Process for preparing nitrosylated propanediol, composition containing the same, pharmaceutical preparation, kit of parts, and combined product

Abstract

The present invention relates to a novel process for synthesizing mono- and bis-nitrosylated propanediols, as well as novel compositions and pharmaceutical formulations containing these compounds. The process proceeds by reacting the corresponding unnitrosylated propanediol with a source of nitrite, optionally in the presence of a suitable acid. When the source of nitrite is an organic nitrite, the reaction step is carried out in a suitable organic solvent; when the source of nitrite is an inorganic nitrite, the reaction step is carried out in a biphasic solvent mixture containing an aqueous phase and a non-aqueous phase. The present invention also relates to methods for treating conditions in which administration of nitric oxide (NO) has a beneficial effect by administering such compounds, compositions, or formulations.

Description

Technical Field

[0001] The present invention relates to a novel process for the synthesis of mono- and bis-nitrosylated propanediols, as well as novel compositions and pharmaceutical formulations comprising such compounds. The present invention also relates to a method of treating a condition in which administration of nitric oxide (NO) has a beneficial effect by administering such a compound, composition or formulation.

Background Art

[0002] The listing or discussion of a document clearly published previously in this specification should not necessarily be taken as an admission that the document is part of the prior art or common general knowledge.

[0003] Pulmonary hypertension (PH) has hitherto been defined as an increase in mean pulmonary artery pressure (mPAP) at rest of 25 mmHg or more, and can be divided into a more slowly progressing chronic form (PH) and acute pulmonary hypertension (aPH). This definition has recently been updated to include an increase in mean pulmonary artery pressure (mPAP) at rest of 20 mmHg or more, together with a Wood unit value of more than 3. In aPH, mPAP rapidly increases due to acutely induced pulmonary vasoconstriction. This can be induced as a reaction to a variety of conditions such as major surgery (e.g., heart surgery), pulmonary embolism, and sepsis. In aPH, right heart adaptation does not develop, so the risk of right heart failure increases, and furthermore, patients usually develop a critical condition due to the condition inducing aPH, and usually have a very low systemic blood pressure. In patients with a more chronic form of PH, aPH can overlap with chronic PH, causing harmful hypertension and leading to right heart failure and death. Acute pulmonary hypertension is a major problem causing numerous deaths and suffering in millions of people worldwide, and diagnosis usually requires right heart catheterization, and the problem is not fully registered or known due to a lack of efficient lung-selective treatment.

[0004] In the normal state, the right heart receives deoxygenated blood from the systemic circulation and pumps it through the lungs. The cardiac output of the pulmonary circulation is equal to the volume of blood circulating through all other body organs. Despite the high flow rate through the lungs, the blood pressure in the pulmonary circulation is only one-fifth of the blood pressure in the systemic circulation. The low resistance in the pulmonary circulation is due to the large cross-sectional area of the pulmonary arteries and the much shorter length of the pulmonary vessels compared to the systemic vessels. The left heart is a powerful pump (working against high pressure) that sends the blood flow of the systemic circulation to, for example, the liver, stomach, kidneys, and the heart itself, and it is a well-known fact that high blood pressure in the systemic circulation can cause many health problems including heart failure, stroke, and kidney disease.

[0005] Many physiological factors affect the complex control of blood flow in the systemic and pulmonary circulations. The blood vessels in the systemic circulation are normally in a state of vasoconstriction (where small muscles in the vessel walls constrict the vessels), while the blood vessels in the pulmonary circulation are under a certain degree of vasodilation (i.e., relaxed, widened vessels), thereby maintaining a very low resistance to blood flow and resulting in a very low blood pressure compared to the systemic circulation.

[0006] In various life-threatening diseases and after major surgeries, pathophysiological reactions with intense inflammatory responses change the physiological state of the systemic and pulmonary blood vessels. These changes generally result in a state where the systemic blood vessels suddenly dilate, causing very low systemic blood pressure (systemic hypotension), which may reduce blood flow to important organs such as the brain, heart, liver, and kidneys. Parallel to this, paradoxically, the pulmonary blood vessels suddenly constrict, causing acute pulmonary hypertension and right heart failure, which further reduces cardiac output and worsens systemic hypotension. These critically ill and hemodynamically unstable patients usually need to be treated in the intensive care unit. In the intensive care unit, it is a challenge to balance drug therapies using vasopressors and inotropic agents to restore systemic blood pressure, as well as pulmonary vasodilators to reduce acute life-threatening pulmonary hypertension.

[0007] Acute PH is often underdiagnosed and treatment is often delayed (Rosenkranz, Stephan et al., European Heart Journal, 37(12), 942 - 954(2016)). The reason aPH is very fatal is that the right heart is a weak pump that normally functions against low pressure, and when the mean pressure in the pulmonary circulation rapidly exceeds 40 mmHg, there is a risk of dysfunction (right heart failure). Acute PH is a distinct critical condition and should not be confused with chronic pulmonary hypertension (Tiller, D et al., PLoS One, 8(3), e59225(2013)). In chronic diseases, when the pressure in the pulmonary circulation gradually increases over time, the right heart adapts and increases in size and strength, and can maintain a much higher outflow pressure. Even healthy people can develop aPH as a complication when they develop an infectious disease, a pulmonary embolism (blood clot in the lung), or undergo major surgery.

[0008] Acute pulmonary hypertension is a huge problem that causes pain, premature death, and suffering for millions of people worldwide. Due to a lack of proper diagnosis and treatment, the problem is not fully registered and is unknown.

[0009] The treatment options for patients with aPH are severely limited today. This is because patients usually show very low systemic blood pressure. When trying to treat with intravenous (i.v.) vasodilators, the drugs available today pass through the lungs (usually <30 seconds) and "leak" into the systemic circulation, often leading to fatal systemic hypotension. Therefore, the optimal intravenous drug for treating aPH must only dilate the pulmonary vessels and not affect the systemic circulation. At present, intravenous pulmonary-selective vasodilators are not available on the market.

[0010] To overcome the systemic side effects of intravenously administered vasodilators, administration by inhalation of nitric oxide or prostacyclin has been developed. Unfortunately, these drugs are often inadequate because, even though they are effective in some cases, they are often inactivated before reaching the target pulmonary vessels. Another major drawback of currently used inhaled drugs is that administration by inhalation is more complex than injecting the drug intravenously. Since the administration of inhaled nitric oxide is very complex, medical staff require special training, and for this reason, many hospitals do not even have the equipment because of the associated costs.

[0011] Nitric oxide (NO) is an important molecule in several biological systems. It is continuously produced in the lungs and can be measured at ppb (parts per billion) levels in exhaled breath. The discovery of endogenous NO in exhaled breath and its use as a diagnostic marker for inflammation dates back to the early 1990s (see, for example, WO93 / 05709 and WO95 / 02181). Today, the importance of endogenous NO is widely recognized, as evidenced by the commercial availability of a clinical NO analyzer (NIOX®, the first tailor-made NO analyzer for daily clinical use in asthmatic patients, manufactured by AEROCRINE AB, Solna, Sweden).

[0012] Since these initial experiments, it has become generally recognized that endogenous nitric oxide (NO) is very important as a mediator of vasodilation in blood vessels. In particular, nitric oxide plays an important role in the regulation of pulmonary vascular tone, optimizing ventilation-perfusion matching in healthy adults (i.e., matching the air reaching the alveoli with the blood reaching the alveoli via the capillaries so that the oxygen provided via ventilation is just sufficient to fully saturate the blood. See, for example, Persson et al., Acta Physiol. Scand., 1990, 140, 449-57). Measurement of NO in exhaled breath is a good way to monitor changes in endogenous NO production or removal in the lungs (Gustafsson et al., Biochem. Biophys. Res. Commun., 1991, 181, 852-7).

[0013] Since ventilation-perfusion mismatch and elevated pulmonary artery pressure are characteristics of pulmonary embolism, inhaled NO has been tested as a potential treatment. For example, US5,670,177 describes a method for treating or preventing ischemia that includes administering to a patient, via an intravascular route, a gaseous mixture containing NO and carbon dioxide in an amount effective to treat or prevent ischemia. US6,103,769 discloses a similar method except that NO-saturated saline is used.

[0014] Furthermore, nitric oxide / oxygen blends are used as a last resort gas mixture in critical care to promote capillary and lung dilation for the treatment of meconium aspiration syndrome associated with primary pulmonary hypertension and congenital defects in neonatal patients (see Barrington et al., Cochrane Database Syst.Rev., 2001, 4, CD000399 and Chotigeat et al., J.Med.Assoc.Thai., 2007, 90, 266-71). Similarly, NO is administered as a rescue therapy to patients with acute right ventricular failure secondary to pulmonary embolism (Summerfield et al., Respir.Care., 2011, 57, 444-8). Inhaled NO is also approved in Europe and Japan as a treatment for acute pulmonary hypertension in cardiac surgery patients.

[0015] Some researchers have investigated the use of compounds that deliver NO instead of providing NO as a gas or dissolved in a solution. For example, WO94 / 16740 describes the use of NO-delivery compounds such as S-nitrosothiols, thionitrites, thionitrates, sydnonimines, froxan, organic nitrates, nitroprusside, nitroglycerin, iron-nitrosyl compounds, etc. for the treatment or prevention of alcoholic liver injury.

[0016] Nitrates are currently used to treat the symptoms of angina (chest pain). Nitrates function by relaxing blood vessels, reducing the heart's workload while increasing the supply of blood and oxygen to the heart. Examples of currently available nitrates include the following. a) Nitroglycerin (glyceryl trinitrate) (1,2,3-propanetriol trinitrate). This is mainly administered sublingually today to suppress acute attacks of angina. However, strong headaches and dizziness due to its rapid and general vasodilatory effects often occur as side effects. Concentrates for nitroglycerin infusion are also available and are diluted with isotonic glucose or saline for intravenous infusion. b) Isosorbide mononitrate (1,4:3,6-dianhydro-D-glucitol-5-nitrate), which is used as a prophylactic drug for angina. The development of tolerance is a problem with long-term treatment regimens. Frequent side effects include headaches and dizziness, as occur with nitroglycerin. c) Isosorbide dinitrate (1,4:3,6-dianhydro-D-glucitol-2,5-dinitrate), which is taken both acutely and prophylactically for angina and heart failure. d) Pentaerythrityl tetranitrate, a group of organic nitrates, is known to exert long-term antioxidant and anti-atherogenic effects by a mechanism that has not yet been identified. Pentaerythrityl tetranitrate has been investigated in the context of the undesirable nitrate tolerance that develops with nitrate therapy and has been experimentally tested in pulmonary hypertension.

[0017] Many of these nitrate compounds, like other nitrate and nitrite compounds, have been tested in vivo and are known to produce NO. For example, in a rabbit model, glyceryl trinitrate, ethyl nitrite, isobutyl nitrate, isobutyl nitrite, isoamyl nitrite, and butyl nitrite have been tested, and a significant correlation has been found between in vivo production of NO and its effect on blood pressure (Cederqvist et al., Biochem. Pharmacol., 1994, 47, 1047-53).

[0018] Accordingly, certain organic nitrites have been suggested to be useful for treating male impotence and erectile dysfunction through topical or intracavernosal administration to the penis (see US5,646,181).

[0019] More recently, the roles of dietary nitrates and nitrites have been re-evaluated because of the discovery of the endogenous production of NO and its role in host defense, particularly in the arginine-nitric oxide system (Larsen et al., N. Engl. J. Med, 2006, 355, 2792-3). Accordingly, L-arginine and its esters such as ethyl-, methyl- and butyl-L-arginine have been used to increase the endogenous production of NO.

[0020] 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 that can form reversible bonds or associations to NO, such as alcohols, carbohydrates and proteins.

[0021] 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 such compounds followed by purging with gaseous nitric oxide (NO).

[0022] Nilsson, K. F. et al., Biochem Pharmacol., 82(3), 248-259 (2011) discuss the formation and identification of novel bioactive organic nitrites.

[0023] Despite recent advances, there are many disadvantages associated with prior art compounds, compositions and methods of preparation.

[0024] For example, among currently available compounds and compositions, many are associated with undesirable properties or side effects such as toxicity problems, delayed action, irreversible action, or long-term effects. One particular problem that frequently occurs when NO-donating compounds are administered in injection form is the formation of methemoglobin (metHb).

[0025] Furthermore, known organic nitrites and their therapeutic use are often associated with problems likely due to impurities and decomposition products present in the composition. Preparing pharmaceutical formulations containing organic nitrites is also difficult because the mixing steps and vehicles used can cause further decomposition.

