formula (II), and it is assumed that the S-isomer is preferably present in an enantiomer excess (ee).

[0103] In certain embodiments, the compound according to formula (II) may have an enantiomer excess of the S-isomer of the compound. That is, more than 50 ee% of the product is the S-isomer, such as 60 ee%, 70 ee%, 80 ee%, 90 ee%, 95 ee%, or 98 ee% or more of the product being the S-isomer.

[0104] In one embodiment where the product is a mononitrosylated compound according to formula (II), more than 50% by weight of the product is nitrosylated at the 2 position (i.e., R 2 (is -NO), approximately 55% to 80% by weight, for example, approximately 55% to 75% by weight, are nitrosylated at position 2.

[0105] The specific compositions that may be mentioned are those in which the composition essentially consists of one or more compounds of formula I, and the corresponding compounds of formula I, but R 1 , R 2 , and R 3 This includes those in which H is represented (i.e., 1,2-propanediol and / or 1,3-propanediol).

[0106] Other specific compositions may include (or, in particular, essentially consist of, or more specifically consist of) one or more compounds of formula II and 1,2-propanediols.

[0107] Similarly, further compositions may comprise (or, in particular, essentially consist of, or more specifically consist of) one or more compounds of formula III and 1,3-propanediol.

[0108] The term "essentially made from" means that at least 90% by weight of the defined feature is present, for example, at least 95%, 96%, 97%, 98%, or 99% by weight of the defined feature is present.

[0109] Furthermore, certain compositions that may be mentioned include those in which the composition contains (or, in particular, essentially consists of, or more specifically consists of) one or more compounds of formulas (II) and (III) together with 1,2-propanediol and 1,3-propanediol.

[0110] The specific compositions that may be mentioned include those that substantially do not contain dissolved nitric oxide.

[0111] The term "substantially contained" means that the composition contains less than 5% by weight, 4% by weight, 3% by weight, 2% by weight, or 1% by weight, for example, less than 0.5% by weight or less than 0.1% by weight of dissolved nitric oxide.

[0112] Furthermore, certain compositions (a) One or more compounds of formula IV, [ka] In the formula, R 4 and R 5 However, each independently represents H or -NO, except R 4 and R 5 One or more compounds of formula IV, provided that at least one of them represents -NO, (b) May contain 1,2-propanediol and

[0113] The composition may be administered alone or via known pharmaceutical compositions / formulations.

[0114] Therefore, the composition may be included in a pharmaceutical preparation, and optionally, the pharmaceutical preparation may contain one or more pharmaceutically acceptable excipients.

[0115] Those skilled in the art will understand that the references herein to pharmaceutical preparations refer to compositions in the form of pharmaceutical preparations and include references to all embodiments and specific forms thereof.

[0116] As used herein, the term pharmaceutically acceptable excipient includes references to vehicles, adjuvants, carriers, diluents, pH adjusters, and buffers, tonicity adjusters, stabilizers, permeability enhancers, wetting agents, and the like. In particular, such excipients may include adjuvants, diluents, or carriers.

[0117] The specific pharmaceutical preparations that may be mentioned include those in which the pharmaceutical preparation contains at least one pharmaceutically acceptable excipient.

[0118] Certain pharmaceutical formulations that may be mentioned include those in which one or more pharmaceutically acceptable excipients are substantially non-aqueous.

[0119] Compounds according to formula (I) can be administered to patients (i.e., subjects) in combination with suitable aqueous buffers, such as non-nucleophilic and weakly basic buffers.

[0120] More specific embodiments that may be mentioned include buffers having 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.

[0121] In particular, the buffer may be a pH 9.2 carbonate buffer, a pH 8.0 phosphate buffer (e.g., 0.154 molar buffer), or a pH 8.0 NaHCO3 buffer.

[0122] To avoid any ambiguity, the references herein to compounds of formula (I) for specific uses may also apply to compositions and pharmaceutical preparations comprising compounds of formula (I), as described herein.

[0123] Whenever the word “approximately” is used herein in the context of the amount of individual components in a composition or the components of a composition (including concentration and ratio), a time frame, and parameters such as temperature, whether absolute or relative (e.g., percentage), such as weight, volume, size, or diameter, it should be understood that such variables are approximate and therefore may vary by ±10%, e.g., ±5%, and preferably ±2% (e.g., ±1%) from the actual figures specified herein. This is true even when such figures are initially presented as percentages (e.g., “approximately 10%” could mean ±10% of the figure 10, which is anywhere between 9% and 11%). [Brief explanation of the drawing]

