Compositions and methods of treating diseases using combinations of nitrovasodilators and nitric oxide compounds - Patents.com

Abstract

Provided herein are compositions and methods for treating vascular or respiratory conditions by administering a therapeutically effective amount of a nitric oxide compound and a nitrovasodilator. The nitric oxide compound can be a nitrite, one or more of several nitro-group-containing compounds, a nitrate-group-containing compound, an NO donor, or an endogenous NO-generating amino acid. The nitrovasodilator can be one or more of nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, nitroprusside, amyl nitrite, nitrosothiol, or dinitrosyl iron complex.

Description

[Technical field]

[0001] Government support This invention was made with Government support under Grant Nos. HL095973 and HD083132 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] The present disclosure relates to compositions and methods of treating diseases using a combination of a nitrovasodilator and a nitric oxide compound. [Background technology]

[0003] Nitric oxide (NO) is a ubiquitous signaling messenger in biological systems. As a chemically reactive free radical, NO has a relatively short half-life. For many years, the effects of endogenous NO were thought to be paracrine only, since it is inactivated within milliseconds in the blood, thereby negating any long-range and persistent endocrine signaling activity. However, early studies of the use of inhaled NO gas to treat pulmonary hypertension surprisingly revealed vasodilation of downstream systemic vascular beds, despite the fact that it takes longer than 10 seconds for blood to travel from the pulmonary capillaries to the peripheral resistance vessels. NO metabolism in biological matrices is a complex web of reactions that result in a variety of products, including nitrite, nitrate, nitrotyrosine, and various molecules with NO bound to thiols or iron. A subset of these NO-containing compounds, including S-nitrosothiols (SNOs), dinitrosyl iron complexes (DNICs) and nitroglycerin (NTG), dilate blood vessels via activation of soluble guanylyl cyclase (sGC) in vascular smooth muscle cells. This class of compounds, defined herein as "nitrovasodilators," has vasodilatory potency comparable to or even greater than that of NO itself, despite poor membrane permeability. These compounds are often thought to achieve vasodilatory potency by releasing their NO moiety to enter vascular smooth muscle cells and activate sGC, but overwhelming experimental findings contradict this understanding. Summary of the Invention

[0004] Systems and methods are provided herein that address these shortcomings in the art and provide other additional or alternative advantages. The disclosure herein provides one or more embodiments of systems and methods for treating vascular conditions or local tissue NO deficiency by administering a therapeutically effective amount of a nitric oxide compound and a nitrovasodilator. The combination of the nitrovasodilator and the nitric oxide compound can be administered as one fixed dose combination or as separate formulations. The nitrovasodilator can be one or more of nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, nitroprusside, amyl nitrite, nitrosothiol, or dinitrosyl iron complex. The nitric oxide compound can be one or more of a nitro group-containing compound, a nitrate group-containing compound, an NO donor, and an endogenous NO-generating amino acid. The nitro group-containing compound can include nitrite, D-NG-nitroarginine methyl ester, or any other nitro group-containing low molecular weight molecule, or a protein, or a lipid. The nitrate group-containing compound may include nitrates, or any nitrate group-containing low molecular weight molecule, or protein, or lipid. The NO donor may be a NONOate, or a hydroxamic acid, such as deferoxamine, acetohydroxamic acid, or hydroxyurea. NONOates are compounds that have an amine functional group, a crosslinked NO - The endogenous NO-generating amino acids are compounds having a terminal nitrosyl group and a terminal nitrosyl group, which release NO under certain biological conditions. The endogenous NO-generating amino acids can be arginine or citrulline or a combination thereof.

[0005] In some embodiments, the present invention includes a method for treating a vascular or respiratory condition in a human subject by administering to the subject a therapeutically effective amount of a stimulator of soluble guanylyl cyclase (sGC stimulator) in addition to a therapeutically effective amount of a nitric oxide compound and a nitrovasodilator. In certain embodiments, the sGC stimulator is riociguat or veruiciguat. In some embodiments, the present invention includes a method for treating a vascular or respiratory condition in a human subject by administering to the subject a therapeutically effective amount of a stimulator of soluble guanylyl cyclase (sGC activator) in addition to a therapeutically effective amount of a nitric oxide compound and a nitrovasodilator. In certain embodiments, the sGC activator is lancaciguat, cinaciguat, ataciguat, or kynurenine. In some embodiments, the present invention includes a method for treating a vascular or respiratory condition in a human subject by administering to the subject a therapeutically effective amount of a cGMP (cyclic guanosine monophosphate)-phosphodiesterase inhibitor in combination with a nitric oxide compound and a nitrovasodilator. In certain embodiments, the cGMP-phosphodiesterase inhibitor is sildenafil, vardenafil, or tadalafil.

[0006] In certain embodiments, the present invention includes a method of treating a vascular or respiratory condition in a human subject by administering to the subject a therapeutically effective amount of a nitric oxide compound and nitric oxide administered via inhalation. In certain embodiments, the nitric oxide is administered via inhalation along with a nebulized thiol. The nebulized thiol can be L-cysteine ​​or glutathione. In some embodiments, the present invention includes a method for treating a vascular or respiratory condition in a subject by administering to the subject a therapeutically effective amount of a first nitrovasodilator in a first formulation for activating nitrovasodilator-activatable intracellular NO stores (NANOS) and a second nitrovasodilator in a second formulation for contributing to NANOS. In certain embodiments, the first nitrovasodilator is nitroglycerin and the first formulation is a sublingual tablet or a lingual spray. In certain embodiments, the second nitrovasodilator is nitroglycerin and the second formulation is a nanoliposome. In certain embodiments, the second nitrovasodilator is dinitrosyl iron complex (DNIC), S-nitroso-glutathione (GSNO), or heme-NO and the second formulation is a nanoliposome.

[0007] A method for treating a vascular or respiratory condition in a subject includes administering to the subject a therapeutically effective amount of a nitric oxide compound and a cGMP-phosphodiesterase inhibitor. The nitric oxide compound can be a nitrate-containing compound or a nitrite compound. The cGMP-phosphodiesterase inhibitor can be sildenafil, vardenafil, or tadalafil. The vascular condition can be erectile dysfunction. The patent or application file contains at least one drawing executed in color. Copies of the patent or application file containing a color drawing will be provided by the Office upon request and payment of the necessary fee.