[0026] Furthermore, the use of inhaled nitric oxide and oxygen has significant problems due to the generation of nitrogen dioxide that needs to be continuously monitored during administration.

[0027] Some prior art preparation methods provide organic nitrites in relatively low concentrations in aqueous solutions. This means that the storage and transport properties of such formulations are often not satisfactory.

[0028] Furthermore, prior art preparation methods result in a significant amount of NO gas and inorganic nitrites dissolved in the solution in addition to the desired organic nitrite. Since NO is highly reactive, care is required in handling and storing the solution to avoid sudden spontaneous decomposition. Also, the NO gas may react with the plastic materials in the storage container.

[0029] Furthermore, the presence of inorganic nitrites increases the methemoglobin fraction of the blood. This is a side effect that limits the dose.

[0030] Therefore, there is a significant and urgent need for methods of preparing NO delivery compounds and compositions thereof that overcome one or more disadvantages associated with prior art preparation methods and compositions. There is also a need for methods that enable the use of the compounds and compositions obtained from such processes.

Summary of the Invention

[0031] The inventors have unexpectedly discovered a process for preparing NO-delivery compounds that overcomes one or more disadvantages associated with prior art preparation methods.

[0032] For example, the process of the present invention provides a relatively high concentration of the compounds of the present invention in solution, thereby facilitating handling and minimizing storage and transportation costs. Furthermore, the process of the present invention does not result in dissolved nitric oxide gas or inorganic nitrite, thus minimizing the risk of sudden spontaneous decomposition and reducing the potential for side effects when the products of the process are used therapeutically. The process of the present invention also produces only very low levels of other impurities.

[0033] Furthermore, the inventors have found that such a process can deliver chemically stable non-aqueous compositions and formulations containing these compounds, enabling convenient transportation and storage prior to therapeutic use. Additionally, the inventors have developed a convenient means for using such compositions and formulations by administering them in combination with a suitable aqueous buffer.

[0034] Process According to a first aspect of the present invention, a process for preparing a composition comprising one or more compounds of formula I,

Chemical formula

[0035] To avoid misunderstanding, the product of the process of the present invention (i.e., the compound of formula I) may also (or alternatively) be referred to as mono- and bis-nitrosylated 1,2-propanediol or 1,3-propanediol (or a mixture of such compounds, i.e., a composition comprising one or more mono- or bis-nitrosylated 1,2- or 1,3-propanediol).

[0036] To avoid misunderstanding, the corresponding compound of formula I, wherein R 1 , R 2 , and R 3 are H, can also be referred to as the corresponding 1,2-propanediol and / or 1,3-propanediol (i.e., corresponding to the structure of the desired product), which can in turn be referred to as the starting material for the process of the present invention. In other words, the corresponding compound of formula I can be a compound according to formula (Ia) as defined below.

Chemical formula

[0037] To avoid misunderstanding, when an integer (n or 1 - n) associated with an oxygen atom is 0, the oxygen atom is absent altogether and the substituent R 1and R 2 (and the corresponding H in the compound of formula (Ia)) are each bonded to the respective carbon.

[0038] Those skilled in the art will understand that references herein to the process of the invention (or similarly "the process of the invention", etc.) include references to all embodiments and their specific features.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] Without departing from the disclosure of the invention, all embodiments and specific features of the invention referred to herein may be employed alone or in combination with any other embodiments and / or specific features referred to herein (thus, more specific embodiments and specific features are described as disclosed herein).

[0041] As used herein, the term "comprising" takes its ordinary meaning in the art, i.e., it indicates that the component includes but is not limited to the relevant features (i.e., among other things, includes). Thus, the term "comprising" includes references to components that are essential to the relevant substance.

[0042] As used herein, unless otherwise specified, the terms "consisting essentially of" and "consists essentially of" refer to related components that are formed of at least 80% (e.g., at least 85%, at least 90%, or at least 95%, e.g., at least 99%) of the specified substance according to the relevant measurements (e.g., by its weight). The terms "consisting essentially of" and "consists essentially of" may each be replaced by "consisting of" and "consists of", respectively.

[0043] To avoid misunderstanding, the term "comprising" also includes references to components consisting of the relevant substance.

[0044] Thus, one skilled in the art will understand that a reference to the preparation of a composition comprising one or more compounds of formula (I) refers, as a component, to the preparation of a composition comprising, optionally together with other compounds, an amount of one or more compounds of a structure as defined in formula I. The process of the present invention may also be referred to as a process for preparing a compound of formula I (i.e., a process for preparing one or more compounds of formula I).

[0045] One skilled in the art will understand that a reference to a process that is a process for preparing a compound of formula I indicates that the process of the present invention may result in the preparation of one or more types of compounds as described respectively by formula I as defined herein (e.g., when two or more such compounds are present as a mixture thereof).

[0046] Thus, one skilled in the art will also understand that the compounds formed in the process of the present invention can take the form of a mixture of each mononitrite and dinitrite product, and the respective relative amounts vary depending on the concentration of the compound of formula I.

[0047] In particular, the process of the present invention concerns R 1 , R 2 and R 3 each independently represent H or -NO, provided that at least 50 wt%, 60 wt%, 70 wt% or 80 wt% (e.g., at least 90 wt% or at least 99 wt%, e.g., at least 99.9 wt%) of the compound of formula I is mononitrosylated such that one of R 1 , R 2 or R 3 represents -NO and the other groups represent H, which may enable the preparation of a composition.

[0048] In particular, the process of the present invention, together with one or more compounds of formula I, concerns the corresponding compounds of formula I, wherein R 1 , R 2 , and R 3Compounds that represent H (i.e., 1,2 - propanediol and / or 1,3 - propanediol, e.g., unreacted 1,2 - propanediol and / or 1,3 - propanediol starting materials), and optionally, the preparation of compositions containing other compounds may occur.

[0049] In certain embodiments, the process of the present invention is a process for preparing a composition consisting essentially of one or more compounds of formula I, and one or more corresponding compounds of formula I, wherein R 1 , R 2 , and R 3 represent H (i.e., 1,2 - propanediol and / or 1,3 - propanediol, e.g., as a mixture thereof).

[0050] One of ordinary skill in the art will understand that the term "react" refers to bringing the relevant components together in such a way that a chemical reaction occurs (e.g., in an appropriate state and medium). In particular, a reference to reacting a starting material (i.e., 1,2 - propanediol and / or 1,3 - propanediol) with a source of nitrite refers to a chemical reaction between the starting material and the nitrite (i.e., the nitrite provided by the source of nitrite).

[0051] One of ordinary skill in the art will understand that a reference to a "source of nitrite" can equivalently refer simply to "nitrite" since it is the nitrite provided by the source of nitrite that undergoes a chemical reaction. Thus, a reference to a source of nitrite is understood to refer to a compound that provides a nitrite moiety (which may exist in either ionic or covalent form depending on the source of nitrite present) for the reaction. Thus, a source of nitrite may sometimes be referred to as a source of reactive (or reactable) nitrite (or nitrite moiety). To avoid confusion, a source of nitrite can be an inorganic nitrite or an organic nitrite.

[0052] As shown herein, when the source of nitrite is an organic nitrite, step (i) is carried out in a suitable organic solvent.

[0053] One of ordinary skill in the art will understand that various organic nitrites, such as alkyl nitrites, can be used in the process of the present invention.

[0054] Specific alkyl nitrites that may be mentioned include ethyl nitrite, propyl nitrite, butyl nitrite and pentyl nitrite. In certain embodiments, the alkyl nitrite is n-butyl nitrite, isobutyl nitrite, or tert-butyl nitrite, such as tert-butyl nitrite.

[0055] When the source of nitrite is an organic nitrite, one of ordinary skill in the art will be able to select a suitable solvent. For example, suitable solvents can include those mentioned herein as suitable organic components of a biphasic solvent system, and mixtures thereof.

[0056] To avoid misunderstanding, unless otherwise specified, references to the processes of the present invention carried out in a suitable organic solvent do not imply the presence of other non-organic solvents, such as water.

[0057] In certain embodiments where the process of the present invention is carried out in a suitable organic solvent, the solvent may be essentially free of water (sometimes referred to as "water-free" or "dry"), which may indicate that the solvent contains less than about 1 wt% (e.g., less than about 0.1 wt%, e.g., less than about 0.01 wt%) of water.

[0058] As used herein, the term "about" is defined to mean that the defined value may deviate by ±10%, such as ±5%, such as ±4%, ±3%, ±2%, or ±1%. The term "about" can be deleted throughout this specification without departing from the teachings of the present invention.

[0059] As shown herein, when the source of nitrite is an inorganic nitrite, step (i) is carried out in a two-phase solvent mixture comprising an aqueous phase and a non-aqueous phase.

[0060] One of ordinary skill in the art will understand that the term "two-phase solvent mixture" as used herein refers to a system composed of two solvents or solvent mixtures that do not mix to form a single solvent phase but instead exist as two separate (i.e., immiscible) phases.

[0061] When such a solvent mixture contains water and an organic solvent (or a mixture of organic solvents), such a solvent system can be said to include an "aqueous phase" and an "organic phase". To avoid misunderstanding, the term two-phase does not indicate that there may be other substances forming phases (i.e., other phases may also exist), such as substances forming solid phases, in addition to the solvent system.

[0062] Specific sources of inorganic nitrite that may be mentioned include metal nitrates such as alkali metal nitrates and alkaline earth metal nitrates. Ionic liquids may also be a suitable source of inorganic nitrite.

[0063] To avoid misunderstanding, the term alkali metal takes its ordinary meaning in the art, i.e., refers to the elements and cations of IUPAC Group 1 including lithium, sodium, potassium, rubidium, cesium, and francium.

[0064] To avoid misunderstanding, the term alkaline earth metal takes its ordinary meaning in the art, i.e., refers to the elements and cations of IUPAC Group 2 including beryllium, magnesium, calcium, strontium, barium, and radium.

[0065] More specific inorganic nitrites that may be mentioned include alkali metal nitrates such as lithium nitrite, sodium nitrite, and potassium nitrite. In certain embodiments, the source of nitrite is sodium nitrite.

[0066] Alternatively, the metal nitrite can be an alkaline earth metal nitrite such as lithium nitrite, magnesium nitrite or calcium nitrite.

[0067] To avoid misunderstanding, those skilled in the art will understand that the non-aqueous phase of the biphasic solvent system can also be an organic solvent and can thus be referred to as the organic phase.

[0068] Those skilled in the art will be able to select a suitable non-aqueous (i.e., organic) solvent based on the properties of the aqueous phase. For example, if the aqueous phase has a particular level of a substance dissolved therein (e.g., an ionic solid such as a salt), a wide range of organic solvents can be selected to form the biphasic solvent system.

[0069] In certain embodiments, the non-aqueous phase consists of a water-immiscible organic solvent. In more particular embodiments, the water-immiscible organic solvent is an aprotic organic solvent.

[0070] Specific water-immiscible organic solvents that may be mentioned (i.e., the specific solvents forming the non-aqueous phase) include ethers (e.g., tert-butyl methyl ether, cyclopentyl methyl ether, methyl tetrahydrofuran, diethyl ether, diisopropyl ether) and dichloromethane (DCM).

[0071] More specific water-immiscible organic solvents that may be mentioned (i.e., the specific solvents forming the non-aqueous phase) include dichloromethane, diethyl ether and tert-butyl methyl ether. In more particular embodiments, the water-immiscible organic solvent is tert-butyl methyl ether.

[0072] In certain embodiments that may be mentioned, the solvent mixture is a compound of formula I, wherein R 1 , R 2 , and R 3may contain an excess of the compound indicating H (i.e., 1,2 - propanediol and / or 1,3 - propanediol). To avoid misunderstanding, in such a situation, 1,2 - propanediol and / or 1,3 - propanediol (i.e., the compound of formula I where R 1 , R 2 , and R 3 indicates H) may be present as either a solvent (e.g., a component of a solvent mixture) or a reagent. Thus, in certain embodiments, the process is a process for preparing a compound of formula I as a solution in the corresponding compound of formula I where R 1 , R 2 , and R 3 indicates H, i.e., 1,2 - propanediol and / or 1,3 - propanediol (e.g., in the form of a mixture containing 1,2 - propanediol and / or 1,3 - propanediol as appropriate). In certain embodiments, when the source of nitrite is an organic nitrite, the solvent may consist essentially of the compound of formula I where R 1 , R 2 , and R 3 indicates H (i.e., 1,2 - propanediol and / or 1,3 - propanediol). That is, the compound of formula I where R 1 , R 2 and R 3 indicates H may act as both a solvent and a reactant.