[0124] [Figure 1a] This is a study protocol for dose-response experiments of intravenous and organ-directed intra-arterial infusion of 1,2-propanediol organic mononitrite (PDNO) in the carotid and femoral arteries (Panel A). [Figure 1b] This is a study protocol for dose-response experiments of intravenous and organ-directed intra-arterial infusion of 1,2-propanediol organic mononitrite (PDNO) in the renal artery and superior mesenteric artery (Panel B). [Figure 2(a)(b)] The effects of organ-directed intra-arterial infusion and intravenous administration of 1,2-propanediol organic mononitrite (PDNO) on local blood flow, mean systemic arterial pressure (MAP), and end-tidal nitric oxide concentration (ETNO) in the left carotid artery (Panels A and B). Data are mean values ​​with standard error of the mean (SEM). * indicates statistical significance (P<0.05) from baseline (BL) in femoral and carotid artery experiments. # indicates statistical significance (P<0.05) from BL during intravenous administration. [Figure 2(c)(d)] The effects of organ-directed intra-arterial infusion and intravenous administration of 1,2-propanediol organic mononitrite (PDNO) on local blood flow, mean systemic arterial pressure (MAP), and end-tidal nitric oxide concentration (ETNO) in the left common femoral artery (Panels C and D). Data are mean values ​​with standard error of mean (SEM). * indicates statistical significance (P<0.05) from baseline (BL) in femoral and carotid artery experiments. # indicates statistical significance (P<0.05) from BL during intravenous administration. [Figure 3(a)(b)]The effects of organ-directed infusion of 1,2-propanediol organic mononitrite (PDNO) and intravenously administered PDNO on local blood flow, mean systemic arterial pressure (MAP), and end-tidal nitric oxide (ETNO) in the superior mesenteric artery (SMA) (Panels A and B). Data are mean values ​​with standard error of the mean (SEM). * indicates statistical significance (p<0.05) from baseline (BL) in the intrarenal artery experiment and from vasopressin (VP) in the SMA experiment. # indicates statistical significance (p<0.05) from BL during intravenous administration. [Figure 3(c)(d)] The effects of organ-directed infusion of 1,2-propanediol organic mononitrite (PDNO) and intravenously administered PDNO on local blood flow, mean systemic arterial pressure (MAP), and end-tidal nitric oxide (ETNO) in the left renal artery (Panels C and D). Data are mean values ​​with standard error of the mean (SEM). * indicates statistical significance (p<0.05) from baseline (BL) in the intrarenal artery experiment and from vasopressin (VP) in the SMA experiment. # indicates statistical significance (p<0.05) from BL during intravenous administration. [Figure 4(a)(b)] This study calculates the dose-ratio of end-tidal nitric oxide (ETNO) between intravenous and intra-arterial administration of 1,2-propanediol organic mononitrite (PDNO) in the left common femoral artery, left common carotid artery, left renal artery, and superior mesenteric artery. The dose-relation is given using logarithmic PDNO, ETNO = B0eB1 dose (Panel A), and natural logarithmic ETNO for linearization, ln ETNO = B1 dose + ln B0 (Panel B). [Figure 4c]Estimated coefficients and standard errors (Panel C). Unless otherwise noted, significance is shown for comparisons with intravenous (iv) reference 1 for carotid and femoral artery infusions, and with intravenous reference 2 for renal artery and superior mesenteric artery infusions. a: Multiple of the dose required to increase log(ETNO) to a similar amount compared to the intravenous dose. : Section for femoral artery infusion compared to carotid artery infusion. #: Significant with P<0.001 compared to both renal artery infusion and intravenous (reference 2). *: P<0.05, **: P<0.01, ***: P<0.001. [Figure 5] These are the arterial fractions of methemoglobin during organ-directed intra-arterial infusion of 1,2-propanediol organic mononitrite (PDNO) and intravenous administration of PDNO. The data are mean values ​​with standard error (SEM) of the mean. [Figure 6] Platelet aggregation in porcine whole blood (heparin as an anticoagulant) was analyzed ex vivo using a Multiplate Aggregator. Coagulated blood clots were intravenously administered to anesthetized pigs (n=5) to induce pulmonary embolism (PE), after which the animals were intravenously administered PDNO at a rate of 160 nmol / kg / min. Arterial blood was collected at specific time points: pre-embolization (baseline), post-embolization, during PDNO infusion, and after cessation of PDNO infusion. The blood was immediately transferred to cuvettes for Multiplate agglutination analysis. Blood was stimulated with the PAR-1 agonist peptide SFLLRN, and platelet aggregation (recorded as an increase in electrical resistance) was tracked for 10 minutes. Data are presented as individual values ​​and median (line). P-values ​​are from the Dunn test analysis. [Figure 7] This study investigated the anti-aggregation efficacy of PDNO. Suspensions of isolated human platelets were activated with the PAR-1 agonist SFLLRN in the absence (blank bars) and presence (filled bars) of various concentrations of PDNO. Platelet aggregation was assessed as an increase in light transmission using a Chronolog aggregater. PDNO was introduced into platelets 2 minutes prior to SFLLRN. The bars represent the mean and standard deviation (n=5). [Figure 8]This shows platelet aggregation in whole blood. Human blood suspensions were stimulated with the PAR-1 agonist SFLLRRN in the presence or absence of either PDNO (black bar) or nitroglycerin (gray bar) (white bar). NO donor drugs were introduced into the blood 2 minutes prior to SFLLRRN. The platelet aggregation response was analyzed as an increase in impedance over 10 minutes and is represented as the area under the curve. The bars represent the mean and standard deviation (n=4). [Figure 9] This involves increased Ser239-specific VASP phosphorylation in platelets. A suspension of isolated human platelets was stimulated with either PDNO or nitroglycerin. Platelets were exposed to the NO donor for 5 minutes, followed by Western immunoblotting for phosphoVASP detection. These experiments were performed on unstimulated and SFLLRN-stimulated platelets. A PAR-1 agonist was added 2 minutes after PDNO / nitroglycerin. Bars represent the mean and standard deviation (n=3). [Figure 10] This study protocol compares intra-arterial injection of 1,2-propanediol monoorganic nitrite (PDNO, n=12) into the left common femoral artery (CFA) with a control state (no injection, n=12) for 6 hours after CFA embolization. [Figure 11a] This shows blood flow in the left common femoral artery (CFA) during local arterial infusion of 1,2-propanediol monoorganic nitrite (PDNO, n=12) or in a control state (no infusion, n=12) for 6 hours after CFA embolization (Panel A). The P-value indicates the difference between groups. Data are presented as mean ± standard error of the mean. [Figure 11b] Femoral vascular resistance (FVR) in the left common femoral artery (CFA) during local arterial infusion of 1,2-propanediol monoorganic nitrite (PDNO, n=12) or in a control state (no infusion, n=12) for 6 hours after CFA embolization (Panel B). P values ​​indicate differences between groups. Data are presented as mean ± standard error of the mean. [Figure 11c]The partial pressure of oxygen in the femoral vein (FV pO2) during local arterial infusion of 1,2-propanediol monoorganic nitrite (PDNO, n=12) in the left common femoral artery (CFA) or in the control state (no infusion, n=12) for 6 hours after CFA embolization (Panel C). The P value indicates the difference between groups. Data are presented as mean ± standard error of the mean. [Figure 11d] Femoral vein oxygen consumption (FV VpO2) during local arterial infusion of 1,2-propanediol monoorganic nitrite (PDNO, n=12) in the left common femoral artery (CFA) or in a control state (no infusion, n=12) for 6 hours after CFA embolization (Panel D). P values ​​indicate differences between groups. Data are presented as mean ± standard error of the mean. [Figure 11e] This image shows femoral vein lactate production (FV lactate production) during local arterial infusion of 1,2-propanediol monoorganic nitrite (PDNO, n=12) in the left common femoral artery (CFA) or in a control state (no infusion, n=12) for 6 hours after CFA embolization (Panel E). The P-value indicates the difference between groups. Data are presented as mean ± standard error of the mean. [Figure 11f] Delta lactate levels (delta lactate) between central arterial blood and femoral vein blood during local arterial infusion of 1,2-propanediol monoorganic nitrite (PDNO, n=12) in the left common femoral artery (CFA) or 6 hours after CFA embolization in a control state (no infusion, n=12) (Panel F). P values ​​indicate differences between groups. Data are presented as mean ± standard error of the mean. [Figure 12a] The mean systemic arterial blood pressure (MAP) is shown during local arterial infusion of 1,2-propanediol monoorganic nitrite (PDNO, n=12) in the left common femoral artery (CFA) or in the control state (no infusion, n=12) for 6 hours after CFA embolization (Panel A). Data are presented as mean ± standard error of the mean. [Figure 12b]End-tidal nitric oxide (ETNO) levels (ETNO) during local arterial infusion of 1,2-propanediol monoorganic nitrite (PDNO, n=12) in the left common femoral artery (CFA) or in a control state (no infusion, n=12) for 6 hours after CFA embolization (Panel B). Data are presented as mean ± standard error of the mean. [Examples]