[0008] Certain embodiments will be readily understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Certain embodiments are illustrated by way of example, and not by way of limitation. [Brief description of the drawings]

[0009] [Figure 1]FIG. 1A (Prior Art) is a diagram of the generation pathways and previously proposed mechanisms of vasodilatory activity for the three prominent stored NO forms, nitrite, DNIC, and SNO. All three are proposed to mediate vasodilatory activity through various pathways of conversion to free NO. DNIC and SNO are largely membrane impermeable and are proposed to release free NO outside of vascular smooth muscle cells, except that SNOs such as S-nitrosocysteine ​​(L-cysNO) or L-cysNO-glycine (L-cysNO-gly) may be selectively transported across the cell membrane via the L-type amino acid transporter (LAT) or dipeptide transporter (PEPT2), respectively. Handover of NO from extracellular SNO to membrane thiols via transnitrosylation and subsequent transport of NO into the cytoplasm has also been proposed. FIG. 1B is a representation of the structure of the mononuclear dinitrosyl iron complex. [Diagram 2] Figure 2 is an illustration of the mechanism of nitrovasodilator-activatable intracellular NO stores (NANOS). Nitrite (NO2-) and nitrovasodilators (NTG, SNO, and DNIC; the latter two may be endogenous NO metabolites) circulate systemically. NO2- contributes to the pool of NO bioactivity within vascular smooth muscle cells. Extracellular nitrovasodilators cause sGC-dependent vasodilation by stimulating the release of NO equivalents (NOe) from NANOS. [Figure 3-1] Figures 3A-3F show MDNIC as a candidate for NANOS (n=5). Figure 3A shows the contribution of NANOS from nanoliposomes containing MDNIC. [Figure 3-2] Figures 3A-3F show MDNIC as a NANOS candidate (n=5). Figure 3B is a graphical representation of the diameter of nanoliposomes encapsulating glut-MDNIC at an average concentration of 0.68 μM. Figure 3C is a graphical representation of the electron paramagnetic resonance (EPR) intensity of control and MDNIC-containing nanoliposomes. Incubation with MDNIC-containing nanoliposomes introduces MDNIC into the artery as measured by electron paramagnetic resonance spectroscopy. Figure 3D is a photographic representation of the uptake of Texas Red-tagged nanoliposomes into the arterial wall as evidenced by fluorescence imaging. [Figure 3-3] Figures 3A-3F show MDNIC as a candidate for NANOS (n=5). Figure 3E is a graphical representation of the arterial response after incubation with nanoliposomes containing MDNIC, demonstrating that reversal of loss of vasodilatory response occurs after repeated exposure to S-nitrosoglutathione (GSNO), but not with nanoliposomes encapsulating GSNO or buffer (empty). Figure 3F is a graphical representation of the arterial response after incubation with nanoliposomes containing MDNIC, demonstrating that incubation does not alter NO-mediated vasodilation for all three samples. [Figure 4] Figures 4A-4D demonstrate the mobilization of NANOS. Figure 4A is a diagram of the protocol. Sheep carotid arteries (8 cm long) were incubated with 10 μM N15-nitrite in DMEM under 10.5% O2 for 6 h to load the vessels with N15-NOx (NO metabolites). Figure 4B is a diagram of the prepared vessels. After extensive washing, the arteries were fully sealed and incubated for 2 h with 100 μM oxyhemoglobin in 3 ml HEPES buffer on the abluminal side and with 5 μM GSNO or control (0.7 ml HEPES buffer containing 0.1 mM DTPA). Figure 4C is a graphical representation of the response of arteries exposed to GSNO. N15-NOx levels were lower in homogenates of arteries exposed to GSNO (p=0.0206). Figure 4D is a graphical representation of the response of arteries exposed to intraluminal GSNO. N15-NOx in extraluminal buffer was higher in arteries exposed to intraluminal GSNO (p=0.0400). N15-NOx was measured by GC-MS. The results are consistent with intraluminal GSNO stimulating NOx efflux from vascular smooth muscle, as manifested by a decrease in intracellular NOx and an increase in extracellular NOx. [Figure 5-1]Figures 5A-5D demonstrate the HNO-like properties of the NO equivalents released from NANOS. Isolated sheep mesenteric arteries were stripped of endothelium and contracted with 10 μM 5-HT (n=5). Figure 5A shows examples of relaxation traces (six arterial strips from one sheep) of NTG in the absence and presence of sGC inhibitor (ODQ) (10 μM) or superoxide dismutase (SOD1) (1000 U / ml, converts HNO to NO), or the extracellular NO scavenger CPTIO (200 μM), or both SOD1 and CPTIO. All traces were normalized for comparison of the degree of relaxation. Figure 5B is a graphical representation of the amplification of the area inside the red circle in "A". [Figure 5-2] Figures 5A-5D demonstrate the HNO-like properties of NO equivalents released from NANOS. Figure 5C is a graphical representation of the effects of SOD1, CPTIO, SOD1+CPTIO, and ODQ on relaxation in response to nitroglycerin. Figure 5D is a graphical representation of the effects of SOD1, CPTIO, and SOD1+CPTIO on relaxation in response to nitrosoglutathione (GSNO). SOD1, through the presumed conversion of HNO to NO, enables the inhibitory effect of CPTIO on relaxation caused by NTG and GSNO. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Nitroglycerin is a nitrovasodilator commonly prescribed for the treatment of angina pectoris. It acts by dilating the coronary arteries, thereby increasing blood flow to the myocardium, and by dilating the systemic venous vessels, thereby decreasing venous return and ventricular preload, thereby decreasing cardiac work. A significant obstacle to the clinical use of nitroglycerin is that tolerance to its vasodilator effect develops after repeated use. The mechanism of this tolerance has yet to be elucidated, despite many studies. This tolerance may be due to the depletion of vascular intracellular stores of NO. Currently, nitrovasodilators such as nitroglycerin are prescribed alone for the treatment of angina pectoris. Provided herein is a method of co-administration of nitro group-containing compounds, such as nitrites, to replenish intracellular stores, thereby attenuating the tolerance that develops to repeated treatment with nitrovasodilators, including NTG.

[0011] Disclosed herein is a method of co-administering one or more nitro-containing chemicals with nitrovasodilators to reduce the tolerance and loss of therapeutic effect that occurs in subjects who require frequent dosing. Nitro-containing chemicals are generally stable and therefore considered to be inert and safe compounds. This class of compounds slowly releases nitric oxide (NO) via Fenton chemistry-related mechanisms or otherwise, enhancing vasodilation and contributing to lowering blood pressure. Nitrite, the endogenous anionic form of the nitro group, also releases NO via Fenton chemistry-related mechanisms. In some embodiments, nitro-containing compounds such as nitrite are provided as useful supplements for beneficial cardiovascular effects via NO-mediated effects on the vasculature. Vascular smooth muscle contains intracellular stores of NO bioactivity that contribute to vasodilation. This intracellular store can be activated by the presence of extracellular compounds classified herein as nitrovasodilators, such as nitrosothiols and nitroglycerin. Repeated activation depletes intracellular stores and impairs vasodilation function. Moreover, in experiments with isolated arteries and live animals, intracellular stores can be replenished by administration of nitro-containing chemicals that act as NO donors. Such treatment restores the vasodilatory effects of nitrovasodilators, e.g., NTG and nitrosothiols. Disclosed herein are embodiments of methods for the treatment of vascular conditions by administering therapeutically effective amounts of nitric oxide compounds and nitrovasodilators, e.g., NTG.

[0012] A method for treating a vascular or respiratory condition in a human subject includes administering a therapeutically effective amount of a nitric oxide compound and a source of exogenous NO or an agent that produces endogenous NO. For example, the exogenous NO can be inhaled NO. In certain embodiments, NO can be inhaled with a nebulized thiol, such as L-cysteine ​​or glutathione.