[0073] In an alternative embodiment, when the source of nitrite is an inorganic nitrite, step (i) can be carried out in a single solvent, which may consist essentially of the compound of formula I where R 1 , R 2 , and R 3 indicates H (i.e., 1,2 - propanediol and / or 1,3 - propanediol). That is, the compound of formula I where R 1 , R 2 and R 3 indicates H may act as both a solvent and a reactant.

[0074] In an alternative embodiment, the process of the invention is carried out with an excess of nitrite with respect to the starting material of formula I, but where R 1 , R 2 , and R 3 represents H (i.e., 1,2-propanediol and / or 1,3-propanediol).

[0075] As used herein, the term "excess" takes its ordinary meaning in the art, i.e., it indicates that a component is present in an amount greater than the stoichiometric amount with respect to a reaction in which the component is a reagent.

[0076] As shown herein, the process of the invention (in particular, the reaction between components) is optionally carried out in the presence of a suitable acid.

[0077] Specific processes of the invention that may be mentioned include processes in which the step of reacting the starting material (i.e., 1,2-propanediol and / or 1,3-propanediol) with a source of nitrite is carried out in the presence of a suitable acid.

[0078] Specific acids that may be mentioned as suitable acids include Bronsted acids (i.e., proton-donating acids), and more specifically, such acids may be referred to as strong acids.

[0079] To avoid misunderstanding, the term "strong acid" takes its ordinary meaning in the art and refers to a Bronsted acid that dissociates substantially completely in an aqueous solution at equilibrium. In particular, reference to a strong acid may refer to a Bronsted acid having a pKa (in water) of less than about 5 (e.g., less than about 4.8). To avoid misunderstanding, in the case of polyprotic acids such as sulfuric acid, the term strong acid refers to the dissociation of the first proton.

[0080] Specific strong acids that may be mentioned include those having a pKa (in water) of less than about 1, such as less than about 0 (e.g., less than about -1 or -2). For example, strong acids that may be mentioned include those having a pKa (in water) of about -3. One of ordinary skill in the art will understand, as is known to those of ordinary skill in the art, that suitable acids may include non-nucleophilic acids.

[0081] Specific suitable acids that may be mentioned include sulfuric acid, phosphoric acid, trifluoroacetic acid, and acetic acid.

[0082] More specific suitable acids that may be mentioned include mineral acids such as sulfuric acid (e.g., strong mineral acids).

[0083] One of ordinary skill in the art will be able to select appropriate amounts of reagents for use in the processes of the present invention within the scope of the teachings of the present invention. For example, for the corresponding compound of formula I, but where R 1 , R 2 , and R 3 are H, the ratio of the compound to nitrite to acid (if present) (i.e., the molar ratio) is from about 1: about 1 to about 5: about 0.5 to about 3.5, such as from about 1: about 1 to about 3: about 0.5 to about 2 (e.g., about 1:4:2.7, or about 1:2:0.95, or about 1:2:1). To avoid misunderstanding, if no suitable acid is present, the ratio between the corresponding compound of formula I, but where R 1 , R 2 , and R 3 are H, and nitrite still applies.

[0084] In certain embodiments, process step (i) is carried out at a temperature of from about -30°C to about 5°C, such as from about -30°C to about 0°C, such as from about -30°C to about -10°C, preferably from about -25°C to about -15°C.

[0085] In certain embodiments, process step (i) is carried out under an inert atmosphere such as a nitrogen or argon atmosphere, preferably an argon atmosphere. Further, in certain embodiments, any step of the process may be carried out under an inert atmosphere such as a nitrogen or argon atmosphere, preferably an argon atmosphere.

[0086] In particular, certain processes of the invention that may be mentioned in which a two-phase solvent system is used are those in which the process, after step (i) (e.g., immediately thereafter), (ii) further comprises removing substantially all of the aqueous phase from the solvent mixture (i.e., removing substantially all of the water).

[0087] One of ordinary skill in the art will understand that the aqueous phase can be removed from the solvent mixture by any suitable process and using any suitable apparatus known in the art (e.g., by using a separatory funnel or similar apparatus).

[0088] As used herein, unless otherwise specified, the term "substantially all" refers to at least 80% (e.g., at least 85%, at least 90%, or at least 95%, e.g., at least 99%) of the specified substance according to the relevant measurement (e.g., by its weight).

[0089] One of ordinary skill in the art will also understand that reference to "removing substantially all of the aqueous phase from the solvent mixture" can be replaced by reference to "removing some or all of the aqueous phase from the solvent mixture" or simply "removing the aqueous phase from the solvent mixture".

[0090] To avoid misunderstanding, in the context of its removal, the term aqueous phase refers to the (separate) phase formed from water and the components dissolved therein.

[0091] In particular, certain processes of the invention that may be mentioned in which a two-phase solvent system is used are those in which the process, after step (i) (e.g., immediately thereafter) (in the order shown), (ii) removing some or all (e.g., substantially all) of the aqueous phase (i.e., water); and (iii) washing the remaining organic phase with one or more additional aqueous phases. (iv) Optionally, a step of repeating step (ii) and (iii) one or more times, further comprising a process.

[0092] A further process of the present invention that may be mentioned, particularly where a biphasic solvent system is used, is that the process, after step (i) (e.g., immediately), in the order shown, (ii) Removing some or all (e.g., substantially all) of the aqueous phase (i.e., water), (iii) Washing the remaining organic phase with one or more further aqueous phases, (iv) Optionally, a step of repeating step (ii) and (iii) one or more times, (v) Optionally, reducing (i.e., decreasing the amount / volume) the organic phase, such as by removing some or substantially all of the water-immiscible organic solvent (e.g., an organic solvent other than 1,2-propanediol and / or 1,3-propanediol), (vi) Optionally, a step of drying the product, further comprising, Steps (ii) to (vi) may be carried out in any order, provided that steps (ii) to (iv) are carried out before steps (v) and (vi).

[0093] In certain embodiments, process steps (ii) to (iv) may be carried out at a temperature of about -20°C to about 5°C, such as, for example, about -10°C to about 5°C.

[0094] In certain embodiments, process step (v) may be carried out at a temperature of about 0°C to about 30°C, such as, for example, about 10°C to about 30°C, such as, for example, about 15°C to about 30°C.

[0095] In certain embodiments, process step (v) is carried out within 6 hours, such as within 5 hours, preferably within 4 hours.

[0096] In certain embodiments, each of steps (ii) to (vi) is carried out, and these steps are carried out in the order shown.

[0097] To avoid misunderstanding, those skilled in the art will understand that the step of washing the remaining organic phase with one or more additional aqueous phases includes adding an additional portion of an aqueous solvent (such as water), mixing (e.g., by stirring and / or shaking together) with the (separate) organic phase, removing substantially all of the aqueous phase, and optionally repeating such steps one or more times.

[0098] Those skilled in the art will understand that step (iii) can be carried out by any suitable process using any suitable apparatus known in the art (e.g., using a separatory funnel).

[0099] Those skilled in the art will understand that step (v) can be carried out by any suitable process using any suitable apparatus known in the art (e.g., by evaporation under reduced pressure).

[0100] In the context of step (v), reference to the removal of some of the organic phase may, as defined herein, refer in particular to the removal of substantially all of the water-immiscible organic solvent. More specifically, the removal of the water-immiscible organic solvent may refer to the removal of at least 99% by weight (e.g., at least 99.5% by weight, 99.9% by weight, or in particular 99.99% by weight) of the water-immiscible organic solvent.

[0101] Such removal of the water-immiscible organic solvent may also refer to removal such that the product after such removal contains less than 1% by weight (e.g., less than 0.5% by weight, less than 0.1% by weight, e.g., less than 0.05% by weight, less than 0.01% by weight) of the water-immiscible organic solvent.

[0102] To avoid misunderstanding, in the context of step (v), reference to the removal of the organic phase, such as a water-immiscible organic solvent, refers to the removal of any solvent as defined herein (e.g., the removal of dichloromethane or tert-butyl methyl ether). If additional organic solvents are present (e.g., those that are not immiscible with water, such as excess 1,2-propanediol and / or 1,3-propanediol that act as solvents), some of such solvents may also be removed (e.g., together with the water-immiscible organic solvent).

[0103] In the context of step (vi), reference to the drying of the product refers to the removal of water from the materials remaining after the previous step. Such removal of water may refer to removal such that the product after such drying contains 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 or less than 0.01% by weight) of water.

[0104] One of ordinary skill in the art will understand that step (vi) can be carried out by any suitable process using any suitable apparatus known in the art (e.g., by contact with a suitable desiccant such as anhydrous sodium sulfate, anhydrous magnesium sulfate for the remaining organic phase and / or by molecular sieves).

[0105] Certain processes of the invention that may be referred to are such that a combination mixture of one or more compounds of formula I and the corresponding compound of formula I, but where R 1 、R 2 、and R 3 are H (i.e., 1,2-propanediol and / or 1,3-propanediol) contains from about 0.01% to about 9% by weight (e.g., from about 0.01% to about 5% by weight, e.g., from about 3% to about 5% by weight, or from about 5% to about 7% by weight) of one or more compounds of the invention, and the process further comprises the step of adding a further amount of the corresponding compound of formula I, but where R 1 、R 2 、and R 3 are H (e.g., after step (i) and, if present, after other steps).

[0106] As outlined above, without departing from the disclosure of the present invention, all embodiments and specific features of the invention referred to herein can be employed alone or in combination with any other embodiments and / or specific features referred to herein (thus, more specific embodiments and specific features are described as disclosed herein).

[0107] For example, process step (i) carried out at a temperature of about -30°C to about 5°C can be combined with the features of process steps (ii) to (iv) carried out at a temperature of about -20°C to about 5°C, the features of process step (v) carried out at a temperature of about 0°C to about 30°C, and / or the features of process step (v) carried out within 6 hours.

[0108] More specific processes that may be mentioned include processes in which the specified parameters follow the examples provided herein.

[0109] A specific product of the process of the present invention is a compound according to formula (II)

Chemical formula

[0110] There are two enantiomers of the compound according to formula (II), which are of the R and S types as shown below.

Chemical formula

[0111] The above two specific processes for the production of the compounds according to formulas (II) and (III) can be carried out together or independently of each other.

[0112] Based on the resulting biphasic nature of the reaction mixture, the optional addition of a phase transfer catalyst (PTC) can support the formation of the product. Common PTCs are, for example, without limitation, tetraalkylammonium ions such as Me4N+, Et4N+, Bu4N+, or Bu3(N+)CH2PHCl, and counterions such as =Cl-, Br-, HSO 4- etc., or other types of alkylammonium PTCs such as Aliquat® 336, in a sub-stoichiometric amount of less than 1 equivalent, for example, not exclusively, in the range of about 0.05 to about 40 mol%, for example about 0.1 to about 30 mol%, for example, about 0.1 to about 20 mol%.

[0113] A further specific product of the process of the present invention is a compound according to formula (IV) as shown below [Chemical formula] (wherein R 4 and R 5 each independently represents H or -NO, provided that at least one of R 4 and R 5 represents -NO).

[0114] Accordingly, a particular process of the present invention is a process for the preparation of a composition comprising one or more compounds of formula (IV), [Chemical formula] wherein R 4 and R 5 each independently represents H or -NO, provided that at least one of R 4 and R 5 represents -NO, the process comprising (i) optionally, reacting 1,2-propanediol with a source of nitrite in the presence of a suitable acid, (a) when the source of nitrite is an organic nitrite, step (i) is carried out in a suitable organic solvent, (b) when the source of nitrite is an inorganic nitrite, step (i) is carried out in a two-phase solvent mixture comprising an aqueous phase and a non-aqueous phase.

[0115] Any of the process steps outlined herein can be combined with the particular process described above with respect to formula (IV), and specific embodiments are outlined below.

[0116] In a particular process, the inorganic nitrite is a metal nitrite, and optionally, the metal nitrite is an alkali metal nitrite or an alkaline earth metal nitrite, preferably an alkali metal nitrite.

[0117] In a particular embodiment, the alkali metal nitrite is sodium nitrite.

[0118] In a further particular embodiment, the organic nitrite is an alkyl nitrite such as tert-butyl nitrite.

[0119] In a particular process, the suitable acid is a strong acid such as a strong mineral acid (e.g., sulfuric acid).

[0120] In certain embodiments, the immiscible phase comprises an immiscible organic solvent such as a water-immiscible aprotic organic solvent.