[0125] The present invention will be illustrated by the following examples, but this is not intended to limit the general scope of the invention.

[0126] Example 1: Composition For the studies reported below, compositions were used that contained a mixture of 1-(nitrosooxy)propan-2-ol and 2-(nitrosooxy)propan-1-ol (hereinafter referred to as PDNO) together with 1,2-propanediol (PD, or propylene glycol). The compositions contained 6.5–7.1 w / w% PDNO, with the remainder being propylene glycol.

[0127] Processes for preparing compounds according to formula (I) as defined above, together with compositions containing these compounds, in particular substantially non-aqueous compositions containing the compounds, and compositions used in the following studies, can be found in WO2020 / 109420, which is incorporated herein by reference.

[0128] To avoid any doubt, the compounds of formula (I) may also be referred to herein by the acronym PDNO, which would indicate that such compounds, including all embodiments and their specific features, are used in the methods and uses described in connection with the present invention. Furthermore, where a composition of PDNO also containing PD is described, PD refers to the propanediol corresponding to the compound of formula (I), i.e., PD is the same compound by formula (I), but in the formula, R 1 , R 2 , and R 3 This represents H.

[0129] Example 2: Intra-arterial administration study PDNO exhibits ultrafast release due to its extremely short half-life, although the half-life has not been determined. The aim of this study was to compare intra-arterial and intravenous infusion of PDNO with respect to local organ blood flow and systemic effects, and to estimate the in vivo half-life of PDNO. A summary of this study, along with detailed methodology, is provided below.

[0130] Intra-arterial infusion of PDNO (0.01-3000 nmol kg) in the left common carotid artery (CCA), left common femoral artery (CFA), left renal artery (RA), and superior mesenteric artery (SMA). -1 min -1 In anesthetized instrumented pigs (n=14), PDNO (3-100 nmol kg) was administered intravenously. -1 min -1 The in vivo half-life of PDNO was estimated in anesthetized instrumented pigs (n=2) using intra-arterial injection in the left CCA and left CFA, as well as central venous injection (PDNO 0.5–4 micromoles). Local blood flow, mean systemic arterial pressure (MAP), end-tidal nitric oxide (ETNO), and arterial blood gases were measured.

[0131] 1 nmol kg -1 min -1 During each intra-arterial infusion, blood flow in the CFA and CCA increased significantly from baseline, and blood flow in the SMA also increased by 30 nmol kg. -1 min -1 Although it increased from RA and intravenous infusion did not affect blood flow. MAP was 10 nmol kg during intravenous infusion. -1 min -1 This was significantly reduced, and this was due to 30 and 100 nmol kg during CCA and CFA injections, respectively. -1 min -1 This corresponds to 3000 nmol kg in the SMA experiment. -1 min -1This corresponded to the following. The dose equivalents of ETNO between intravenous administration (reference) and CFA, CCA, RA, and SMA infusions were 2.7, 2.9, 2.6, and 47, respectively. Using the measured circulation time between the intra-arterial infusion / injection site and the intravenous infusion / injection site, as well as the dose equivalents from the infusion and injection experiments, the in vivo half-life was estimated to be 5.5–6.1 seconds (infusion) and 3.0–3.9 seconds (injection).

[0132] Organ-directed intra-arterial (CCA, CFA, and SMA) infusion of PDNO, in contrast to RA and intravenous infusion, dose-dependently increased organ-specific blood flow and had little to no systemic effect at effective doses. Comparing intra-arterial and intravenous infusion, there was a substantial right shift in the dose-response effect on ETNO concentration, likely due to the rapid degradation of PDNO in the blood. The in vivo half-life of PDNO was estimated to be in the range of 3.0–6.1 seconds.

[0133] research group Sixteen domestically bred, three-month-old pigs (Swedish native breed, Hampshire, and Yorkshire crossbreed, average weight 30 kg [range 27-34 kg], sex ratio 1:1) were included in the study. The experiment was conducted at the animal laboratory of Öreble University Hospital in Öreble, Sweden, from March 7-18, 2022, and from May 9-13, 2022, and on September 15, 2023. Ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden, approval number 00259-2022). This study was conducted in accordance with Directive 2010 / 63 / EU on the protection of animals used for scientific purposes, and followed the ARRIVE guidelines as closely as possible.

[0134] Surgical preparation and measurement The animals were anesthetized and mechanically ventilated 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). Basic instrumentation (intravenous access for fluids and anesthetics, pulmonary artery catheter, urinary catheterization) was performed as described above (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). Arterial access was obtained by ultrasound-guided placement of introducers on both sides of the brachial artery (5Fr on the right side for catheter access (Cordis Corporation, Florida, USA), and 4Fr on the left brachial artery for arterial blood pressure measurement and arterial blood gas analysis (Cordis Corporation, USA)).

[0135] Preparation of the femoral and carotid arteries Flow probes (3 mm, Transonic System Inc., New York, USA) were attached to the femoral artery and common carotid artery. Angiography catheters (Soft-Vu Berenstein 5Fr, Queensbury, New York, USA or SOFT-VU Sos Omni® Selective 5Fr, Queensbury, New York, USA) were positioned in the left carotid artery or left femoral artery via a 5Fr introducer (Cordis Corporation, USA) in the right brachial artery using a guidewire (Terumo Radiofocus® STIFF type angle 0.035, Terumo Corporation, Tokyo, Japan). Positioning was confirmed by angiography. A microcatheter (Terumo Progreat®, 2.7Fr, Terumo Cooperation, Tokyo, Japan) was inserted into the angiography catheter and positioned proximal to the flow probe. Next, the angiography catheter was withdrawn to the brachiocephalic trunk in the carotid artery experiment and to the external iliac artery in the femoral artery experiment. In the femoral artery experiment, peripheral venous access was secured to the superficial veins of the distal lower limb (BD Venflon Pro Safety, 0.9 mm, Switzerland).