[0013] A method for treating a vascular or respiratory condition includes administering a therapeutically effective amount of two different formulations of a nitrovasodilator. A particular method for treating a vascular or respiratory condition in a human subject includes administering to the subject a therapeutically effective amount of a nitrovasodilator for activating NANOS in a first formulation and a nitrovasodilator for contributing to NANOS in a second formulation. For example, in one embodiment, NTG is administered as a sublingual tablet or a lingual spray with another administration of NTG as nanoparticles. In one embodiment, the nanoparticles can be NTG encapsulated in liposomes. In certain embodiments, the first formulation can be a sublingual tablet or a lingual spray and the second formulation is nitroglycerin, DNIC, GSNO, or heme-NO encapsulated in liposomes. A therapeutically effective amount of a nitric oxide compound or a nitrovasodilator can be entrapped, encapsulated, and / or absorbed in liposomes, lipid or polymer nanoparticles, vesicles, or microemulsions used as delivery vehicles. Biopolymers such as polylactide, polyglycolide, poly(lactide-co-glycolide), polyhydroxybutyric acid, polycaprolactone, and polydioxanone can be used to make subunit and / or composite nanoparticles. Natural hydrophilic polysaccharides such as hyaluronic acid or inulin are useful for making subunit and / or composite nanoparticles. In some embodiments, the nitric oxide compounds or nitrovasodilator compounds described herein can be coupled to polymers used in nanoparticles, such as polystyrene particles, PLGA particles, PLA particles, or other nanoparticles.

[0014] Specific vascular conditions that may be treated and / or prevented by the compositions disclosed herein include, but are not limited to, coronary and peripheral artery disease, asthma, chronic rejection, vasculopathy associated with diabetes, congestive circulatory conditions, peripheral edema, scleroderma, glaucoma, systemic hypertension, pulmonary hypertension, wound healing, anal fissures, vulvodynia, erectile dysfunction, post-hemorrhagic vasospasm, pre-eclampsia, Raynaud's phenomenon, and heparin overdose. Specific respiratory conditions that may be treated and / or prevented by the compositions disclosed herein include, but are not limited to, apnea, chronic obstructive pulmonary disease, and pulmonary hypertension.

[0015] The combination of the nitrovasodilator and the nitric oxide compound can be administered as one fixed dose multi-drug combination or as separate formulations. The nitric oxide compound can be one or more of a nitro group-containing compound, a nitrate group-containing compound, an NO donor, and an endogenous NO-generating amino acid. Nitro group-containing compounds include nitrites, N 5 -[Imino(nitroamino)methyl]-D-ornithine (DN G -nitroarginine methyl ester) or any other low molecular weight molecule containing a nitro group, or a protein, or a lipid. The nitrate group-containing compound can include nitrate, or any low molecular weight molecule containing a nitrate group, or a protein, or a lipid. The NO donor can be a NONOate or a hydroxamic acid. NONOates are compounds with an amine function, a bridging NO-group, and a terminal nitrosyl group, which release NO under certain biological conditions. Hydroxamic acids include, but are not limited to, deferoxamine, acetohydroxamic acid, and hydroxyurea. The endogenous NO-generating amino acid can be arginine or citrulline, or a combination thereof.

[0016] The present disclosure relates to compositions for the treatment of vascular or respiratory conditions by increasing the effective amount of NO available to affected cells and / or tissues. The term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject suffering from a condition, will have the intended therapeutic effect, e.g., alleviation, amelioration, mitigation or elimination of one or more symptoms of the condition in the subject. The therapeutically effective amount will vary depending on the subject and condition being treated, the subject's weight and age, the severity of the condition, the salt, solvate, or derivative of the active drug moiety selected, the particular composition or excipient selected, the dosing regimen to be followed, the timing of administration, the mode of administration, etc. The complete therapeutic effect may not necessarily occur by administration of a single dose, but only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0017] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes herein, beneficial or desired results include, but are not limited to, inhibiting and / or suppressing the occurrence and / or onset of a disease or condition responsive to a composition described herein, or reducing the severity of such a disease or condition, e.g., reducing the number and / or severity of symptoms associated with a disease or condition, improving the quality of life of a person suffering from a disease or condition, reducing the dose of other medications required to treat the disease or condition, enhancing the effectiveness of another medication that an individual is taking for a disease or condition, and extending the survival of an individual with a disease or condition. As used herein, "preventing" refers to reducing the probability of developing a disorder or condition in an individual who does not have the disorder or condition but is at risk of developing the disorder or condition.

[0018] In some embodiments, the therapeutic dose of the nitrovasodilator, or a pharma- ceutically acceptable salt or solvate thereof, administered to the subject is sufficient to produce a mean serum concentration of about 100 pg / mL to about 1 μg / mL, or any subrange or subvalue therebetween. In one embodiment, the nitrovasodilator, such as NTG, is administered at a dose ranging from 0.1 mg to about 0.6 mg each time. In one embodiment, the nitrovasodilator, such as isosorbide mononitrate, is administered at a dose ranging from 10 mg to about 300 mg, which may contain about 120 mg per tablet. In one embodiment, the dose of the nitrovasodilator, or a pharma-ceutically acceptable salt or solvate thereof, administered to the subject is about 0.1 mg to 0.6 mg. In some embodiments, the therapeutic dose of the nitric oxide compound is in the range of about 1 mg to about 10 g. In some embodiments, the nitrovasodilator and the nitric oxide compound are administered to the subject in a ratio ranging from 1:1 to 1:33, or 1:1 to 1:333, or 1:1 to 1:3333, or 1:1 to 1:33333. In some embodiments, the nitrovasodilator and the nitrate compound are administered to the subject in a ratio ranging from 1:1 to 1:34, or 1:1 to 1:333, or 1:1 to 1:3333, or 1:1 to 1:33333. The nitrate compound can be a pharmaceutically acceptable salt, for example, a sodium salt, or a solvate thereof. In some embodiments, the nitrovasodilator and the nitrite compound are administered to the subject in a ratio ranging from 1:1 to 1:33, or 1:1 to 1:333, or 1:1 to 1:3333. The nitrite compound can be a pharmaceutically acceptable salt, for example, a sodium salt, or a solvate thereof. In some embodiments, the therapeutically effective amounts of the nitric oxide compound and nitroglycerin are administered as a fixed dose combination. In some embodiments, the therapeutically effective amounts of the nitric oxide compound and nitroglycerin are administered as a free dose combination.

[0019] The term "about," when used before a numerical designation including a range, e.g., temperature, time, amount, concentration, etc., indicates an approximation that may vary by (+) or (-) 10%, 5% or 1%, or any subrange or subvalue therebetween.

[0020] Any route of administration can be used, for example, oral, topical, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into a body compartment. The compositions herein can be administered by direct bloodstream delivery, for example, sublingual, intranasal, or pulmonary administration. Administration can be via transdermal patch, gum, lozenge, sublingual tablet, intranasal, intrapulmonary, oral, or other administration. "Pharmaceutically acceptable salts" refers to pharma- ceutically acceptable salts, including pharma-ceutically acceptable partial salts, of a compound, which are derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, chloride, hydrobromide, phosphoric acid, sulfuric acid, methanesulfonic acid, phosphorous acid, nitric acid, perchloric acid, acetic acid, tartaric acid, lactic acid, succinic acid, citric acid, malic acid, maleic acid, aconitic acid, salicylic acid, phthalic acid, embonic acid, enanthic acid, oxalic acid, and the like, and, in cases where the molecule contains an acidic functional group, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like.