[0121] In one embodiment, the water-immiscible organic solvent is dichloromethane.

[0122] In certain processes, the solvent mixture further comprises excess 1,2-propanediol.

[0123] In a further particular process, after step (i), the process further comprises (ii) removing substantially all of the aqueous phase from the solvent mixture.

[0124] In one embodiment, after step (i), the process further comprises (ii) removing some or all (e.g., substantially all) of the aqueous phase (i.e., water), (iii) washing the remaining organic phase with one or more additional aqueous phases, (iv) optionally repeating steps (ii) and (iii) one or more times, (v) optionally reducing the organic phase (i.e., reducing the amount / volume), (vi) optionally drying the product, and steps (ii) to (vi) can be performed in any order, provided that steps (ii) to (iv) are performed before steps (v) and (vi).

[0125] In certain embodiments, the process further comprises adding an additional amount of 1,2-propanediol such that the combination mixture of one or more compounds of Formula I and 1,2-propanediol comprises from about 0.01 wt% to about 9 wt% of one or more compounds of Formula IV.

[0126] Products and Compositions In a second aspect of the present invention, there is provided a product prepared using the process of the present invention (i.e., manufactured according to the first aspect of the present invention, including all embodiments and specific features therein), for example, a product obtained or obtainable by the process of the present invention. Hereinafter, these products may be referred to as "compounds of the present invention".

[0127] The compounds of the present invention may contain asymmetric carbon atoms as outlined above and will thus exhibit optical isomerism. The various stereoisomers can be isolated by conventional methods, for example, by separating a racemic mixture or other mixtures of the compounds using fractional crystallization or HPLC techniques. Alternatively, the desired optical isomer can be prepared by reacting an appropriate optically active starting material under conditions that will not cause racemization (i.e., the "chiral pool" method), reacting an appropriate starting material with a "chiral auxiliary" that can be removed at an appropriate stage by subsequent derivatization (i.e., resolution including kinetic resolution), for example, using a homochiral acid, and then separating the diastereomeric derivatives by conventional means such as chromatography or by reacting with an appropriate chiral reagent or chiral catalyst under conditions known to those skilled in the art. All stereoisomers and their mixtures are included within the scope of the present invention.

[0128] The process of the present invention advantageously enables the preparation of a substantially non-aqueous composition comprising one or more compounds of the present invention. In particular, the process of the present invention allows for a relatively high concentration of one or more compounds of the present invention in the composition, thereby facilitating handling and minimizing storage and transportation costs.

[0129] Accordingly, in a third aspect of the present invention, there is provided a substantially non-aqueous composition comprising (a) one or more compounds of formula I as defined herein, and (b) one or more corresponding compounds of formula I, wherein R 1 , R 2 , and R 3A composition comprising a compound that exhibits H (e.g., 1,2 - propanediol and / or 1,3 - propanediol) is provided. This composition may hereinafter be referred to as "the substantially non - aqueous composition of the present invention".

[0130] Those skilled in the art will understand that references herein to the substantially non - aqueous composition of the present invention include references to all its embodiments and specific forms.

[0131] As used herein, reference to "substantially non - aqueous" refers to a component containing 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.

[0132] Certain substantially non - aqueous compositions of the present invention that may be mentioned include those in which the composition contains from about 0.01% to about 9% by weight (e.g., from about 0.01% to about 5% by weight, e.g., from about 3% to about 5% by weight, or from about 5% to about 7% by weight) of one or more compounds of the present invention (i.e., the compound of formula I).

[0133] Certain substantially non - aqueous compositions of the present invention 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 of the S - type.

[0134] The S - type of the compound according to formula (II) is preferred because it has a higher metabolic rate than the R - type. Furthermore, the S - type has different metabolic degradation pathways and produces metabolites with lower toxicity than the R - type.

[0135] Certain substantially non - aqueous compositions of the present invention that may be mentioned include those in which the composition contains a compound according to formula (III).

[0136] Preferably, the compound according to formula (II) is of the S - type, but the product is a mixture of both the S - type and R - type of formula (II), and the S - type is preferably present in enantiomeric excess (ee).

[0137] In certain embodiments where the product of the process is a compound according to formula (II), the compound according to formula (II) can be in the S - enantiomeric excess of the compound. That is, 50 ee% or more of the product is of the S - type. For example, 60 ee% or more, 70 ee% or more, 80 ee% or more, 90 ee% or more, 95 ee% or more, 98 ee% or more of the product is of the S - type.

[0138] To achieve the S - enantiomeric excess of the compound (II), in one embodiment, the starting material (i.e., 1,2 - propanediol) can be present in the S - enantiomeric excess. That is, 50 ee% or more of the starting material (i.e., 1,2 - propanediol) is of the S - type. For example, 60 ee% or more, 70 ee% or more, 80 ee% or more, 90 ee% or more, 95 ee% or more, or 98 ee% or more of the starting material is of the S - type.

[0139] In embodiments 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), for example, from about 55% to about 80% by weight, for example, from about 55% to 75% by weight is nitrosylated at the 2 - position.

[0140] Certain substantially non - aqueous compositions of the invention that may be mentioned are those in which the composition consists essentially of one or more compounds of formula I and the corresponding compounds of formula I, but where R 1 , R 2 and R 3 represent H (i.e., 1,2 - propanediol and / or 1,3 - propanediol).

[0141] In particular, when the starting material is 1,2 - propanediol, the substantially non - aqueous composition of the invention can contain (or, in particular, consist essentially of, or more specifically, consist of) one or more compounds of formula II and 1,2 - propanediol.

[0142] Similarly, when the starting material is 1,3-propanediol, the substantially non-aqueous composition of the present invention may contain (or, in particular, consist essentially of, or more specifically, consist of) one or more compounds of formula III and 1,3-propanediol.

[0143] The term "consisting essentially of" means that at least 90% by weight, for example at least 95% by weight, 96% by weight, 97% by weight, 98% by weight, or 99% by weight, of the defined characteristics are present.

[0144] Furthermore, specific substantially non-aqueous compositions of the present invention that may be mentioned include those in which the composition contains (or, in particular, consists essentially of, or more specifically, consists of) one or more compounds of formulas (II) and (III) together with 1,2-propanediol and 1,3-propanediol.

[0145] Specific substantially non-aqueous compositions of the present invention that may be mentioned include those in which the composition substantially does not contain dissolved nitric oxide.

[0146] The term "substantially does not contain" means that the non-aqueous composition of the present invention contains less than 5% by weight, 4% by weight, 3% by weight, 2% by weight, or less than 1% by weight, for example less than 0.5% by weight or less than 0.1% by weight, of dissolved nitric oxide.

[0147] Furthermore, specific substantially non-aqueous compositions of the present invention (a) one or more compounds of formula IV

Chemical formula

[0148] The compounds of the present invention and the substantially non-aqueous compositions of the present invention are useful as pharmaceuticals. Such compounds can be administered alone or via known pharmaceutical compositions / formulations.

[0149] Accordingly, in a fourth aspect, there are provided pharmaceutical formulations comprising the substantially non-aqueous compositions of the present invention and optionally one or more pharmaceutically acceptable excipients, which formulations may hereinafter be referred to as "the pharmaceutical formulations of the present invention".

[0150] Those skilled in the art will understand that references herein to the pharmaceutical formulations of the present invention include references to all embodiments and specific forms thereof.

[0151] As used herein, the term "pharmaceutically acceptable excipient" includes references to vehicles, adjuvants, carriers, diluents, pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, etc. In particular, such excipients may include adjuvants, diluents, or carriers.

[0152] Specific pharmaceutical formulations of the present invention that may be mentioned include those in which the pharmaceutical formulation comprises at least one pharmaceutically acceptable excipient.

[0153] Specific pharmaceutical formulations of the present invention that may be mentioned include those in which one or more pharmaceutically acceptable excipients are substantially non-aqueous.

[0154] To avoid misunderstanding, references herein to the compounds of the present invention for a particular use (and likewise uses and methods of use relating to the compounds of the present invention) also apply to compositions and pharmaceutical formulations comprising the compounds of the present invention described herein.

[0155] Surprisingly, it has been found that the compounds of the present invention can be administered to a patient (i.e., a subject) in combination with a suitable aqueous buffer. In particular, the compounds of the present invention are suitably stable in an aqueous buffer (e.g., present without significant degradation for at least 15 minutes) when the aqueous buffer, more specifically when the aqueous buffer is non-nucleophilic and weakly basic.

[0156] According to a fifth aspect of the present invention, there is provided a kit of parts comprising: (A) a pharmaceutical formulation of the present invention (i.e., as in the fourth aspect of the present invention, including all embodiments and specific features therein), and (B) a suitable aqueous buffer, wherein components (A) and (B) are provided in a form suitable for administration to each other, providing a kit of parts. Hereinafter, this kit of parts will be referred to as the "kit of parts of the present invention".

[0157] The specific embodiments referred to include those in which the buffer is non-nucleophilic and weakly basic.

[0158] More specific embodiments that may be mentioned include those in which the buffer has a pH of about 7.1 to about 10 (e.g., about 8 or about 9.2), such as a carbonate buffer (e.g., NaHCO3 which may have a pH of about 7.4 or about 8.0) or a physiological phosphate buffer (optionally at pH 8), or a mixture thereof. Physiological saline can also be used as a buffer.

[0159] In particular, the buffer can be the buffer used in Example 5 described below herein, such as a carbonate buffer at pH 9.2 or a phosphate buffer at pH 8.0 (e.g., 0.154 molar buffer), or a NaHCO3 buffer at pH 8.0.

[0160] According to a sixth aspect of the present invention, there is provided a combination product comprising: (A) a pharmaceutical formulation of the present invention (i.e., as in the fourth aspect of the present invention, including all embodiments and specific features therein), and (B) Provide a combined product formed by mixing with a suitable aqueous buffer defined in the fifth aspect of the present invention (including all embodiments and specific features therein), Hereinafter, this combined product is referred to as the "combined product of the present invention".

[0161] According to the seventh aspect of the present invention, a process for preparing the combined product of the present invention (i.e., the one in the sixth aspect of the present invention including all embodiments and specific features therein), (A) A pharmaceutical preparation of the present invention (i.e., the one in the fourth aspect of the present invention including all embodiments and specific features therein), (B) A step of mixing with a suitable aqueous buffer defined in the fifth aspect of the present invention (including all embodiments and specific features therein), to provide a process.

[0162] Specific kits of parts and combined products of the present invention that may be mentioned include those in which the volume ratio of the pharmaceutical preparation of the present invention to the suitable aqueous buffer is about 1:2 to 1:99, or about 3:7 to 1:99 (e.g., about 1:3 to 1:99).

[0163] Alternatively, the kits of parts of the present invention that may be mentioned include those in which the kit includes instructions for mixing the components (e.g., before administration, e.g., by using the techniques described herein) such that the volume ratio of the pharmaceutical preparation of the present invention to the suitable aqueous buffer is about 3:7 to 1:99 (e.g., about 1:3 to about 1:99).

[0164] Medical use As discussed above, the compounds of the present invention, the non-aqueous compositions of the present invention, the pharmaceutical preparations of the present invention, and thus the kits of parts and combined products containing them are useful for the treatment of conditions in which the administration of NO has beneficial effects.

[0165] According to an eighth aspect of the present invention, there is provided a non-aqueous composition as defined above (i.e., in the third aspect of the present invention including all embodiments and specific features therein), a pharmaceutical preparation as defined above (i.e., in the fourth aspect including all embodiments and specific features therein), a kit of parts as defined above (i.e., in the fifth aspect of the present invention including all embodiments and specific features therein), or a combined product as defined above (i.e., in the sixth aspect of the present invention including all embodiments and specific features therein) for use in the treatment of a condition in which administration of NO has a beneficial effect.

[0166] In an alternative eighth aspect of the present invention, there is provided a method of treating a condition in which administration of NO has a beneficial effect, the method comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical preparation of the present invention or a combined product of the present invention.

[0167] In a further alternative eighth aspect of the present invention, there is provided a method of treating a condition in which administration of NO has a beneficial effect, the method comprising administering to a patient in need thereof a therapeutically effective amount of components, (A) a pharmaceutical preparation of the present invention (i.e., that in the fourth aspect of the present invention including all embodiments and specific features therein), and (B) a suitable aqueous buffer as defined in the fifth aspect of the present invention (including all embodiments and specific features therein).

[0168] Specific methods that may be mentioned include methods in which the mixing of components (A) and (B) is carried out immediately prior to administration to the patient, such as by co-administration thereof.