[0136] In two animals, PDNO was repeatedly injected via microcatheter into the left common femoral artery (1-4 micromoles), left carotid artery (0.5-2 micromoles), and superior vena cava central ventricle (CVC) (0.5-4 micromoles). In addition, 10 ml of 5% sodium bicarbonate solution was injected into these sites.

[0137] Preparation of the renal artery and superior muscular aorta A 3mm flow probe (Transonic System, USA) was inserted into the renal artery through a retroperitoneal incision, and the superior muscular aorta (SMA) was surrounded by a 6mm flow probe (Transonic System, USA) through an abdominal incision. Microcatheters (Rebar™-18 microcatheter, Microtherapeutics, Inc., California, USA) were inserted into the targeted arteries as described above, and the angiography catheters were withdrawn to the aorta in both the renal and SMA experiments.

[0138] After instrumentation, 5000E heparin (Leo Pharma A / S, Denmark) was administered, and no intervention was performed for the following hour. The animals were covered with a heat blanket to maintain normal body temperature. After 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.

[0139] Experiments on the femoral artery and carotid artery PDNO was injected with a carrier solution (1.4% bicarbonate) using a syringe pump, and then administered via a microcatheter to the left common femoral artery and left carotid artery in eight doses (0.01, 0.1, 1, 3, 10, 30, 100, and 300 nmol kg). -1 min -1 The drug was administered over 20 minutes per dose, and block randomization was applied (Figure 1A (carotid and femoral arteries) and Figure 1B (renal and superior mesenteric arteries)). For reference, four doses (3, 10, 30, and 100 nmol kg) were administered between local intra-arterial infusion routes. -1 min -1 Intravenous infusion via central venous catheter was administered at a rate of 10 minutes per dose. A 60-minute washout period was observed between PDNO administrations.

[0140] Experiments on the renal artery and superior mesenteric artery (SMA) Eight doses of PDNO (0.01, 0.1, 1, 3, 10, 30, 100, and 300 nmol kg) -1 min-1 The drug was administered via a microcatheter into the left renal artery in 20 minutes per dose. In SMA, seven doses (3, 10, 30, 100, 300, 1000, and 3000 nmol kg) were administered. -1 min -1 (20 minutes per dose) Vasopressin (Empressin®, OrPha Swiss, Switzerland), 0.06E kg -1 min -1 (40 IU 2ml -1 This was performed during the simultaneous injection of ( ). Intravenous injection was given between local intra-arterial injections, with a washout period of 60 minutes in between.

[0141] Data collection Hemodynamic and ventilatory parameters, including local blood flow in the target organ and fractions of exhaled nitric oxide (FENO; CLD 77, ECO PHYSICS, Switzerland), were continuously recorded (Acqknowledge Software, BIOPAC® Systems Inc, California, USA). Arterial blood gas analysis was collected at the end of all doses (GEM5000, Werfen, Massachusetts, USA).

[0142] Data analysis and statistics The primary outcomes were the effects on local blood flow, systemic blood pressure, and end-tidal nitric oxide (ETNO). These data are presented as mean and standard error (SEM) of the mean. Normality was determined using the Shapiro-Wilk normality test, a linear mixed model was implemented, and changes from baseline were evaluated using SPSS (version 27, IBM Corp., Armonk, New York, USA). Graphs were generated using GraphPad Prism 9.4.1 (GraphPad Software, Inc., San Diego, California, USA). Secondary measures included hemodynamic and blood gas measurements. Intra-arterial versus intravenous dose comparisons were performed. A linear mixed model was fitted, with fixed effects including administration route (categorical), dose (continuous), and dose-route interaction, and a random effect of subject ID (using lme from nlme v.3.1-16 in R4.2.2(16)). For dose comparison, estimated coefficients (estimated using lstrends from emmeans v.1.8.4-1) were compared using the ANOVA function and adjusted using Tukey's HSD test. Circulation time from intra-arterial injection / infusion site (carotid and femoral artery) to intravenous injection / infusion site was calculated by subtracting the time to the first exhalation with an increase in ETNO and end-tidal CO2 when PDNO or sodium bicarbonate was injected into intra-arterial and central venous catheters, respectively. The in vivo half-life was then estimated by linear regression analysis (ln N(t) = k × t + ln(N(0)) and T 1 / 2 (=ln2 / k). A p-value < 0.05 was considered statistically significant.

[0143] Hemodynamic results During intra-arterial PDNO administration at each injection site, blood flow in the left femoral artery and left carotid artery was 1 nmol kg. -1 min -1 It began to increase, and the blood flow to the SMA was 30 nmol kg. -1 min -1The levels began to increase (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 experiment, but increased to 30 nmol kg in the carotid artery experiment. -1 min -1 When the value exceeded a certain threshold, it increased significantly (P<0.05, Figure 2A). Mean systemic arterial pressure (i.e., MAP) was 30 and 100 nmol kg in the carotid and femoral artery experiments, respectively. -1 min -1 It began to decrease at 3000 nmol kg (P<0.05, Figures 2B and 2D, respectively), and in the SMA experiment, 3000 nmol kg -1 min -1 It began to decrease (P<0.05, Figure 3B). The corresponding effect of intravenously administered PDNO on MAP was 10 nmol kg -1 min -1 This was observed (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 indicate that intra-arterial administration of PDNO can achieve increased local therapeutic efficacy at higher doses than intravenous administration, without overall systemic effects. Furthermore, unexpectedly, in contrast to intra-arterial infusion, intravenous PDNO infusion did not affect organ blood flow, highlighting the need for an intra-arterial infusion route to effectively treat systemic organs.

[0144] ETNO End-tidal nitric oxide (ETNO) levels were 30 nmol kg in experiments using the femoral artery, carotid artery, and kidney. -1 min -1 (P<0.05 compared to baseline), and in the SMA experiment, 3000 nmol kg -1 min -1 The levels began to increase (P<0.05 compared to baseline, Figures 2B, 2D, 3B, and 3D). The corresponding increase in intravenous experiments was 10 nmol kg. -1 min -1This was the case (P<0.05 compared to baseline, Figures 2 and 3).