[0021] As used herein, the term "solvate" is taken to mean a solid form of a compound that crystallizes with one or more molecules of a solvent trapped therein. A few examples of solvents that can be used to make solvates, e.g., pharmaceutically acceptable solvates, include, but are not limited to, water, methanol, ethanol, isopropanol, butanol, C1-C6 alcohols in general (may be substituted), tetrahydrofuran, acetone, ethylene glycol, propylene glycol, acetic acid, formic acid, water, and solvent mixtures thereof. Other such biocompatible solvents that can be useful for making pharmaceutically acceptable solvates are well known in the art and are applicable to the present disclosure. Furthermore, various organic and inorganic acids and bases can be added or even used alone as solvents to make the desired solvates. Such acids and bases are well known in the art. When the solvent is water, the solvate can be referred to as a hydrate. In addition, the compounds of the present disclosure may absorb moisture when left in the air or recrystallized, and the crystals formed may contain one or more water molecules, thus forming hydrates. Even when such hydrates are formed, they are included in the term "solvate".

[0022] The nitric oxide compound administered with the nitrovasodilator may be one or more metabolites of NO. These metabolites of NO are classified based on structural characteristics as nitrogen oxoanions, e.g., nitrite (NO2 - ) and nitrates (NO3 -NO metabolites can be divided into classes including NO sequestered in hydrophobic regions of NO, S-nitrosothiols (SNO), iron nitrosyl species (FeNO), such as nitrosyl-heme (heme-NO) and dinitrosyl iron complexes (DNICs; non-heme-iron nitrosyl species), nitrated lipids, and proteins. Importantly, some members of each of these categories of NO metabolites are stable enough to circulate systemically and also have NO-like biological activity at physiological concentrations. These NO metabolites mediate their effects by protecting NO from irreversible metabolism during its transit through the circulation and subsequently releasing the same NO moiety at its remote site of action. As such, these compounds are often referred to as "stored NO" forms, or reservoirs of NO vasoactivity. The class of NO metabolites has a wide range of molecular weights, stabilities, and membrane permeabilities due to their various chemical structures. However, despite these differences, this diverse group of NO metabolites, including many SNOs and DNICs, exhibits remarkably similar vasodilatory activity with potency comparable to that of free NO itself. These compounds are collectively termed "nitrovasodilators" (Table 1) based on five functional criteria. First, like NO, all nitrovasodilators dilate blood vessels via direct or indirect activation of soluble guanylate cyclase (sGC) and the canonical cGMP vasodilatory pathway. Second, nitrovasodilators have an initial vasodilatory capacity comparable to or even greater than that of free NO itself. Third, unlike the transient vasodilatory effect of free NO on isolated arteries, nitrovasodilators produce a vasodilatory response that is significantly more sustained. Fourth, they are relatively stable, either not releasing measurable free NO or releasing free NO so slowly that their vasodilatory effect cannot be explained by free NO release. Finally, nitrovasodilators have a lower membrane permeability than that of NO itself. This last feature applies to almost all molecules, given that NO is a small, non-polar, uncharged molecule that is 3-4 times more soluble in lipids than in aqueous solutions.However, highlighting this feature of nitrovasodilators is important because it highlights the fact that the release of NO moieties from nitrovasodilators cannot achieve the intracellular free NO concentrations that would be necessary to explain their ability to activate sGC with a potency comparable to NO itself. Indeed, the latter four features of nitrovasodilators pose a challenge to the commonly held belief that they dilate blood vessels simply by releasing NO moieties that activate sGC. The present disclosure utilizes an alternative explanation for the source of NO involved in nitrovasodilator-mediated sGC activation.

[0023] [Table 1]

[0024] Vasodilator vasodilation involves the release of NO moieties from stores inside vascular smooth muscle cells (Figure 2), which in some ways resembles the activation of NO-mediated vasodilation in response to exposure to light. Nitrovasodilators cause vasodilation by an uncharacterized pathway upstream of sGC activation, involving the release of NO equivalents from preformed stores inside vascular smooth muscle cells, termed nitrovasodilator-activated store NO (NANOS). It is believed that NO synthesized in the endothelium by NO synthase can diffuse randomly into adjacent vascular smooth muscle, where it can bind to the heme of sGC, resulting in cGMP-dependent vasodilation. It can also react in blood and tissues to form a variety of stable but bioactive metabolites, of which nitrite, SNO, and DNIC are the three most prominent metabolites.

[0025] Figure 1A (Prior Art) summarizes the current general understanding of their generation pathways and vasodilatory mechanisms. Figure 1A shows the generation pathways and vasodilatory mechanisms of three prominent stored NO forms: nitrite, DNIC, and SNO. All three are proposed to mediate vasodilatory activity through various conversion pathways to free NO. DNIC and SNO are largely membrane impermeable and are proposed to release free NO outside of vascular smooth muscle cells, except that SNOs such as S-nitrosocysteine ​​(L-cysNO) or L-cysNO-glycine (L-cysNO-gly) may be selectively transported across the cell membrane via the L-type amino acid transporter (LAT) or dipeptide transporter (PEPT2), respectively. Delivery of NO from extracellular SNO to membrane thiols via transnitrosylation and subsequent transport of NO to the cytoplasm has also been proposed.

[0026] Until the mid-1990s, nitrite was thought to be a by-product of NO metabolism that is inactive at physiological concentrations and simply excreted in urine. However, it is becoming increasingly clear that nitrite, although orders of magnitude less vasoactive than NO, can serve as a source of NO bioactivity. A growing body of literature reports that increasing circulating nitrite concentrations through dietary intake leads to decreased systemic vascular resistance and improved exercise capacity. One proposed mechanism for the vascular effects of nitrite is based on the ability of deoxyhemoglobin to reduce nitrite to NO. Through this reaction, deoxyhemoglobin, which is abundant in hypoxic tissues, facilitates the conversion of nitrite to NO, leading to vasodilation and helping to restore adequate O2 delivery to hypoxic tissues. Consistent with this understanding, it has been demonstrated that the vasodilatory effects of nitrite are enhanced under hypoxic conditions. However, because red blood cell hemoglobin scavenges free NO at a rate approximately one million times faster than deoxyhemoglobin produces NO from nitrite, it remains unclear how the NO produced by this reaction escapes from red blood cells and reaches the vessel wall. Alternatively, some experiments have presented evidence that the vasodilatory effect of nitrite is not facilitated by deoxyhemoglobin. For example, infusion of nitrite into the relatively deoxygenated blood of the pulmonary or femoral arteries during systemic hypoxia does not result in a decrease in pulmonary or femoral vascular resistance until intravascular nitrite concentrations reach several orders of magnitude above the physiological range. Similarly, although fetal hemoglobin reduces nitrite to NO twice as quickly as adult hemoglobin, and normal fetal oxyhemoglobin saturation favors the reduction of nitrite to NO, infusion of nitrite into chronically instrumented fetal sheep does not produce cerebral vasodilation until plasma nitrite concentrations exceed 100 times the physiological range.