[0169] The inventors have found that administration of the compounds of the present invention may damage blood cells via hemolysis due to osmotic stress, and that these effects can be managed or avoided by administering the compounds of the present invention in a suitable aqueous buffer.

[0170] More specific methods that may be mentioned include those in which the mixing is carried out by a mixing flow process that occurs, for example, at the time of administration to the patient. Even more specific methods that may be mentioned include those in which the mixing flow process is an intravenous infusion using a Y-site connector.

[0171] One of ordinary skill in the art will understand that references to "treating" a particular condition (or similarly, treating that condition) are in their ordinary meaning in the medical field. In particular, this term may refer to achieving a reduction in the severity of one or more clinical symptoms and / or signs associated with the above condition. For example, in the case of pulmonary embolism, this term may refer to achieving a reduction in the severity of chest pain, shortness of breath, and / or pulmonary hypertension via vasodilation.

[0172] As used herein, references to a patient will refer 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 refer to animals (e.g., mammals) such as household pets (e.g., cats, and particularly dogs), livestock, horses, etc.

[0173] As used herein, the term effective amount refers to the amount of a compound that confers a therapeutic effect on a treated patient. The effect may be objective (i.e., measurable by a certain test or marker) or subjective (i.e., the subject gives an indication of the effect and / or feels the effect).

[0174] As shown herein, the pharmaceutical formulations of the present invention may be useful in the treatment of conditions in which the administration of NO has a beneficial effect.

[0175] The specific conditions that may be mentioned include pulmonary hypertension of various origins, including acute pulmonary vasoconstriction of various origins, primary hypertension and secondary hypertension; conditions of various origins that require vasodilation; systemic hypertension of various origins; localized vasoconstriction of various origins; local vasoconstriction of various origins; acute heart failure (regardless of the presence or absence of preserved ejection fraction (HFpEF)); coronary heart disease; myocardial infarction; ischemic heart disease; angina pectoris; unstable angina pectoris; cardiac arrhythmia; acute pulmonary hypertension in cardiac surgical patients; acidosis; airway inflammation; cystic fibrosis; COPD; immotile cilia syndrome; lung inflammation; pulmonary fibrosis; adult respiratory distress syndrome; acute pulmonary edema; acute mountain sickness; asthma; bronchitis; hypoxia of various origins; stroke; cerebrovascular vasoconstriction; gastrointestinal inflammation; gastrointestinal dysfunction; gastrointestinal complications; IBD; Crohn's disease; ulcerative colitis; liver disease; pancreatic disease; urethral and bladder inflammation; skin inflammation; diabetic ulcer; diabetic neuropathy; psoriasis; inflammation of various origins; wound healing; organ protection in ischemia-reperfusion states; organ transplantation; tissue transplantation; cell transplantation; acute kidney disease; uterine relaxation; cervical relaxation; eye diseases such as glaucoma and conditions that require relaxation of smooth muscle.

[0176] More specific conditions that may be mentioned are pulmonary hypertension of various origins, including primary hypertension and secondary hypertension, acute heart failure (regardless of the presence or absence of preserved ejection fraction (HFpEF)). For example, the condition may be pulmonary hypertension caused by surgery.

[0177] Pulmonary hypertension is defined as an increase in mean pulmonary artery pressure (mPAP) of 20 mmHg or more at rest and a combination with a Wood unit value exceeding 3.

[0178] One of ordinary skill in the art will be able to determine how the pharmaceutical formulations as described herein can be administered in treatment, together with a suitable buffer. In particular, such combinations of the pharmaceutical formulations and buffers described herein can be administered intravenously or intraarterially.

[0179] One of ordinary skill in the art can determine the appropriate dosage of the active ingredient used in the treatment based on the nature of the formulation used (e.g., the combination of pharmaceutical formulations and appropriate buffers described herein), the condition being treated, and the condition of the patient (e.g., the disease state). For example, when administered intravenously or intraarterially to an adult human, an appropriate dosage can be a compound of formula I at about 0.5 to about 3,000 nmol / kg / min, e.g., about 1 to about 3,000 nmol / kg / min, e.g., about 5 to about 3,000 nmol / kg / min. Such dosages can be administered by infusion (either continuous or pulsed), such as an infusion over a long period (e.g., 1 to 2 hours or up to 1 week), or as a single (bolus) dose (a one-time dose or a single dose for each therapeutic intervention such as a single dose as needed or a single dose every 24 hours during treatment).

[0180] One of ordinary skill in the art will also understand that the temperature at which the formulation of the present invention (i.e., the pharmaceutical composition containing the compound of formula I) is formed and / or administered during treatment (i.e., administered to a subject) can be the temperature of the environment in which the administration is carried out (i.e., room temperature), or the temperature can be controlled. For example, such a formulation can be formed and / or administered at room temperature or a low temperature (i.e., a temperature lower than room temperature), e.g., about 0 to about 25 °C.

[0181] In certain embodiments, the compound of formula (II) is particularly important for use in humans, and the compound of formula (III) is particularly important for use in veterinary applications.

[0182] Without wishing to be bound by theory, it is believed that when administered to a patient, the compound of formula I is hydrolyzed to release nitric oxide, which provides the desired therapeutic effect. The processes described herein are unexpectedly believed to enable the preparation of an appropriate concentrated and stable composition containing the required active ingredient. Furthermore, it has been unexpectedly found that certain types of buffers enable the safe administration of such compositions without significantly degrading the active ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0183]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

Examples

[0184] The present invention will be described by the following examples, but is not intended to be limited.

[0185] Abbreviations aq aqueous conc concentration GC gas chromatography NMR Nuclear Magnetic Resonance equiv. Equivalent rel.vol. Relative volume

[0186] General procedure The starting materials and chemical reagents specified in the following preparations are commercially available from numerous suppliers such as Sigma Aldrich.

[0187] All NMR experiments were performed at 298 K on a Bruker 500 MHz AVI instrument equipped with a QNP probe head with Z gradient using Bruker Topspin 2.1 software. Unless otherwise specified, signals were referenced to residual CHCl3 at 7.27 ppm.

[0188] Stability assay The assay of stability samples was performed by GC / FID under the following conditions. 1,4-Dioxane was used as the internal standard (IS; about 0.50 mg / ml in CH3CN).

[0189] GC column: Rxi-5Sil MS, 20 m × 0.18 mm, 0.72 μm Carrier gas: Helium Inlet: 200 °C, split ratio 30:1 Constant flow rate: 1.0 ml / min Oven temperature profile: 40 °C (3 min), 10 °C / min, 250 °C (3 min) FID: Temperature 300 °C; H2 flow rate 30 ml / min, air flow 400 ml / min, makeup flow (N2) 25 ml / min

[0190] In vivo study Prior to the experiments, ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden, approval number 953). Recently, the anesthesia management, surgical instruments, and measurement methods have been described (Dogan et al. 2018, Sadeghi et al. 2018).

[0191] Briefly, eight male and female pigs (crossbred between Swedish domestic breeds, Hampshire and Yorkshire, 3 - 4 months old, average body weight 27 kg, range 21 - 34 kg) were pre - medicated with azaperone on the farm and transported to the laboratory. In the laboratory, anesthesia was induced with a mixture of tiletamine, zolazepam, and azaperone (intramuscular injection). Propofol was administered via a peripheral intravenous catheter in the auricular vein as needed. Bolus doses of atropine and cefuroxime were administered intravenously. The animals were tracheally intubated and mechanically ventilated (positive end - expiratory pressure of 5 cm H2O, minute ventilation was adjusted to normal ventilation). General anesthesia was maintained with propofol and fentanyl via continuous intravenous infusion, and additional bolus administrations were performed as needed. To compensate for water loss, a solution of Ringer's acetate glucose was continuously administered intravenously. After the use of surgical instruments, heparin was given as an intravenous bolus dose. After the experiment, the animals were killed under general anesthesia by propofol injection followed by rapid intravenous injection of potassium chloride (40 mmol), and cardiac arrest was confirmed.

[0192] Animals were equipped with an arterial catheter in the right carotid artery for measurement of systemic arterial blood pressure and heart rate, as well as for arterial sampling. A sheath was placed in the right external jugular vein for introduction of a pulmonary artery catheter. This catheter was used for continuous measurement of pulmonary artery blood pressure, semi-continuous cardiac output, and intermittent pulmonary artery wedge pressure. A central venous catheter was inserted into the left external jugular vein for drug and fluid administration. All fluid and drug administrations were performed by an electric syringe or a drip pump. A catheter was inserted into the bladder. Respiratory gases, pressures, and volumes were measured with an endotracheal tube. Respiratory and hemodynamic variables were measured by Datex AS / 3 (Helsinki, Finland), and data were collected by a computerized system (MP150 / Acknowledge 3.9.1, BIOPAC system, Goleta, California, USA). Blood gas and methemoglobin concentrations were measured by a blood gas apparatus (GEM 4000, Instrumental Laboratory, Lexington, Massachusetts, USA). Pulmonary and systemic vascular resistances were calculated with standard formulas. After the use of surgical instruments, a period of 1 hour without intervention followed.

[0193] Data were presented as median and interquartile range because of non-normal distribution. The ppm dose of inhaled NO was converted to a dose in nmol kg -1 min -1 using the ideal gas law and minute ventilation, assuming complete uptake of NO into the lungs. In the drug data, analysis was performed by Friedman's test using Wilcoxon's signed-rank test for post hoc multiple comparisons. The Mann-Whitney U test was used to compare drugs at doses of NO delivery similar to the maximal dose (45 nmol kg -1 min -1 of PDNO and 5 ppm of inhaled NO). A critical P value of 0.05 was used and adjusted by the Benjamini-Hochberg step-up procedure for multiple comparisons.

[0194] Example 1 - Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane using sodium nitrite. 1,2-Propanediol (15 mL, 205 mmol), water (100 mL), dichloromethane (200 mL), and sodium nitrite (57 g, 826 mmol) were added to a 500 mL three-necked round-bottom flask. The mixture was cooled to 0 °C in an ice bath. Concentrated sulfuric acid (30 mL, 546 mmol) and water (30 mL) were added to a dropping funnel and cooled to 5 °C in a refrigerator. The funnel was fitted to the round-bottom flask and the acid was added to the nitrite mixture over 2 hours. The mixture was stirred with a magnet for 20 minutes and then poured into a separatory funnel together with more dichloromethane (100 mL) and water (100 mL). The organic phase was separated, dried over sodium sulfate, and reduced on a rotary evaporator to obtain a mixture of 1,2-propanediol (3 wt%), 1-(nitrosooxy)-propan-2-ol (23 wt%), 2-(nitrosooxy)-propan-1-ol (13 wt%) and 1,2-bis(nitrosooxy)propane (57 wt%).

[0195] Example 2 - Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane using sodium nitrite 1,2 - propanediol (20 mL, 273.4 mmol), water (60 mL), dichloromethane (120 mL) and sodium nitrite (37.72 g, 546.7 mmol) were added to a 0.5 reactor equipped with a stirrer, flushed with nitrogen, and maintained under nitrogen during the following reaction. By cooling the mantle to 0 °C, the mixture was cooled to below 5 °C. Concentrated sulfuric acid (26.3 g, 260.1 mmol) and water were added to a dropping funnel. The funnel was attached to the reactor and the acid was added to the nitrite mixture over 33 minutes. The mixture was stirred for 54 minutes and then poured into a flask containing saturated aqueous sodium bicarbonate (100 mL). The mixture was transferred to a separatory funnel and the organic phase was washed. The aqueous phase was discarded and the organic phase was washed with additional saturated aqueous sodium bicarbonate (100 mL). The organic phase was dried over magnesium sulfate and then transferred to a 1 L round-bottom flask together with 1,2 - propanediol (120 mL, 1640 mmol). The solution was reduced on a rotary evaporator under reduced pressure until dichloromethane was removed. The removal of dichloromethane was monitored by NMR. A clear solution containing 1,2 - propanediol (82.8 wt%), 1 - (nitrosooxy) - propan - 2 - ol (10.4 wt%), 2 - (nitrosooxy) - propan - 1 - ol (6 wt%) and 1,2 - bis(nitrosooxy)propane (0.8 wt%) was obtained.