[0145] The estimated coefficients between administration routes using logETNO, and consequently the estimated dose ratios of equivalent effect compared to intravenous infusion, were 2.7, 2.9, 2.6, and 47 for femoral, carotid, renal, and SMA infusions, respectively, using intravenous PDNO infusion as a reference (Figure 4).

[0146] These results are consistent with the results shown in hemodynamic analysis.

[0147] Arterial blood gas The methemoglobin fraction was significantly higher in the SMA experiment at the two highest doses compared to baseline (P<0.05, Figure 5). The methemoglobin fraction did not change with other administration routes, which was also particularly surprising and unexpected. SaO2 was 30 and 100 nmol kg in the carotid and femoral artery experiments, respectively. -1 min -1 This was significantly reduced (P<0.05 compared to baseline).

[0148] Estimation of circulation time and half-life from artery to venous region. The median (25th–75th percentile) circulating time from the carotid and femoral artery injection sites, measured to the first exhalation, increased to 13.5 seconds (12.3–15.4, n=13) and 13.7 seconds (12.9–15.2, n=9) for ETNO, respectively, and to 5.0 seconds (4.3–5.9, n=29) and 5.3 seconds (3.3–5.8, n=9) for the central venous injection site. The circulating time from the carotid and femoral artery injection sites to the central venous injection site was calculated to be 8.5 seconds and 8.4 seconds, respectively, as shown in Table 1 below. By using the increase in ETNO integral caused by intra-arterial and central venous PDNO injection, as well as the circulating time from each injection site, in vivo summary half-life of PDNO was estimated to be 3.6 seconds (3.0–4.4, 95% CI; see Table 2 below for half-life estimates at each dose). Using the dose equivalents estimated in carotid, femoral, and central venous infusion experiments (Figures 4A and 4B) with the same equation, the in vivo half-life of PDNO was estimated to be an average of 5.8 seconds.

[0149] Table 1: Estimated half-lives obtained from injection and infusion experiments with PDNO in two anesthetized pigs. Additionally, the circulation time between the intra-arterial and intravenous injection sites was estimated after injecting 10 ml of 5% sodium bicarbonate solution. Dose ratios (DRs) were obtained from experiments using intra-arterial and intravenous infusion (Figures 1-5). [Table 1]

[0150] conclusion Organ-directed intra-arterial (carotid, femoral, and superior mesenteric) infusion of PDNO increased organ-specific blood flow in a dose-dependent manner, in contrast to renal and intravenous infusion. The effective doses required to increase blood flow in the carotid, superior mesenteric, and femoral arteries had little to no effect on systemic blood pressure. Comparing intra-arterial and intravenous infusions, there was a substantial rightward shift in the dose-response effect on end-tidal NO concentration, likely due to the rapid breakdown of PDNO in the blood. Methemoglobin levels increased to a maximum of 300 nmol kg.-1 min -1 The half-life of PDNO during arterial organ-directed infusion of the dose was less than 3%. The half-life of PDNO in vivo was estimated to vary slightly depending on the experimental setting, ranging from 3.0 to 6.1 seconds.

[0151] Example 3: Antiplatelet aggregation effect of intravenous PDNO administration method research group Five domestically bred, three-month-old pigs (a crossbreed of Swedish native, Hampshire, and Yorkshire breeds) were used. They were provided with free access to food and water until the morning of the experiment. Ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden, approval number 00259~2022). This experiment was conducted in the animal laboratory of Öreble University Hospital in Öreble, Sweden, in accordance with Directive 2010 / 63 / EU on the protection of animals used for scientific purposes.

[0152] Animal preparation and monitoring Anesthesia and ventilation were established and maintained according to Stene Hurtsen 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), with the exception of premedication using Zoletil forte (50 mg ml each). -1 ) and Medetomidin (1 mg ml -1 0.05 ml kg of Domitor Vet -1The mixture was changed to this. Basic instrumentation (arterial line, pulmonary artery catheter, urinary catheterization) was performed as described above (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 the arterial line. -1 The blood was drawn into a 50 ml syringe, and an intermediate dose of thrombin (0.1 U ml) was administered for subsequent pulmonary embolism. -1 Coagulation in the syringe was accelerated using a urinary catheter. A 20 Fr urinary catheter was inserted into the right jugular vein to inject the coagulated blood. Intravenous access for fluids and anesthetics was secured in the left external jugular vein via a 7 Fr introducer (Cordis Corporation, USA). Nitric oxide (NO) in exhaled breath was measured via an endotracheal tube (Eco Physics, Duernten, Switzerland). The animals were covered with a heat blanket 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.

[0153] Experimental protocol After a period without intervention, pulmonary embolism was induced by injecting clotted blood into the right external jugular vein to a target mean pulmonary artery pressure of 45–55 mmHg. Concurrently, intravenous norepinephrine infusion was initiated and titrated to maintain a mean systemic arterial pressure above 60 mmHg. The inspired oxygen (FiO2) fraction was increased to maintain normal arterial oxygenation, and the respiratory rate was increased to partially correct hypercapnia. Approximately 15 minutes later, 160 nmol kg was administered. -1 min -1Intravenous infusion of PDNO was initiated and continued for approximately 15 minutes, after which it was discontinued. After the experiment, the animals were euthanized by a bolus dose of 200 mg of propofol followed by rapid intravenous injection of 40 mM potassium chloride. Cardiac arrest was confirmed by hemodynamic and respiratory measurements.

[0154] Hemodynamic and ventilatory parameters, including end-tidal NO concentration, were continuously recorded (Acqknowledge Software, BIOPAC® Systems Inc, California, USA). Baseline (pre-pulmonary embolism), post-pulmonary embolism, PDNO 160 nmol kg. -1 min -1 Arterial and mixed venous blood samples were collected during administration and 15 minutes after discontinuation of PDNO infusion. The arterial and mixed venous blood samples were analyzed using a blood gas analyzer (GEM5000), and the arterial blood samples collected with a heparinized syringe were immediately transferred to a cuvette for multiplate agglutination analysis. The blood was stimulated with the PAR-1 agonist peptide SFLLRN, and platelet aggregation (recorded as an increase in electrical resistance) was tracked for 10 minutes and calculated as the area under the curve (see the method in Example 4 for details).