[0027] As an alternative to the hemoglobin reductase hypothesis, the vasodilatory effect of nitrite appears to result from its direct action on the vascular wall.As shown by the data herein, the vasodilatory effect of treatment with nitrite is found to persist even after plasma nitrite levels return to baseline.The mechanism of these effects remains unclear, but may involve the reduction of nitrite to NO or some NO adducts within vascular smooth muscle cells by several different proposed pathways.

[0028] Interestingly, measurements of nitrite concentrations also indicate that it may play a special role in vascular smooth muscle. For example, concentrations measured in most tissues, including blood, are well below 1 μM, whereas concentrations in the walls of animal aortas range from 5 to 25 μM. The presence of such high concentrations is not inconsistent with the presence of NANOS in the vascular wall. Concentrations measured in the aorta are not necessarily the same as those found in downstream resistance vessels, where vascular tone is more important in controlling blood pressure.

[0029] S-nitrosothiols, a category of NO adducts with potent vasodilatory effects, are formed by the addition of NO to thiols of molecules ranging in size from small molecules such as thionitrite (HSNO) and nitrosoglutathione (GSNO) to high molecular weight compounds, e.g., nitrosylated cysteine ​​residues of large proteins such as hemoglobin and albumin. In addition, SNOs can be generated by the "handover" of an NO moiety from one SNO to another thiol, a reaction called transnitrosylation (R1-SNO+R2-SH⇔R1-SH+R2-SNO, where R1 and R2 are different molecules). As a result of transnitrosylation, the various SNOs in plasma exist in equilibrium with each other, with the more stable high molecular weight (HMW) forms, such as albumin-SNO, predominating. For example, intravascularly infused low molecular weight (LMW) SNOs, such as S-nitroso-cysteine ​​(cysNO) or S-nitroso-glutathione (GSNO), are converted to HMW plasma SNOs within one systemic transit time. Advancing the understanding that SNOs may be the circulating form of NO bioactivity, Stamler et al. proposed that the cysteine ​​thiol on the beta subunit of hemoglobin (β-93) is an important carrier of NO in blood. Although not without controversy, there is evidence that the NO moiety of hemoglobin-SNO can be exported from red blood cells in the form of GSNO through a process facilitated by the conformational R-to-T transition that occurs upon deoxygenation. As discussed in the next section, GSNO and other membrane-impermeable SNOs are potent vasodilators. It remains to be elucidated how exported GSNO, which is membrane-impermeable and does not release free NO, can cause vasodilation.

[0030] The mechanism underlying SNO-mediated vasodilation has puzzled researchers in the field for over 40 years. Like NO, SNO causes vasodilation primarily through activation of sGC, but can signal through protein transnitrosylation, resulting in posttranslational modification of protein function. However, the mechanism by which extracellular SNO activates cytoplasmic sGC remains unclear. Due to their different thiol ligands, SNOs can have a wide variety of stability and membrane permeability (Table 1). For example, L-cysNO and its stereoisomer D-cysNO are 10-fold less stable than GSNO and therefore more likely to release NO, but L-cysNO releases less than 10% of its NO moiety over a sustained period of 1 h in the presence of metal chelators in PBS. On the other hand, Hogg et al. demonstrated that L-cysNO can be taken up into cells via the L-type amino acid transporter (LAT), whereas D-cysNO and GSNO (whose membrane permeability is 200-fold lower than that of L-cysNO) are largely membrane impermeable. Based on these properties, it can be expected that the vasodilatory potencies of these three compounds are significantly different, with L-cysNO being the most potent and GSNO being the least potent. However, despite their different degradation rates, conformations, and membrane permeability, all three of these SNOs have vasodilatory potencies comparable to that of NO itself. In other words, although most of the NO moiety of GSNO remains outside the cell, GSNO can activate sGC with potency comparable to that of L-cysNO and even free NO itself (both of which enter the cell much more rapidly). Although multiple mechanisms have been proposed to explain this phenomenon (Figure 1A), no consensus has been reached to support any one of these pathways.

[0031] Dinitrosyl iron complexes are a class of FeNO complexes that contain NO and ferrous non-heme iron (Figure 1B) and usually thiol ligands, although other ligands have been described. Figure 1B is a representation of the structure of mononuclear dinitrosyl iron complexes. These compounds contain one (mononuclear) or two (binuclear) Fe(NO)2 nuclei (MDNIC or BDNIC, respectively). Like SNOs, DNICs can transfer their Fe(NO)2 nuclei to thiols on other molecules, resulting in a wide variety of DNICs with different molecular weights, structures and stabilities. DNICs have been found to dilate isolated precontracted arteries at submicromolar concentrations, and this effect is blocked by inhibition of sGC. For example, glutathione-ligand-bound DNICs have vasodilatory ECs on the order of about 1 μM. 50 , which is comparable to that of free NO itself. As with SNO, the comparable vasodilatory potency of glutathione ligand-bound DNIC and free NO raises mechanistic challenges as to how membrane-impermeable and stable DNIC activates intracellular sGC as potently as free NO itself, and how DNIC and SNO produce a more pronounced and sustained vasodilatory response than free NO.

[0032] Although DNICs are clearly potent vasodilators with pharmacological potency, their physiological relevance remains unclear due to the lack of assay methods with sufficient selectivity and sensitivity to measure DNICs in blood and tissues. Electron paramagnetic spectroscopy (EPR), the gold standard method, can distinguish DNICs from other NO metabolites. However, the lower detection limit of EPR is above the vasoactive concentration of DNIC, and therefore EPR cannot unequivocally determine whether DNICs are present at vasoactive concentrations. Ozone-based chemiluminescence offers the ability to detect DNICs at concentrations within or below the vasoactive range. However, this method requires sample handling and pretreatment, which may result in degradation of DNIC to form other NO metabolites before it reaches the assay instrument. Despite these interconversions, cell culture experiments using both exogenous and endogenous NO sources suggest that DNIC is the most abundant intracellular NO adduct. DNICs are the "working forms" of NO in cells and tissues; high molecular weight DNICs are the major reservoir of intracellular NO bioactivity, while the less stable LMW DNICs shift NO bioactivity to heme- or thiol-containing targets.

[0033] As outlined above, the presence of photosensitive stored NO in vascular muscles is well established. For example, precontracted endothelium-denuded arteries relax upon exposure to light, particularly UV light, and this photorelaxation is due to NO-mediated activation of sGC, despite the absence of endothelial NO production. Moncada et al. demonstrated that photoactivated stored NO can be depleted by repeatedly exposing arteries to light, and that photoactivated stored NO can also be gradually replenished if endothelium-derived NO production is preserved or if endothelium-denuded arteries are incubated with a NO donor. The activation mechanism of photosensitive stored NO has not been fully elucidated. Previous reports supported UV light irradiation as an activator of stored NO. However, recent studies have found that far-infrared, near-infrared, and visible light can also activate photorelaxation. Indeed, the nature of the vasodilatory response differs depending on the wavelength of light used for stimulation. For example, visible light induces a transient, reversible relaxation that is endothelium-dependent, whereas relaxation induced by UV light involves an early, transient response superimposed on a sustained response that is endothelium-independent. This finding led to the proposal that there are two or more light-sensitive stores of NO with distinct chemical properties, one in the endothelium and the other in vascular smooth muscle cells.