[0196] 1 H - NMR, δ ppm: 5.61 (br s 1H), 4.75 - 5.58 (m, 2H), 4.11 (br s, 1H), 3.90 - 3.87 (m, 1H), 3.83 - 3.69 (m, 2H), 3.60 (dd, J = 3.0, 11.2 Hz, 1H), 3.38 (dd, J = 7.9, 11.2 Hz, 1H), 1.47 (d, J = 6.6 Hz, 3H), 1.39 (d, J = 6.4 Hz, 3H), 1.26 (D, J = 6.4 Hz, 3H), 1.15 (D, J = 6.3 Hz, 3H). The CH and CH2 signals of 1,2 - bis(nitrosooxy)propane were below the detection limit.

[0197] Example 3 - Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane using tert-butyl nitrite tert-Butyl nitrite (2 mL, 15.1 mmol) was added to a round-bottom flask together with 1,2-propanediol (11 mL, 150.3 mmol), and the resulting solution was stirred at ambient temperature. Next, 1 mL of the reaction solution was mixed with 7.5 mL of 1,2-propanediol.

[0198] Example 4 - Stability of non-aqueous mixtures of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-propanediol Three different concentrations of 1-(nitrosooxy)-propan-2-ol and 2-(nitrosooxy)-propan-1-ol in 1,2-propanediol were prepared and stored both in a refrigerator (5 °C) and a freezer (-20 °C). Aliquots of each solution were taken periodically and analyzed by GC to measure the concentrations of 1-(nitrosooxy)-propan-2-ol and 2-(nitrosooxy)-propan-1-ol.

[0199] The results of the GC analysis are shown in the following table (column: Rxi-5Sil MS, 20 m x 0.18 mm, 0.36 film thickness, carrier: He, inlet: 250 °C, split ratio 100:1, constant flow rate: 1.0 mL / min; oven temperature profile: 40 °C (3 min), 10 °C / min, 80 °C (0 min), 30 °C / min, 250 °C (3 min); FID: 300 °C, H2 flow rate 30 mL / min, air flow 400 mL / min, makeup flow (N2) 25 mL / min; internal standard: 1,1,1,3,5,5,5-heptamethyltrisiloxane): [Table 1]

[0200] Example 5 - Stability of buffered aqueous solutions of 1-(nitrosooxy)-propan-2-ol and 2-(nitrosooxy)-propan-1-ol (PDNO), and 1,2-propanediol (PD) 100 μl of the stability sample was added to a GC vial. 400 μl of solution PD / buffer (1:9) and 400 μl of CH3CN were added. Next, 500 μl of CH2Cl2 was added and the mixture was extracted by gently shaking for 1 minute. 500 μl of the organic phase (lower phase) was transferred to another GC vial and 50 μl of the IS was added. The extract was analyzed by GC / FID according to the above conditions.

[0201] Calibration curves for 1-nitrite and 2-nitrite were prepared respectively. The peak area ratio (nitrite / IS) was plotted against the amount of nitrite. A high-concentration stock solution of PDNO / PD was used for the preparation of the standards. The concentration of nitrite expressed as % w / w was calculated.

[0202] The results obtained are shown in the following table and Figure 1. [Table 2]

[0203] Example 6 - In Vivo Study After collecting baseline data, stable pulmonary hypertension was induced by continuous intravenous infusion of thromboxane A2 mimetic 9,11-dideoxy 9α,11α-methano PGF 2α (U46619, Cayman Chemical, Michigan, USA; supplied in methyl acetate and diluted in 0.9% NaCl, final concentration 30 μg ml -1 ; up to a target mean pulmonary artery pressure of 35 - 45 mmHg, 60 - 150 ng kg -1 min -1 ). Thereafter, in a non-random crossover design with a washout period of 30 minutes between drugs, incremental doses of PDNO (15, 30, 45, and 60 nmol kg -1 ; pH approximately 8; Fresenius Kabi, Uppsala, Sweden; infusion rate 10 times the PDNO infusion rate) in a carrier flow of sodium bicarbonate solution (50 mg ml -1 min -1) continuous intravenous infusion or inhalation of NO (5, 10, 20, and 40 ppm; delivered to the inspiratory limb of a Servo 300 ventilator equipped with an inhaled NO dosing unit from a 1000 ppm tank in nitrogen [Siemens - Elema, Stockholm, Sweden]). The exact dose of inhaled NO was checked with an NO analyzer. Each dose was administered for 5 - 10 minutes. Hemodynamic and respiratory data were extracted, and arterial blood was sampled at the last moment of each administration.

[0204] Intravenous infusion of U46619 was performed before NO inhalation and PDNO injection, with mean pulmonary artery pressure of 43 (37 - 48) mmHg and 43 (41 - 46) mmHg, and pulmonary vascular resistance of 8.3 (6.7 - 11.7) mmHg min -1 and 9.8 (7.7 - 12.5) mmHg min -1 ) to induce stable pulmonary hypertension. Both inhaled NO and intravenous injection of PDNO significantly decreased pulmonary artery pressure and vascular resistance. However, PDNO decreased mean pulmonary artery pressure more efficiently (steeper slope) than inhaled NO, and PDNO decreased pulmonary vascular resistance more significantly compared to an equivalent dose of inhaled NO (as shown in Figure 2). There was no drug that significantly affected mean arterial pressure and systemic vascular resistance, but in the PDNO group, systemic vascular resistance was slightly lower compared to the inhaled NO group at an equivalent dose (Figure 2). Both drugs significantly decreased the pulmonary - to - systemic vascular resistance ratio, and at the highest dose, this ratio was slightly lower in the inhaled NO group compared to the PDNO group (data not shown). Cardiac output decreased slightly with U46619, but there was no drug that significantly changed cardiac output (data not shown). There was no drug that significantly affected methemoglobin concentration, but there was a tendency to increase in the inhaled NO group, and methemoglobin concentration seemed to be related to the dose of NO delivered (Figure 2). Arterial partial pressure of oxygen decreased slightly with U46619, and both drugs increased this variable towards normal (data not shown).

[0205] Example 7 Solvent-free Preparation of 1-(Nitrosooxy)-propan-2-ol, 2-(Nitrosooxy)-propan-1-ol, and 1,2-Bis(nitrosooxy)propane Using Sodium Nitrite Water (30 mL) and sodium nitrite (19.01 g, 272.8 mmol) were added to a 100 mL three-necked round-bottom flask, flushed with nitrogen, and cooled to 1 °C in a water bath cooled with an external cooler. 1,2-Propanediol (10 mL, 136.7 mmol) was added. Concentrated sulfuric acid (7 mL, 127.4 mmol) and water (20 mL) were pre-cooled to room temperature and added dropwise via a dropping funnel over 1 hour. During the addition, the aqueous layer formed a thick slurry and a green second layer was formed. Before the addition of the acid was complete (5 mL remaining), the flask was removed from the cold bath, the green layer was decanted into a separatory funnel, and washed with 2 × saturated aqueous NaHCO3. The green layer faded to yellow, and after separation, it was dried over Na2SO4 and filtered through a syringe filter (Acrodisc® 13 mm, 0.45 μ MSUPOR®) to obtain 1.1 g of a mixture of approximately 0.25 / 0.1 / 1 1-(Nitrosooxy)-propan-2-ol / 2-(Nitrosooxy)-propan-1-ol / 1,2-Bis(nitrosooxy)propane. 1,2-Propanediol, the starting material, could not be detected within the range of NMR sensitivity.

[0206] 1 H-NMR, δ ppm: 5.81 - 5.76 (m, br, 1.0H), 5.63 (br, 0.1H), 4.93 (br, 2.08H), 4.73 - 4.65 (br, m, 0.47H), 4.14 (br, 0.19H), 3.84 - 3.77 (br, m, 0.22H), 1.49 - 1.48 (br, m, 3.21H), 1.43 (br, 0.51H), 1.28 (br, 0.72H).

[0207] Example 8 Preparation of (2S)-1-(Nitrosooxy)-propan-2-ol, (2S)-2-(Nitrosooxy)-propan-1-ol, and (2S)-1,2-Bis(nitrosooxy)propane (S)-1,2 - Propanediol (5 mL, 66.97 mmol), water (15 mL), dichloromethane (30 mL) and sodium nitrite (9.34 g, 134 mmol) were added to a 100 mL three - necked round - bottom flask, flushed with nitrogen and cooled to 1 °C on a water bath cooled with an external cooler. Concentrated sulfuric acid (3.5 mL, 63.69 mmol) and water (10 mL) were pre - cooled to room temperature and added dropwise over 1 hour using a syringe pump. After addition, the mixture was stirred for an additional 60 minutes. After separating the two layers, the DCM layer was diluted with additional DCM (15 mL), washed with saturated aqueous NaHCO3 (15 mL), followed by brine (15 mL), then dried over Na2SO4, filtered through a sintered glass filter and reduced in vacuo. The residue was taken up again in 30 mL of DCM, washed with 1.4% w / w aqueous bicarbonate solution, then dried over Na2SO4, filtered through a sintered glass filter and reduced under vacuum to give 1 g of a product mixture. Based on NMR, the mixture consisted of (2S)-1,2 - propanediol (3%), (2S)-1 - (nitrosooxy) - propan - 2 - ol (23%), (2S)-2 - (nitrosooxy) - propan - 1 - ol (14%) and (2S)-1,2 - bis(nitrosooxy)propane (60%).

[0208] 1 1H - NMR, δ ppm: 5.83 - 5.74 (m, 1.0H), 5.66 - 5.57 (br, 0.22H), 4.99 - 4.85 (br, 1.98H), 4.76 - 4.59 (br, 0.77H), 4.17 - 4.07 (br, 0.38H), 3.86 - 3.73 (br, 0.40H), 1.8 - 1.6 (br, 0.97H), 1.48 (d, J = 6.7 Hz, 3.12H), 1.40 (d, J = 6.6 Hz, 0.63H), 1.28 (d, J = 6.5 Hz, 1.15H).

[0209] Example 9 - Preparation of (2R)-1 - (nitrosooxy) - propan - 2 - ol, (2R)-2 - (nitrosooxy) - propan - 1 - ol and (2R)-1,2 - bis(nitrosooxy)propane (R)-1,2-Propanediol (5 mL, 66.97 mmol), water (15 mL), dichloromethane (30 mL), and sodium nitrite (9.34 g, 134 mmol) were added to a 100 mL three-necked round-bottom flask, flushed with nitrogen, and cooled to 1 °C on a water bath cooled with an external cooler. Concentrated sulfuric acid (3.5 mL, 63.69 mmol) and water (10 mL) were pre-cooled to room temperature and added dropwise over 1 hour with a syringe pump. After addition, the mixture was stirred for an additional 55 minutes. After separating the two layers, the DCM layer was diluted with additional DCM (10 mL), washed with saturated aqueous NaHCO3 (20 mL), then dried over Na2SO4, filtered through a sintered glass filter, and reduced in vacuo. Based on NMR, the mixture consisted of (2R)-1,2-propanediol (17%), (2R)-1-(nitrosooxy)-propan-2-ol (16%), (2R)-2-(nitrosooxy)-propan-1-ol (7%), and (2R)-1,2-bis(nitrosooxy)propane (59%).

[0210] 1 H-NMR, δ ppm: 5.83 - 5.74 (m, 1.0H), 5.66 - 5.57 (br, 0.12H), 4.99 - 4.85 (br, 2.10H), 4.76 - 4.59 (br, 0.53H), 4.17 - 4.07 (br, 0.24H), 3.86 - 3.73 (br, 0.28H), 2.4 - 2.1 (br, 0.38H), 1.48 (d, J = 6.8 Hz, 3.20H), 1.40 (br, 0.56H), 1.28 (br(d), 0.88H).

[0211] Example 10 - Preparation of 1-(nitrosooxy)propan-3-ol and 1,3-bis(nitrosooxy)propane 1,3 - Propanediol (2.5 g, 32.86 mmol), water (7 mL), dichloromethane (15 mL) and sodium nitrite (4.53 g, 65.7 mmol) were added to a 100 mL round bottom flask, flushed with nitrogen, and cooled to 0 °C for 15 minutes on a water bath cooled with an external cooler. Concentrated sulfuric acid (1.7 mL, 31.2 mmol) and water (5 mL) were pre-cooled to room temperature and added dropwise over 5 minutes. After addition, the mixture was stirred at 0 °C for an additional 60 minutes. Next, the two layers were separated, the organic phase was diluted with additional DCM (10 mL), washed with saturated aqueous NaHCO3 (2 X 25 mL), dried over MgSO4, and filtered through a sintered glass filter. Finally, 1,3 - propanediol (16.4 g 216 mmol) was added to the organic phase, and then DCM was removed in vacuo. Based on NMR, the mixture (18.1 g) contained 1,3 - propanediol (86.9 wt%), 1 - (nitrosooxy) - propan - 3 - ol (11.8 wt%), and 1,3 - bis(nitrosooxy)propane (1.3 wt%).