[0155] statistical analysis The primary outcome was the change in PAR-1 agonist peptide SFLLRN-induced platelet aggregation between pulmonary embolism at measurement points and intravenous PDNO infusion (paired comparison [Dunn test] where a Friedman test including all four time points found a P-value < 0.05). Secondary outcomes were changes in hemodynamic variables, end-tidal nitric oxide (NO) levels, and blood gas values ​​comparing embolism with and without PDNO infusion. Statistical analysis of secondary outcomes was not performed.

[0156] result The animals had normal hemodynamic and respiratory variables prior to pulmonary embolism. Pulmonary embolism induced acute pulmonary hypertension with systemic hypotension (improved with norepinephrine infusion), arterial deoxidation (improved with increased FiO2), hypercapnia (partially improved with increased respiratory rate), and increased end-tidal nitric oxide (NO). Intravenous PDNO infusion caused a decrease in pulmonary and systemic vascular resistance as well as an increase in end-tidal NO. These effects improved when PDNO infusion was discontinued. Intravenous PDNO infusion during pulmonary embolism statistically significantly reduced PAR-1 agonist peptide SFLLRN-induced platelet aggregation compared to before PDNO infusion (P=0.0143, Figure 6).

[0157] conclusion The data show that intravenous PDNO in pulmonary embolism surprisingly inhibits platelet aggregation (measured ex vivo), demonstrating its effectiveness as a treatment for thromboembolic diseases. When combined with local effects achieved through PDNO administration, such as vasodilation, the present invention can further provide improved treatments for thromboembolic diseases.

[0158] Example 4: Further antithrombotic research Platelet aggregation in whole blood The potential antithrombotic effect of PDNO in vitro was evaluated by analyzing its aggregation response to the thrombin-mimicking hexapeptide SFLLRN. This peptide is an agonist of the major thrombin receptor designated as protease-activated receptor-1 (PAR-1). The inhibitory effect of PDNO was elucidated in isolated suspensions and whole blood of human platelets.

[0159] Heparinized human or porcine blood (0.5 ml) was diluted 1:1 with Krebs-Ringer glucose (KRG) buffer (isotonic saline, pH 7.4). The diluted blood was transferred to an analytical cuvette and placed in a multiplate aggregater. Platelet aggregation in vitro (human blood) and ex vivo (porcine blood) was measured as an increase in electrical impedance between two platinum electrodes. Measurements were performed at 37°C under agitation conditions.

[0160] In in vitro experiments, PDNO or nitroglycerin (used as a control drug) was introduced 2 minutes before the hexapeptide SFFLRN. This hexapeptide acts as an agonist for the main thrombin receptor designated as protease-activated receptor-1 (PAR-1). In ex vivo experiments, PDNO was administered intravenously to anesthetized pigs. After blood collection, diluted blood was transferred to a Multiplate instrument and stimulated with SFFLRN. The agglutination response was recorded for 10 minutes and expressed as the area under the curve.

[0161] The potential anti-aggregating activity of PDNO was investigated by measuring the increase in light transmission in aliquots of isolated platelets after the addition of SFLLRN (by Bone aggregation assay). As shown in Figure 7, PDNO inhibited SFLLRN-induced platelet aggregation in a concentration-dependent manner, demonstrating antithrombotic activity.

[0162] Aggregation of isolated platelets Platelet aggregation in whole blood (human blood containing heparin as an anticoagulant) was analyzed using a multiplate aggregater as an increase in electrical resistance (change in impedance between two platinum electrodes).

[0163] Heparinized blood was obtained from healthy volunteers and immediately mixed with acidic dextrose citrate (ACD) solution in a volume ratio of 5 parts blood to 1 part ACD. Platelets were centrifuged at 220xg for 20 minutes to obtain platelet-rich plasma. Platelets were then pelletized by a second centrifugation (480xg for 20 minutes) and gently resuspended in KRG buffer. 0.3 ml of isolated platelet suspension (2.5 × 10⁸ platelets per ml) was placed in a Chrono-log aggregater and analyzed at 37°C under agitation. Platelets were stimulated with PDNO or nitroglycerin for 2 minutes and then activated with SFLLRN. Platelet aggregation was assessed as a percentage increase in light transmission through the cuvette (light transmission in platelet-free KRG represents 100%).

[0164] Figure 8 shows that PDNO significantly inhibited SFLLRN-induced platelet aggregation under these experimental conditions, while nitroglycerin did not. This finding indicates that PDNO exerts antithrombotic effects both in whole blood and in the presence of hemoglobin.

[0165] Detection of protein phosphorylation by Western blotting In platelets and vascular smooth muscle cells, cytoskeletal connective protein vasodilator-stimulated phosphorylated protein (VASP) is a major molecular target of the NO / cyclic GMP signaling pathway. Ser239-specific phosphorylation of VASP after NO exposure in platelets is thought to be an important molecular mechanism underlying the anti-aggregating / antithrombotic effects of NO. The effect of PDNO on Ser239-specific VASP phosphorylation was analyzed by Western (immuno) blotting.

[0166] Aliquots of isolated platelet suspensions were exposed to either PDNO or nitroglycerin for 5 minutes. This series of experiments was performed on both resting platelets and SFLLRN-stimulated platelets.

[0167] Specifically, NO / cyclic GMP-induced phosphorylation of Ser239 on vasodilator-stimulated phosphorylated protein (VASP) was detected by Western blotting. Isolated platelet suspensions (0.2 ml) were stimulated with PDNO or nitroglycerin for 5 minutes in the absence or presence of SFLLRN. Hexapeptide was added 2 minutes after NO donor. Platelets were lysed by adding 50 μl of sodium-dodecyl sample buffer, and the proteins were further denatured at 95°C for 5 minutes. Platelet proteins were separated by electrophoresis and then blotted on polyvinylidene fluoride membranes. To determine VASP phosphorylation, the membranes were incubated with a Ser239-specific VASP antibody, followed by a horseradish peroxidase antibody, and visualized using a Fuji LAS chemiluminometer.

[0168] As shown in Figure 9, PDNO caused a significant increase in Ser239-specific VASP phosphorylation, while nitroglycerin did not. These data indicate that PDNO induces significant NO / cyclic GMP signaling in human platelets, which is associated with a significant anti-aggregation effect (Figures 7 and 8).