[0034] In addition to light, low millimolar levels of LMW thiols, well above physiological levels, can also activate stored NO to mediate endothelium-independent vasodilation, although it is unclear whether this thiol-sensitive NO store is identical to the light-sensitive NO store. Muller et al. demonstrated that membrane-permeable N-acetyl-L-cysteine ​​(NAC) relaxes rat aortas pretreated with LPS and L-arginine to form putative intracellular NO stores, but not those pretreated with LPS or L-arginine alone, or with LPS, L-arginine, and L-NAME. It has been proposed that LMW thiols promote the dissociation of NO from the intracellular NO store form, resulting in vasodilation. The chemical nature of the light-sensitive NO stores is only partially characterized. SNO, heme-bound NO, and DNIC are all capable of photolytic release of NO under irradiation with UV, red, and infrared light. In contrast, the photolytic instability of nitrite is relatively low, and unphysiologically high intracellular concentrations (>1 mM) of nitrite appear to be required for photorelaxation.

[0035] Several lines of observation suggest a role for SNO as an intracellular NO store. For example, exposure of isolated arteries to compounds that deplete SNO attenuates both light- and thiol-mediated relaxation. Conversely, incubation of blood vessels with membrane-permeable SNO, increasing the concentration of NO adducts in the vessel wall, increases thiol-mediated relaxation. In addition, exposure of arteries to red light stimulates vascular release of vasodilators with the characteristics of SNO, strongly indicating a close relationship between SNO and intracellular NO stores. There are also findings supporting the role of DNIC as an intracellular NO store. Preincubation of arteries with a combination of LPS and L-arginine or with NO donors leads to increased intracellular stores of DNIC with protein ligands, which are mobilized by membrane-permeable LMW thiols to release NO in the form of LMW thiol-liganded DNIC, resulting in vasodilation. However, SNO, DNIC, and other NO adducts are interconvertible and likely exist in equilibrium, so they may all play a role in the function of the intracellular NO store.

[0036] Although most of the body's vascular smooth muscle is shielded from the amount of light required to activate NO release from photosensitive NO stores, both photosensitive NO stores and thiol-sensitive NO stores in the vasculature are indirectly linked to cardiovascular function and pathology, suggesting that they are involved in vascular smooth muscle function even in the absence of light. For example, in young spontaneously hypertensive rats, the function of thiol-sensitive NO stores declines in parallel with the development of endothelial dysfunction and hypertension. On the other hand, acclimation with mild to moderate hypoxia enhances the function of the stores and also prevents endothelial dysfunction and hypertension. In humans, Feelisch et al. suggested that UV light modulates systemic NO bioavailability by mobilizing NO stores in the skin, and that this may contribute to latitudinal and seasonal variations in blood pressure and cardiovascular disease.

[0037] Based on experiments intended to determine the mechanism underlying SNO-mediated vasodilation, the inventors realized that nitrovasodilators utilize intracellular stores of NO, herein "NANOS," to bring about vasodilation. Previous hypotheses were based on the assumption that these NO adducts release their NO moiety, which then directly activates sGC (FIG. 1A). Many pathways have been proposed by which NO from extracellular SNO could reach intracellular sGC. However, the experiments herein show that none of these pathways play a significant role. Instead, these results raised another possibility that SNO causes vasodilation via mobilization of NO moieties from sources other than extracellular SNO itself, essentially from existing intracellular stores of NO reminiscent of light-sensitive ones.

[0038] As shown diagrammatically in Figure 2, this NANOS model shows that preformed intracellular stores of NO are mobilized by extracellular nitrovasodilators to mediate vasodilation. Inhibition of endogenous NOS and / or repeated stimulation with these nitrovasodilators attenuates the vasodilatory response to subsequent nitrovasodilator exposure via depletion of NANOS. Furthermore, incubation of arteries with compounds that may contribute to the store enhances nitrovasodilator-mediated vasodilation. One prediction consistent with the NANOS concept is that its content can be depleted and its function correspondingly reduced, as has been repeatedly demonstrated for light-sensitive NO stores. Indeed, consistent with depletion, isolated vessels lose approximately 50% of their initial vasodilatory response to GSNO after repeated exposure to GSNO, despite remaining fully responsive to NO itself. This finding is consistent with a use-dependent loss of GSNO-mediated vasodilation due to attenuation of pathway components upstream of sGC activation. Initial exposure of arteries to GSNO induces the recruitment of NANOS, whose depletion leads to attenuated responses to subsequent GSNO exposures.

[0039] Based on the understanding that endogenous eNOS activity is required to maintain NANOS homeostatically, treatment with L-NAME was evaluated for its ability to attenuate the vasodilatory response to GSNO in intact rats and sheep. In both cases, femoral vasodilation in response to GSNO, D-cysNO, and L-cysNO was greatly reduced or even abolished in animals treated with L-NAME. Interpreted in the context of the NANOS model, these results suggest that inhibition of endogenous NO production by NOS results in the abrogation of NANOS-dependent vasodilation caused by SNO.

[0040] Moreover, according to the NANOS model, enhancing the stores should enhance the vasodilatory response to nitrovasodilators. To experimentally increase the stores, isolated ovine femoral arteries were pre-exposed to 1 μM nitrite, a concentration 2-3 orders of magnitude lower than the concentration at which nitrite alone causes vasodilatation. Such treatment was found to enhance the vasodilatory response to GSNO. In addition, the attenuation of the vasodilatory response to GSNO that occurs in isolated arteries after repeated exposure to GSNO to deplete NANOS can be reversed by subsequent incubation of the arteries with 1 μM nitrite for 30 min. Similarly, intravenous infusion of nitrite at subvasodilatory concentrations into intact rats and sheep pretreated with L-NAME resulted in enhanced vasodilatory responses to subsequent infusion of SNO in femoral arteries that would normally not respond to SNO. Interpreted in the context of the NANOS model, these findings suggest that nitrite can replenish stores of NO after they have been depleted by inhibition of endogenous NOS activity.

[0041] Natural examples of a reduction in NO stores are found in newborn humans and lambs. In both species, their plasma nitrite concentrations decrease by more than 50% within minutes of birth to levels even lower than those observed after NOS inhibition. Treatment of newborn lambs with low micromolar concentrations of intravenous nitrite during this postnatal period has been found to enhance GSNO-mediated vasodilation. This finding is also consistent with the understanding that nitrite plays a role in replenishing NANOS. Surprisingly, under some conditions, L-NAME can contribute independently to NANOS. In contrast to acute administration of L-NAME, pretreatment of rats with L-NAME for 4 days enhanced mesenteric vasodilator responses to nitrovasodilators, but not to NO itself, a finding suggestive of upregulation of pathway components upstream of sGC activation and reminiscent of an expansion of NO stores. L-NAME independently contributes directly to NANOS in mesenteric arteries, and L-NAME increased concentrations of NO-containing compounds in the mesenteric artery wall. Furthermore, isotopically labeled L-NAME (R-NAME) with a nitro group enhanced NANOS in mesenteric arteries. 15 In vitro and in vivo application of NO2) demonstrated that metabolic conversion of L-NAME releases NO from its nitro group.