[0212] 1H - NMR, δ 4.76 - 4.88 (m, 2H), 3.83 (t, J = 5.7 Hz, 2H), 3.73 (t, J = 6.1 Hz, 2H), 2.79 (s, 1H), 2.18 (quintet, J = 6.3 Hz, 2H), 1.99 (quintet, J = 6.2 Hz, 2H), 1.80 (quintet, J = 5.7 Hz, 2H).

[0213] Example 11 - Scale - up process for the preparation of 1 - (nitrosooxy) - propan - 2 - ol, 2 - (nitrosooxy) - propan - 1 - ol and 1,2 - bis(nitrosooxy)propane using sodium nitrite 11.1 Chemicals Used The starting materials were purchased from the list of suppliers in the following table. Unless otherwise specified, the chemicals were used as received without further purification.

Table 3

[0214] 11.2 General procedure for synthesizing PDNO using DCM as a solvent (starting process) The round-bottom flask was equipped with a stirrer and a dropping funnel. Water (3.0 equivalents) was added, and sodium nitrite (2.0 equivalents) was placed in the flask. The solution was cooled (0 °C), and PD (1.0 equivalent) and DCM (6 relative volumes) were also added. While further cooling, a sulfuric acid solution (1.0 equivalent H2SO4, 2.0 relative volumes of water) was prepared. While maintaining the reaction mixture between 0 °C and 5 °C, the sulfuric acid solution was further added dropwise to the reaction mixture. After the acid was completely added, the solution was stirred for an additional 1 hour to complete the reaction.

[0215] Next, the reaction was quenched with saturated NaHCO3 solution (6.0 relative volumes). The phases were separated, and the organic layer was further washed with NaHCO3 solution (6.0 relative volumes). The organic phase was dried over MgSO4, filtered, diluted with PD, and concentrated under reduced pressure using a rotary evaporator (water bath temperature 40 °C).

[0216] The product was obtained as a slightly yellowish liquid.

[0217] 11.3 General synthesis of PDNO using TBME as a solvent The round-bottom flask was equipped with a stirrer and a dropping funnel. Argon was flushed for several minutes. Dilute sulfuric acid solution (1.0 equivalent H2SO4, 2.0 relative volumes of water) was prepared in advance and pre-cooled (-30 °C). Water was added to the flask (3.0 relative volumes). Sodium nitrite (2.0 equivalents) was added to water. TBME (7.5 relative volumes) was added. Propanediol (1.0 equivalent) was added, and the reaction mixture was cooled (-20 °C) and continuously flushed with argon. The reaction mixture was stirred well while adding the pre-cooled sulfuric acid dropwise. The reaction temperature was constantly monitored during the addition of the acid. After the addition, the reaction mixture was stirred further at low temperature (-20 °C) for 30 - 60 minutes. Then, the reaction mixture was allowed to warm up (-5 °C). The reaction was stopped by quenching with saturated NaHCO3 solution (6.0 relative volumes). The phases were separated. The organic layer was washed further with saturated NaHCO3 solution until a pH value of 7 - 8 was obtained. Next, the organic phase was dried over MgSO4. The crude PDNO solution was diluted with PD (3 relative volumes) and further concentrated at ambient temperature (25 °C) under reduced pressure.

[0218] The crude PDNO solution was further purified using a vertical tube evaporator.

[0219] PDNO was obtained as a slightly yellowish liquid.

[0220] 11.4 Detailed synthesis of PDNO using TBME as a solvent This process was designed to produce approximately 7.5 L of 7% PDNO solution in one synthesis (one "run"). The synthesis was carried out several times to obtain the desired batch size. GC analysis was used for purity measurement for each run. Runs within the specification range of organic related compounds could be mixed to produce one batch. Next, the entire crude PDNO batch was purified. After purification, the strong PDNO solution was further diluted with PD to obtain the target concentration (usually 7% PDNO solution).

[0221] A suitable double-walled reactor (60 L) was equipped with a specific "cup stirrer", a dropping funnel, and an attachment for argon. The reactor was flushed with a constant argon flow for 5 to 10 minutes. Water (3.0 L) was added to the reactor. Sodium nitrite (2.0 equivalents, 1886 g) was added through the reactor. The reactants were further stirred until all the salts were dissolved, 1,2-propanediol (1.0 equivalent, 1040 g, 1 L) was added, followed by tert-butyl methyl ether (7.5 relative volume, 7.5 L). Next, the reaction mixture was cooled to an internal reaction temperature of -20 °C with continuous stirring and an argon flow. On the other hand, sulfuric acid (1.0 equivalent, 1340 g, 728 mL) was diluted with water (2.0 L) and cooled to -30 °C. After reaching an internal reaction temperature of -20 °C, the diluted acid was added dropwise to the reaction mixture with vigorous stirring.

[0222] The stirring speed was changed during the addition of the acid. It started at about 350 rpm and was reduced to a slow stirring speed (about 180 rpm) by the end of the reaction. This variation in the stirring speed was due to the two-phase reaction system and the slow precipitation of sodium sulfate (due to the addition of more and more sulfuric acid) as the reaction proceeded further.

[0223] The reaction temperature was constantly monitored during the addition of sulfuric acid. The temperature should ideally be in the range of (-20 ± 3) °C. Furthermore, the reactants were stirred at (-20 ± 3) °C for 30 to 60 minutes.

[0224] The reactants were warmed from -5 °C to 0 °C. The reaction was stopped by adding a saturated NaHCO3 solution (6.0 relative volume, 6.0 L), followed by water (10 L). The phases were separated, and the organic layer was transferred to another double-walled reactor and cooled to 0 °C to 5 °C. The organic layer was washed several times (about 2 to 3 times) with a saturated NaHCO3 solution (4.0 relative volume, 4.0 L). The pH value of the aqueous phase was monitored after each washing step. The pH value was about 7 to 8. The aqueous phase was discarded. The organic layer was dried over MgSO4 and filtered through Whatman filter paper.

[0225] Furthermore, by adding PD (3.0 relative volume, 3.0 L), the crude PDNO (solution in TBME) was diluted. This crude PDNO was transferred to a rotary evaporator and concentrated under reduced pressure. The water bath temperature during evaporation was maintained at a maximum temperature of 25 °C. The evaporation of the major amount of TBME was removed in the time range of 1.5 hours to 2.0 hours.

[0226] Next, using a high vacuum pump, the evaporation of the organic solvent could be continued for several hours at a water bath temperature of (0 ± 2) °C (during development, the PDNO purity was monitored under these conditions and the product purity was not affected over 6 hours).

[0227] 11.5 Further purification of the crude PDNO solution The final purification of the PDNO solution was carried out by vertical tube evaporation. The PDNO solution was distilled under high vacuum using a continuous thin vapor of PDNO at 0 °C. The storage tank of the "crude" PDNO solution was cooled to 0 °C. The entire distillation was carried out at 0 °C. The storage tank of the "purified" PDNO was also cooled to -10 °C to 0 °C. Each time the evaporation of the entire batch of PDNO was carried out, the residual organic solvent (TBME) could be confirmed via GC. This evaporation was continued until the desired limit of the residual solvent was achieved. For PDNO, the limit of the residual solvent is 1000 ppm.

[0228] 11.6 Preparation of the final dilution After purification, the PDNO was further diluted to reach the desired concentration. The first step was to filter the PDNO solution through a Whatman filter into a clean glass bottle. Furthermore, the assay of the PDNO solution was determined by q-NMR. The amount of PD for dilution could be calculated. The PD was first filtered through a Whatman filter. The final dilution could be carried out at ambient temperature. The calculated amount of PD was added to the PDNO solution (or vice versa). The resulting mixture was shaken for several minutes to obtain a homogeneous solution. The final PDNO solution was filled into the product bottle.

[0229] PDNO (7.5 kg; 7% solution) was obtained as a slightly yellowish liquid.

[0230] Example 12 - Hemodynamic Effects of Intravenous PDNO: Influence of Various Carrier Solutions in Anesthetized Pigs The influence of various carrier solutions on the hemodynamic effects of the organic mononitrite 1,2 - propanediol (PDNO) administered intravenously to anesthetized pigs was studied.

[0231] Prior to the experiment, ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden, approval number 953). This study was conducted in accordance with Directive 2010 / 63 / EU on the protection of animals used for scientific purposes. Two healthy domestic 3 - month - old pigs (crossbred between Swedish breeds Hampshire and Yorkshire, weighing 26 kg and 27 kg) were included in the study.

[0232] The animals were pre - medicated with azaperone on the farm and transported to the laboratory. In the laboratory, anesthesia was induced with a mixture of tiletamine, zolazepam, and azaperone (intramuscular injection). Propofol was administered to the peripheral venous catheter of the ear vein as needed. Bolus doses of atropine and cefuroxime were administered intravenously. The animals were tracheally intubated and mechanically ventilated (positive end - expiratory pressure at 5 cm H2O, minute ventilation was adjusted to normal ventilation). General anesthesia was maintained with propofol and fentanyl via continuous intravenous infusion, and additional bolus administrations were performed as needed. To compensate for water loss, a Ringer's acetate glucose solution was continuously administered intravenously. After the use of surgical instruments, heparin was administered as an intravenous bolus dose. After the experiment, the animals were killed under general anesthesia by propofol injection followed by a rapid intravenous injection of potassium chloride (40 mmol), and cardiac arrest was confirmed.

[0233] The animals were equipped with an arterial catheter in the right carotid artery to measure systemic arterial blood pressure and heart rate. A sheath was placed in the right external jugular vein for introducing a pulmonary artery catheter. This catheter was used for continuous measurement of pulmonary artery blood pressure and semi - continuous cardiac output. A central venous catheter was inserted into the left external jugular vein for drug and fluid administration. All fluid and drug administrations were performed by an electric syringe or a drip pump. A catheter was inserted into the bladder. Hemodynamic variables were measured by Datex AS / 3 (Helsinki, Finland), and data were collected by a computerized system (MP150 / Acknowledge 3.9.1, BIOPAC system, Goleta, California, USA). After the use of surgical instruments, a period of at least 1 hour without intervention followed.

[0234] Intravenous infusion of PDNO was administered at a rate of 30 nmol kg -1 min -1 for 15 minutes into the carrier flow at a rate 9 times the PDNO infusion rate in sodium bicarbonate (14 mg ml-1; pH 7.4 or 8.0), pH 8 saline or saline (Research Institutes of Sweden, Södertälje, Sweden). A carrier solution was generated using standard chemicals. Hemodynamic effects were measured at the end of each infusion.

[0235] The results are shown in the following table. Baseline values before each intravenous combination of PDNO and carrier solution were normal in healthy anesthetized pigs. Intravenous PDNO in a carrier solution of bicarbonate buffer at pH 8 decreased mean systemic arterial pressure (MAP) and mean pulmonary artery pressure (MPAP) by - 11 ± 1.2 mmHg and - 2.4 ± 0.8 mmHg, respectively, while intravenous PDNO in a carrier solution of saline decreased MAP and MPAP by - 6.9 ± 2.5 mmHg and - 2.4 ± 0.1 mmHg. Intravenous PDNO combined with bicarbonate buffer at pH 7.4 and physiological phosphate buffer at pH 8 affected MAP and MPAP in the same way as saline. Heart rate and semi - continuous cardiac output were affected only slightly by the infusions.

[0236] In combination with various carrier solutions at 30 nmol kg -1 min -1 Hemodynamic variables (n = 2 per carrier solution) in anesthetized and mechanically ventilated pigs that received repeated intravenous infusions of PDNO.

Table 4

[0237] Data are presented as mean and standard deviation.

[0238] Mean arterial blood pressure (MAP), mean pulmonary artery pressure (MPAP), heart rate (HR), semi-continuous cardiac output (CCO).

[0239] Intravenous PDNO in combination with a carrier solution of bicarbonate buffer at pH 8 produced a greater hemodynamic effect compared to carrier solutions of physiological saline, physiological phosphate buffer, and bicarbonate buffer at pH 7.4.