[0169] Example 5: Intra-arterial infusion of PDNO to treat acute lower extremity embolism research group Twenty-four domestically bred, three-month-old pigs (a crossbreed of Swedish native, Hampshire, and Yorkshire breeds (sex ratio 1:1, weight 32±4kg)) were used. They were provided with free access to food and water until the morning of the experiment. Ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden, approval number 00259~2022). This experiment was conducted from September 12-30, 2022 and January 24-31, 2023, in the animal laboratory of the University Hospital in Örebl, in accordance with Directive 2010 / 63 / EU on the protection of animals used for scientific purposes.

[0170] Animal preparation and monitoring Anesthesia and ventilation were established and maintained for the first 14 animals according to Stene Hurtsen et al. (Stene Hurtsen et.al. 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), but for the last 10 animals, premedication was administered at 0.05 ml kg. -1 Zoletil forte (250 mg + 250 mg) and Medetomidin (1 mg ml) -1The mixture was changed to a combination of Domitor Vet. Basic instrumentation (arterial line, pulmonary artery catheter, urinary catheterization) was performed as described above (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 subsequent embolization, 1 ml kg was administered through the arterial line. -1 Blood was drawn into a 5 ml syringe (Cordis Medical ApS, Denmark). Venous access for fluids and anesthetics was secured in the left external jugular vein via a 7 Fr introducer (Cordis Corporation, USA). A 5 Fr catheter (Soft-Vu Berenstein 5Fr, Queensbury, New York, USA) was placed through the left venous access and advanced to the common femoral vein at the branch to the deep vein. Similarly, a 5 Fr catheter (Soft-Vu Berenstein, USA) was inserted through the brachial artery via a 5 Fr (Cordis Corporation, USA) introducer and advanced to the common femoral artery (CFA). The position was confirmed by fluoroscopy. Flow probes (Transonic Systems Inc., New York, USA) were placed on both sides of the CFA, 3 mm to the left and 6 mm to the right. A laser Doppler probe (Perimed, Sweden) was placed in the peroneal muscle of the hind limb, and a microdialysis catheter (M Dialysis AB, Sweden) was placed in the tibialis anterior muscle. Nitric oxide in exhaled breath was measured through an endotracheal tube (Eco physics, Duernten, Switzerland). The animals were covered with a heat blanket 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.

[0171] Experimental protocol After a period without intervention, a 1 ml / kg autologous blood clot was extracted from the left common femoral artery via a 5 Fr catheter (Berenstein, USA). -1 (Minimum 2 hours of coagulation time) 5ml min -1 Selective embolization was performed. One hour after embolization, PDNO3 nmol kg -1 min -1 Block randomization to either the treatment group or the control group (no infusion) was performed (Figure 10). NO donors were administered via a syringe pump, and the carrier solution (1.4% bicarbonate) was inserted through a microcatheter (Terumo Progreat®, 2.7Fr, Terumo cooperation, Tokyo, Japan) via a 5Fr catheter (Berenstein, USA) into the right brachial artery and directed towards the common femoral artery. In both groups, the 5Fr catheter (Berenstein, USA) was withdrawn to the aortic bifurcation. Reperfusion time was followed for 6 hours. At the end of the protocol, bilateral muscle biopsies of the tibialis anterior muscle were taken and placed in formalin (Solveco AB, Rosersberg, Sweden) for blinded histopathological evaluation.

[0172] Hemodynamic and ventilatory parameters, including local blood flow, were continuously recorded in CFA (Acqknowledge Software, BIOPAC® Systems Inc, California, USA). Laser Doppler signals, exhaled nitric oxide (FENO, apparaten) fractions, arterial and local venous blood samples, and blood gases were collected according to protocol (Figure 10).

[0173] The primary outcome was left femoral artery blood flow. Secondary outcomes included ischemia index scores at 6 hours (concentrations of lactate, potassium, aspartate aminotransferase, lactate dehydrogenase, and creatine kinase (reference 19667877)), lower extremity microcirculation (via laser Doppler), and histopathology of lower extremity muscle biopsies. Adverse events (methemoglobinemia, systolic blood pressure) were measured. Measurements were collected according to Figure 10.

[0174] calculation Femoral venous resistance (FVR): femoral artery blood flow / MAP.

[0175] Delta lactate (Δ lactate): Central arterial lactate level - femoral vein lactate level.

[0176] Lactic acid production: femoral artery blood flow × Δlactate × -1.

[0177] Saturation levels of the central artery and femoral vein: (partial pressure of oxygen [pO2] 2.94) / (pO2 2.94 + (4.76 × (10(-0.441 × (pH-7.4)))^2.94).

[0178] O2 content of the central artery and femoral vein: ((saturation level × Hb) / 10 × 1,31 + 0,025 × pO2) × 10.

[0179] Femoral vein oxygen consumption: (Arterial oxygen content - Femoral vein oxygen content) × (Femoral artery blood flow / 1000).

[0180] statistical analysis The normal distribution was analyzed using the Shapiro-Wilk test. Normally distributed data were analyzed using a linear mixed model with group and time as the primary factors, followed by their interactions and then multiple interactions. Normally distributed data are presented as mean ± standard error of the mean. Data deviating from the normal distribution were analyzed using the Mann-Whitney U test and presented as the median (interquartile range, 25th–75th percentile). All statistics were performed using SPSS (version 27, IBM Corp., Armonk, New York, USA), and graphs were generated using GraphPad Prism 9.4.1 (GraphPad Software, Inc., San Diego, California, USA).

[0181] result The PDNO group and the control group were similar at baseline, except for cardiac output (CO) and femoral vein oxygen consumption (FV O2), which were higher in the control group at baseline. Common femoral artery (CFA) blood flow disappeared 15 minutes post-embolization in both groups, and was significantly higher in the PDNO group after infusion initiation (Figure 11). Similarly, femoral vein resistance (FVR) was significantly lower in the PDNO group 1 hour after infusion (Figure 12). Heart rate, CO, and MAP were similar between the groups.

[0182] Femoral vein (FV) pO2, FV VO2, and FV lactate production were all higher in the PDNO group compared to the control group, but not statistically significant (Figure 11). Femoral vein CO2 levels were significantly higher in the control group after 3 hours of infusion, and FV O2 levels were generally higher in the PDNO group, but not statistically significant. Similarly, ETNO and MHb levels were higher and pO2 was generally lower in the PDNO group, but not statistically significant (Figure 12).