[0042] As further evidence that the enhancing effect of L-NAME is due to the release of NO and not due to NOS inhibition, its stereoisomer D-NAME, which does not inhibit NOS but releases NO from its nitro group, also enhanced nitrovasodilator-mediated vasodilation. Moreover, this enhancement was not observed after treatment with L-NMMA, an NOS inhibitor that does not contain a nitro group. In addition, the enhancing effect of L-NAME was found to be increased in the presence of oxidative stress, which favors the release of NO from the nitro group. Thus, there is evidence that chronic treatment with L-NAME can enhance the mesenteric vasodilation of nitrovasodilators through the contribution of its nitro group to stored NO.

[0043] Nitrite likely contributes to NO stores via its reduction to NO, but the underlying mechanism remains unclear. Several different metalloproteins, such as hemoglobin (Hb), myoglobin, xanthine oxidase, cytochrome C oxidase, and eNOS, have been proposed to be involved in the reduction of nitrite to NO. However, in most cases, O2 competes very effectively with nitrite for the metal active sites on these proteins, so that their ability to reduce nitrite to NO is largely inhibited under physiological O2 partial pressures. Furthermore, the oxygenated forms of many of these metalloproteins avidly capture NO and convert it to nitrate. Thus, the importance of these pathways for the conversion of nitrite to NO under physiological conditions is questionable, especially in the walls of resistance vessels where PO2 is close to arterial levels.

[0044] At low pH, nitrite can also be converted to NO via dismutation. However, the pKa of nitrite is 3.3, and therefore the contribution of this pathway to nitrite reduction is also very limited at physiological pH. Oxidative stress promotes NO release and the NANOS enhancing properties of the nitro group of L-NAME.

[0045] DNIC and nitroglycerin appear to cause vasodilation in a manner similar to SNO, despite exhibiting a wide range of chemical reactivity, molecular size, and stoichiometry. These NO moiety-containing compounds cause vasodilation through activation of sGC with potency comparable to that of NO itself, but the mechanism by which they activate sGC remains unclear. Although these compounds are often thought to be NO donors, most SNOs and DNICs differ from bona fide NO donors such as NONOates in that they do not readily release free NO and also because they cause sustained relaxation in isolated vessels, unlike the more transient vasodilation induced by NO donors. Similarly, NTG is thought to activate sGC by releasing the NO moiety from its nitrate group via the action of enzymes such as aldehyde dehydrogenase-2. However, this conversion produces nitrite rather than NO, especially at therapeutic NTG concentrations (<1 μM). On the other hand, ECs below 0.1 μM 50 In this study, the vasodilatory sensitivity of NTG is approximately one order of magnitude greater than that of NO itself when tested in isolated arteries. Due to these similarities, we have reclassified these NO moiety-containing vasodilators as nitrovasodilators, a name that does not include a bona fide NO donor such as NONOate. This term also serves to classify these compounds based on their common role as stimulators of NANOS, as suggested above for SNO. Consistent with this classification, vasodilator effects in the rat mesenteric vasculature by GSNO, glut-BDNIC, and NTG are all potentiated by chronic pretreatment with L-NAME and D-NAME, which contain nitro groups that may contribute to stored NO, but not by pretreatment with L-NMMA or L-arginine, both of which lack the nitro group. In addition, all potentiating effects are increased under oxidative stress, which may favor the contribution of nitro groups to stored NO via the release of NO. This finding further justifies the reclassification of SNO, DNIC, and NTG as nitrovasodilators and supports their role as stimulators of intracellular NO stores.

[0046] NTG induces tolerance, i.e., loss of responsiveness, after repeated use. Interestingly, NTG tolerance is often accompanied by cross-tolerance to other nitrovasodilators and even endothelium-dependent vasodilators. Cross-tolerance also occurs with some so-called NO donors, such as sodium nitroprusside, but these chemicals are actually classified in the nitrovasodilator category in that they do not release significant amounts of free NO. No cross-tolerance is observed with bona fide donors of NO, such as DEA-NONOate. Moreover, NTG tolerance can be gradually reversed by periods of drug-free use. These characteristics of tolerance to nitrovasodilators such as NTG are consistent with depletion of intravascular NANOS.

[0047] In the NANOS working model, the endothelium provides a constant source of NO and nitrite for incorporation into the NANOS of adjacent vascular smooth muscle. Thus, chronic inhibition of endothelial NOS results in depletion of NANOS and attenuated vasodilation mediated by nitrovasodilators. There is also evidence that the endothelium itself may release SNOs and DNICs in addition to free NO. Important aspects of research in this field are consistent with the NANOS model. For example, vasodilation in response to bradykinin is diminished with repeated use, and this decline occurs more rapidly in the presence of NOS inhibition. A use-dependent loss of vasodilation has also been observed for acetylcholine-mediated vasodilation in the presence of NOS inhibition, despite no change in the vasodilatory response to NO donors. Furthermore, recent studies have demonstrated that endothelium-dependent flow-mediated vasodilation in humans is improved by dietary nitrite and nitrate, which may contribute to intracellular NO stores. Taken together, these facts raise the possibility that endothelium-dependent vasodilation is at least partially dependent on NANOS. NANOS include contributing factors, nitrovasodilators, transmembrane signaling to activate the stores, components of the stores themselves, NO equivalents, and mechanisms for uptake, retention, and release of NO bioactivity from the stores.

[0048] As shown in Figure 2, direct contributors to the store include NO, nitrite, and nitro-group-containing compounds generated by endothelial NOS. Nitrovasodilators, including SNO, DNIC, and NTG, have been shown to be well-established vasodilators that stimulate store NO. Furthermore, the endothelium has been shown to act as a source of nitrovasodilators in addition to releasing free NO. Transmembrane signaling mechanisms are included to account for the transmembrane vasodilatory signaling of nitrovasodilators that are membrane impermeable and stable. Such membrane receptors / transporters are likely dependent on the recognition of the NO moiety, as they are common to all nitrovasodilators.

[0049] This NANOS model is reminiscent of intracellular calcium stores, where actual calcium cations constitute the reserve and mechanisms exist for their uptake, retention, and intracellular release to facilitate vasoconstriction. However, the molecular components of NANOS and those in the light- and thiol-sensitive NO stores have not been fully characterized. As NO is a highly reactive, freely diffusible gas, it is unlikely that it is stored and regulated by itself. Instead, stored NO is likely to be more stable NO adducts, such as SNO and DNIC. Taking advantage of a cell-based model amenable to surrogate genetic approaches to modulate cellular GSNO levels via overexpression or knockdown of GSNO reductase, intracellular GSNO was evaluated for its role in mediating the cGMP-dependent pathway. To test whether intracellular stored NO is related to GSNO, GSNO encapsulated in nanoliposomes was used to preload the arterial wall with GSNO, avoiding potential mobilization of stored NO by extracellular stimuli. Preloading arteries with liposomal GSNO did not restore the use-dependent loss of vasodilatory response to GSNO, suggesting that GSNO is unlikely to be a candidate for stored NO. On the other hand, parallel experiments using glutathione-ligand-conjugated MDNIC encapsulated in nanoliposomes increased DNIC levels in arteries and restored the use-dependent resistance to GSNO-mediated vasodilation without altering NO-mediated vasodilation (Figures 3A-3F). These findings support the idea that intracellular DNIC is a candidate for NANOS.