[0240] Example 13 - Pharmacological investigation of 1,2-PDNO-R, 1,2-PDNO-S, and 1,3-PDNO in anesthetized pigs Before the experiment, ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden, approval number 953). Briefly, two male and two female pigs (Swedish domestic breeds, crossbred between Hampshire and Yorkshire, 3 - 4 months old, 24 - 26 kg) were pre - medicated with azaperone on the farm and transported to the laboratory. In the laboratory, anesthesia was induced with a mixture of tiletamine, zolazepam, and azaperone (intramuscular injection). Propofol was administered to the peripheral vein catheter of the auricular vein as needed. Bolus doses of atropine and cefuroxime were administered intravenously. The animals were intubated tracheally and mechanically ventilated (positive end - expiratory pressure at 5 cm H2O, minute ventilation was adjusted to normal ventilation). General anesthesia was maintained with propofol and fentanyl via continuous intravenous infusion, and additional bolus administrations were performed as needed. To compensate for water loss, Ringer's acetate glucose solution was continuously administered intravenously. After the use of surgical instruments, heparin was given as an intravenous bolus dose. After the experiment, the animals were killed under general anesthesia by propofol injection followed by rapid intravenous injection of potassium chloride (40 mmol), and cardiac arrest was confirmed.

[0241] The animals were equipped with an arterial catheter in the right carotid artery for measurement of systemic arterial blood pressure and heart rate. A sheath was placed in the right external jugular vein for introduction of a pulmonary artery catheter. This catheter was used for continuous measurement of pulmonary artery blood pressure, semi - continuous cardiac output, and intermittent pulmonary artery wedge pressure. A central venous catheter was inserted into the left external jugular vein for administration of drugs and fluids. All administrations of fluids and drugs were performed by an electric syringe or a drip pump. A catheter was inserted into the bladder. Respiratory gases, pressure, and volume were measured with an endotracheal tube. Respiratory and hemodynamic variables were measured by Datex AS / 3 (Helsinki, Finland), and data were collected by a computerized system (MP100 or MP150 / Acknowledge 3.9.1, BIOPAC system, Goleta, California, USA). After the use of surgical instruments, a period of 1 hour without intervention followed.

[0242] After collecting baseline data, 10 - 15 minutes of intravenous infusion of 1,2-PDNO-R (43 nmol kg -1 min -1 ), 1,2-PDNO-S (43 nmol kg -1 min -1 ), and 1,3-PDNO (30 nmol kg -1 min -1 ) was administered in a carrier flow of sodium bicarbonate solution (14 mg ml -1 ; pH approximately 8; infusion rate was 9 times the PDNO infusion rate). Hemodynamic and respiratory data were extracted at the end of each administration.

[0243] 1,2-PDNO-R, 1,2-PDNO-S, and 1,3-PDNO decreased mean systemic and pulmonary artery pressures. This is shown in Figure 3. The conclusion is that 1,2-PDNO-R, 1,2-PDNO-S, and 1,3-PDNO cause systemic and pulmonary vasodilation and thus they exhibit vasodilatory capacity.

Claims

1. A process for the preparation of a composition comprising one or more compounds of formula I, wherein 【Chemical 1】 wherein, R １ , R ２ , and R ３ each independently represents H or -NO, n is 0 or 1, When n is 0, R １ is H, When n is 1, R ２ is H, R １ , R ２ , and R ３ At least one of them represents -NO, the process comprises (i) a compound of formula I wherein R １ , R ２ , and R ３ are each H, with a source of nitrite; when the source of the nitrite is an organic compound that provides a nitrite moiety, step (i) is carried out in a suitable organic solvent, when the source of the nitrite is an inorganic compound that provides a nitrite moiety, step (i) is carried out in a two-phase solvent mixture comprising an aqueous phase and a non-aqueous phase, One or more compounds of formula I and a compound of formula I wherein R １ , R ２ , and R ３ is H, are combined such that the mixture contains 0.01% to 9% by weight of one or more compounds of formula I, and a further amount of the compound of formula I wherein R １ , R ２ , and R ３ is H is added. Process.

2. The process according to claim 1, wherein step (i) is carried out in the presence of a suitable acid.

3. The process according to claim 1 or 2, wherein the inorganic compound that provides the nitrite moiety is a metal nitrite.

4. The process according to claim 3, wherein the metal nitrite is an alkaline earth metal nitrite.

5. The process according to claim 3, wherein the metal nitrite is an alkali metal nitrite.

6. The process according to claim 5, wherein the alkali metal nitrite is sodium nitrite.

7. The process according to claim 1 or 2, wherein the organic compound that provides the nitrite moiety is an alkyl nitrite.

8. The process according to claim 7, wherein the alkyl nitrite is tert-butyl nitrite.

9. The process according to any one of claims 2 to 8, wherein the suitable acid is a strong acid.

10. The process according to claim 9, wherein the strong acid is a strong mineral acid.

11. The process according to claim 10, wherein the strong mineral acid is sulfuric acid.

12. The process according to any one of claims 1 to 11, wherein the non-aqueous phase comprises a water-immiscible organic solvent.

13. The process according to claim 12, wherein the water-immiscible organic solvent is a water-immiscible aprotic organic solvent.

14. The process according to claim 12 or 13, wherein the water-immiscible organic solvent is dichloromethane.

15. The process according to claim 12 or 13, wherein the water-immiscible organic solvent is tert-butyl methyl ether.

16. The solvent mixture is a compound of formula I wherein R １ , R ２ , and R ３ is H, further comprising an excess of the compound, the process according to any one of claims 1 to 15.

17. After step (i), the process according to any one of claims 1 to 16 further comprises (ii) removing all of the aqueous phase from the solvent mixture.

18. After step (i), the process according to any one of claims 1 to 16 further comprises (ii) removing a portion of the aqueous phase (i.e., water). The process according to any one of claims 1 to 16, further comprising the step of washing the remaining organic phase with one or more further aqueous phases.

19. The process according to claim 18, wherein in step (ii), all of the aqueous phase (i.e., water) is removed.

20. The process according to claim 18 or 19, wherein after step (iii), steps (ii) and (iii) are repeated one or more times.

21. After step (iv), The process according to claim 20, further comprising the step of reducing the organic phase (i.e., reducing the amount / volume).

22. After step (v), The process according to claim 21, further comprising the step of drying the product, and steps (ii) to (vi) can be carried out in any order, provided that steps (ii) to (iv) are carried out before steps (v) and (vi).

23. A composition comprising: One or more compounds of formula I, n is 0 or 1, 【Chemical 2】 wherein, R １ , R ２ , and R ３ each independently represents H or -NO, The composition contains 0.01 wt% to 9 wt% of one or more compounds of formula I, and the total amount of components (a) and (b) is present in at least 80 wt%. When n is 0, R １ is H, When n is 1, R ２ is H, R １ , R ２ , and R ３ at least one of which represents -NO, (b) A compound of formula I wherein R １ , R ２ , and R ３ are H, and

24. The composition according to claim 23, wherein the composition contains less than 1 wt% of water.

25. A composition comprising: One or more compounds of formula I, n is 0 or 1, The composition contains 0.01 wt% to 9 wt% of one or more compounds of formula I, and the composition contains less than 1 wt% of water. [Chemical 3] wherein, R １ , R ２ , and R ３ each independently represents H or -NO,

26. When n is 0, R １ is H, When n is 1, R ２ is H, R １ , R ２ , and R ３ at least one of which represents -NO, (b) A compound of formula I wherein R １ , R ２ , and R ３ are H, and The composition according to any one of claims 23 to 25, wherein the composition does not contain dissolved nitric oxide.

27.

28. A pharmaceutical formulation comprising the composition according to any one of claims 23 to 27.

29. The composition comprises one or more compounds of formula I and a compound of formula I wherein R １ , R ２ , and R ３ is H, the composition according to any one of claims 23 to 26.

30. The pharmaceutical formulation according to claim 28, wherein the pharmaceutical formulation contains one or more pharmaceutically acceptable excipients, and the one or more pharmaceutically acceptable excipients are non-aqueous.

31.

32. A kit of parts comprising: The pharmaceutical formulation according to claim 28 or 29, and A suitable aqueous buffer, and components (A) and (B) are provided in a form suitable for administration to each other.

33.

34. A combination product comprising: The pharmaceutical formulation according to claim 28 or 29, and (A)The pharmaceutical formulation according to claim 28 or 29, and A combined product formed by mixing together (B) a suitable aqueous buffer solution.

32. The kit of parts according to claim 30, or the combined product according to claim 31, wherein the volume ratio of the pharmaceutical preparation to the suitable aqueous buffer solution is 3:7 to 1:

99.

33. The kit of parts according to claim 30 or 32, or the combined product according to claim 31 or 32, wherein the suitable aqueous buffer solution is non-nucleophilic and weakly basic.

34. The kit of parts according to any one of claims 30, 32 and 33, or the combined product according to any one of claims 31 to 33, wherein the suitable aqueous buffer solution maintains a pH of 7.1 to 10.

35. The kit of parts according to any one of claims 30 and 32 to 34, or the combined product according to any one of claims 31 to 34, wherein the suitable aqueous buffer solution is a carbonate buffer solution or a phosphate buffer solution, or a mixture thereof.

36. A process for preparing the combined product according to any one of claims 31 and 33 to 35, comprising the step of (A) mixing together the pharmaceutical preparation according to claim 28 or 29, and (B) a suitable aqueous buffer solution as defined in any one of claims 33 to 35.

37. The composition according to any one of claims 23 to 27, the pharmaceutical preparation according to claim 28 or 29, the kit of parts according to any one of claims 30 and 32 to 35, or the combined product according to any one of claims 31 to 35 for use in the treatment of a condition in which administration of NO has a beneficial effect.

38. A pharmaceutical preparation or combined product for use in the treatment of a condition in which administration of NO has a beneficial effect, comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical preparation or the combined product, the pharmaceutical preparation according to claim 28 or 29, or the combined product according to any one of claims 31 to 35.

39. A kit of parts or combined product for use in the treatment of a condition in which administration of NO has a beneficial effect, comprising administering to a patient in need thereof a therapeutically effective amount of components (A) the pharmaceutical preparation according to claim 28 or 29, and (B) a suitable aqueous buffer solution as defined in any one of claims 32 to 35. The kit of parts according to any one of claims 30 and 32 to 35, or the combined product according to any one of claims 31 to 35, wherein the components (A) and (B) are mixed before administration.

40. The state is acute pulmonary vasoconstriction of various origins; pulmonary hypertension of various origins including primary hypertension and secondary hypertension; states of various origins requiring vasodilation; systemic hypertension of various origins; localized vasoconstriction of various origins; local vasoconstriction of various origins; acute heart failure (regardless of the presence or absence of preserved ejection fraction (HFpEF)); coronary heart disease; myocardial infarction; ischemic heart disease; angina pectoris; unstable angina pectoris; cardiac arrhythmia; acute pulmonary hypertension in cardiac surgery patients; acidosis; airway inflammation; cystic fibrosis; COPD; immotile cilia syndrome; lung inflammation; pulmonary fibrosis; adult respiratory distress syndrome; acute pulmonary edema; acute mountain sickness; asthma; bronchitis; hypoxia of various origins; stroke; cerebrovascular vasoconstriction; gastrointestinal inflammation; gastrointestinal dysfunction; gastrointestinal complications; IBD; Crohn's disease; ulcerative colitis; liver disease; pancreatic disease; inflammation of the urethra and bladder; skin inflammation; diabetic ulcer; diabetic neuropathy; psoriasis; inflammation of various origins; wound healing; organ protection in ischemia-reperfusion state; organ transplantation; tissue transplantation; cell transplantation; acute kidney disease; uterine relaxation; cervical relaxation; and states requiring relaxation of smooth muscle, selected from the group consisting of The composition according to claim 37, the pharmaceutical preparation according to claim 37 or 38, the kit of parts according to claim 37 or 39, or the combined product according to any one of claims 37 to 39.

41. The kit of parts according to claim 39 or 40, or the combined product according to claim 39 or 40, wherein the mixing of the components (A) and (B) is carried out immediately before administration to the patient.

42. The kit of parts according to claim 41, or the combined product according to claim 41, wherein the administration is carried out by co-administration.

43. The kit of parts according to any one of claims 39 to 42, or the combined product according to any one of claims 39 to 42, wherein the mixing is carried out by a mixing flow process.

44. The kit of parts according to claim 43, or the combined product according to claim 43, wherein the mixing flow process is carried out at the time of administration of the components to the patient.

45. The composition according to claim 40, the pharmaceutical preparation according to claim 40, the kit of parts according to claim 40, or the combined product according to claim 40, wherein the state is selected from the group consisting of pulmonary hypertension of various origins including primary hypertension and secondary hypertension, and acute heart failure (regardless of the presence or absence of preserved ejection fraction (HFpEF)).

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