[0183] conclusion The data show that femoral artery infusion in experimental acute limb embolism in pigs increases femoral artery blood flow compared to controls without systemic side effects. Further analysis will indicate whether this vasodilatory effect also leads to improved metabolism and reduced lower limb injury.

Claims

1. A compound of formula (I), 【Chemistry 1】 In the formula, R 1 , R 2 , and R 3 However, each independently represents either H or -NO. n is 0 or 1, If n is 0, R 1 However, it is H, If n is 1, R 2 However, it is H, However, R 1 , R 2 , and R 3 The condition is that at least one of them represents -NO, It is for use in the treatment of a disease state, and the compound of formula (I) is administered to a patient in need thereof via intra-arterial infusion at a dose of about 0.01 to 3000 nmol kg -1 min -1 of the compound of formula (I).

2. The compound of formula (I) is present in quantities of approximately 0.01 to approximately 300 nmol / kg. -1 min -1 For example, approximately 1 to approximately 300 nmol kg -1 min -1 For example, approximately 1 to approximately 100 nmol kg -1 min -1 , or approximately 10 to approximately 3000 nmol kg -1 min -1 The compound for use according to claim 1, administered in the dose of [amount].

3. The compound of formula (I) is approximately 1 to approximately 30 nmol kg. -1 min -1 A compound for use according to any one of the prior claims, administered in a dose of [amount].

4. The compound for use according to any one of the prior claims, wherein the compound of formula (I) is administered optionally over a period of up to 14 days, for example, over a period of 7 days.

5. The compound for use according to any one of the prior claims, wherein the compound of formula (I) is administered to the femoral artery and / or carotid artery via intra-arterial injection.

6. The compound of formula (I) is injected into the femoral artery and / or carotid artery via intra-arterial injection at a rate of approximately 1 to approximately 300 nmol / kg. -1 min -1 For example, 1 to approximately 60 nmol kg -1 min -1 For example, 1 to approximately 50 nmol kg -1 min -1 For example, 1 to approximately 30 nmol kg -1 min -1 A compound for use according to any one of claims 5, administered in the dose of [amount].

7. The compound for use 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 injection.

8. The compound of formula (I) is injected into the superior mesenteric artery via intra-arterial injection at a rate of approximately 3 to 3000 nmol / kg. -1 min -1 For example, approximately 30 to approximately 1000 nmol kg -1 min -1 For example, approximately 100 to 1000 nmol kg -1 min -1 The compound for use according to claim 7, administered in the dose of [amount].

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

10. The compound of formula (I) is injected into the renal artery via intra-arterial injection at a rate of approximately 0.01 to approximately 300 nmol kg. -1 min -1 For example, approximately 1 to approximately 30 nmol kg -1 min -1 The compound for use according to claim 9, administered in the dose of [amount].

11. The compound for use according to any one of the prior claims, wherein the compound is for use in the treatment of a condition in which NO has a beneficial effect.

12. The aforementioned pathological condition Acute pulmonary vasoconstriction of different causes, pulmonary hypertension of different causes including primary and secondary hypertension, preeclampsia, eclampsia, conditions of different causes requiring vasodilation, erectile dysfunction, systemic hypertension of different causes, localized vasoconstriction of different causes, regional vasoconstriction of different causes, acute heart failure (with or without ejection fraction preservation (HFpEF)), coronary heart disease, myocardial infarction, ischemic heart disease, angina pectoris, unstable angina, cardiac arrhythmias, acute pulmonary hypertension in cardiac surgery patients, acidosis, airway inflammation, cystic fibrosis, COPD, cilia syndrome, lung inflammation, pulmonary fibrosis, acute lung injury (ALI), adult respiratory distress syndrome, acute pulmonary edema, acute altitude sickness, asthma, trachea Compounds for use as described in any one of the prior claims, selected from the group consisting of bronchitis, hypoxia of different causes, ischemic disease of different causes, stroke, cerebral vasoconstriction, inflammation of the gastrointestinal tract, gastrointestinal dysfunction, gastrointestinal complications, IBD, Crohn's disease, ulcerative colitis, liver disease, pancreatic disease, inflammation of the bladder of the urinary tract, inflammation of the urinary tract, bladder and ureters, skin inflammation, diabetic ulcers, diabetic neuropathy, psoriasis, inflammation of different causes, wound healing, organ protection in ischemia-reperfusion conditions, organ transplantation, tissue transplantation, cell transplantation, acute kidney disease, uterine laxity, cervical laxity, thromboembolic diseases including thromboembolic diseases such as various hematological disorders, arterial occlusion such as arterial embolism, and conditions requiring smooth muscle relaxation.

13. The compound for use according to claim 12, wherein the pathological condition to be treated is selected from the group consisting of ischemic diseases of different causes, thromboembolic diseases including diseases accompanied by thromboembolism such as various blood disorders, arterial thrombosis, peripheral ischemia (ischemia of the limbs), thromboembolic stroke, pulmonary embolism, acute mesenteric ischemia (occlusion of the mesenteric artery), 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), 【Chemistry 2】 In the formula, R 1 , R 2 , and R 3 However, each independently represents either H or -NO. n is 0 or 1, If n is 0, R 1 However, it is H, If n is 1, R 2 However, it is H, However, R 1 , R 2 , and R 3 The condition is that at least one of them represents -NO, A compound of formula (I) intended for use in the treatment of thromboembolic diseases.

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

16. The above compound is present in an amount of approximately 0.01 to 3000 nmol / kg. -1 min -1 The compound for use according to claim 14 or 15, administered in the dose of [amount].

17. The above compound is present in an amount of approximately 0.01 to approximately 300 nmol / kg. -1 min -1 For example, approximately 1 to approximately 300 nmol kg -1 min -1 A compound for use according to any one of claims 14 to 16, administered in the dose of [amount].

18. The aforementioned compound is approximately 1 to approximately 300 nmol / kg -1 min -1 The compound for use according to claim 17, administered in the dose of [amount].

19. The above compound is present in an amount of about 1 to about 10 nmol kg. -1 min -1 The compound for use according to claim 17, administered in the dose of [amount].

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

21. The compound for use 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 (ischemia of the limbs), thromboembolic stroke, pulmonary embolism, acute mesenteric ischemia (occlusion of the mesenteric artery), acute renal artery occlusion, arterial stenosis, arterial occlusion, and arterial embolism.

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