[0050] Figures 3A-3F show MDNIC as a candidate for NANOS (n=5). Figure 3A shows the contribution of NANOS from nanoliposomes containing MDNIC. Figure 3B is a graphical representation of the diameter of nanoliposomes encapsulating glut-MDNIC at an average concentration of 0.68 μM. Figure 3C is a graphical representation of the electron paramagnetic resonance (EPR) intensity of control and MDNIC-containing nanoliposomes. Incubation with MDNIC-containing nanoliposomes introduces MDNIC to the artery as measured by electron paramagnetic resonance spectroscopy. Figure 3D is a photographic representation of the uptake of Texas Red tagged nanoliposomes into the arterial wall as evidenced by fluorescent images. Figure 3E is a graphical representation of the arterial response following incubation with nanoliposomes containing MDNIC, demonstrating the reversal of the loss of vasodilatory response following repeated exposure to S-nitrosoglutathione (GSNO), but not nanoliposomes encapsulating GSNO or buffer (empty). FIG. 3F is a graphical representation of the arterial response following incubation with nanoliposomes containing MDNIC, demonstrating that incubation does not alter NO-mediated vasodilation for all three samples.

[0051] DNIC functions as an intracellular store of NO. NO can be stored as DNIC in ferritin, a cytoplasmic protein that stores and releases iron in a regulated manner. Nitrite, a contributor to stored NO, leads to significant amounts of cytoplasmic DNIC in isolated tissues and whole animal experiments. Incubation of cells with GSNO, a putative NANOS activator, stimulates mobilization of intracellular DNIC, a complex process involving glucose metabolism, iron release, glutathione efflux, and the coordination of DNIC carriers and transporters. Consistent with this, a general mobilization of arterial intracellular NO-containing species (NOx) upon extracellular stimulation with nitrovasodilators has recently been observed. As shown in Figures 4A-4D, incubation with GSNO leads to the efflux of NOx from the arterial wall, providing physical evidence consistent with the NANOS model. Figures 4A-4D demonstrate the mobilization of NANOS. Figure 4A is a diagram of the protocol. Sheep carotid arteries (8 cm long) were incubated with 10 μM N 15 -Incubate with nitrite for 6 hours under 10.5% O2 to inject N 15 The arteries were loaded with 100 μM oxyhemoglobin in 3 ml of HEPES buffer on the abluminal side and 5 μM GSNO or control (0.7 ml of HEPES buffer containing 0.1 mM DTPA) on the luminal side for 2 h. Figure 4C is a graphical representation of the response of the arteries exposed to GSNO. 15 -NOx levels were lower in homogenates from arteries exposed to GSNO (p=0.0206). Figure 4D is a graphical representation of the response of arteries exposed to intraluminal GSNO. N in extraluminal buffer 15 -NOx was higher in arteries exposed to intraluminal GSNO (p=0.0400). 15 -NOx was measured by GC-MS. The results are consistent with intraluminal GSNO stimulating NOx efflux from vascular smooth muscle, as manifested by a decrease in intracellular NOx and an increase in extracellular NOx.

[0052] As discussed above, there is reason to doubt that NO itself is the mediator of sGC activation in the NANOS model. Rather, NANOS activation may release an equivalent of NO. Although the debate continues, it has been suggested that HNO, the redox counterpart of NO, is an alternative activator of sGC. NO equivalents have HNO-like properties. As shown in Figures 5A-5D, CPTIO, a membrane-impermeable NO scavenger, does not affect either NTG- or GSNO-mediated relaxation, whereas SOD1, which converts HNO to NO, allows inhibition of vasodilation by CPTIO. Furthermore, a similar effect of SOD1 on CPTIO was observed for glut-BDNIC and the HNO donor Angeli salt (sodium trioxodisodium salt). Finally, chemicals that convert HNO to NO but do not themselves affect vasodilation, such as SOD1, TEMPOL, and CuCl, also allow the inhibitory effect of CPTIO. Figures 5A-5D demonstrate the HNO-like properties of the NO equivalents released from NANOS. Isolated sheep mesenteric arteries were denuded of endothelium and contracted with 10 μM 5-HT (n=5). Figure 5A shows examples of relaxation traces (six arterial strips from one sheep) of NTG in the absence and presence of sGC inhibitor (ODQ) (10 μM) or superoxide dismutase (SOD1) (1000 U / ml, converts HNO to NO), or the extracellular NO scavenger CPTIO (200 μM), or both SOD1 and CPTIO. All traces were normalized to compare the degree of relaxation. Figure 5B is a graphical representation of the amplification of the area inside the red circle in "A". Figure 5C is a graphical representation of the effect of SOD1, CPTIO, SOD1+CPTIO, and ODQ on relaxation in response to nitroglycerin (NTG). FIG 5D is a graphical representation of the effects of SOD1, CPTIO, and SOD1+CPTIO on relaxation in response to nitrosoglutathione (GSNO). SOD1, presumably through the conversion of HNO to NO, potentiates the inhibitory effect of CPTIO on relaxation caused by NTG and GSNO.

[0053] As discussed above, the endothelium may play a role in NANOS through the production of nitrovasodilators as stimulators, and NO and nitrite as contributors. This correlation adds another dimension to the implication of endothelial function, one of the most valuable predictors of future cardiovascular events. Conversely, this also has major physiological and pathological implications for the NANOS model. Consistent with this rationale, it has been suggested that plasma levels of nitrite, which correlate with stored NO function, reflect the degree of endothelial function. Extrapolating from endothelial NOS, NO sources such as inducible and neuronal NOS and dietary nitrite and nitrate may also play a role in the NANOS model. Through their effects on these NO sources, various physiological and pathological conditions may be relevant to NANOS. It is worth noting that physiological and pathological conditions may not simply affect the size of NANOS. As has been suggested for thiol-sensitive NO stores, the size, capacity, and efficiency of NO stores may all vary depending on conditions.

[0054] The existence of NANOS also provides the opportunity to improve medical treatment.Examples of NANOS regulation that may have therapeutic potential include a wide variety of hypertensive disorders, hypercoagulability treatment, and exercise performance improvement, for which dietary nitrite has already proven effective.A specific example of where increasing NANOS may have significant benefits is to avoid resistance to nitroglycerin, a century-old problem in the treatment of angina pectoris.

[0055] The proposed involvement of NANOS in the regulation of vascular tone is a novel idea that challenges the basic paradigm of NO signaling. In contrast to the conventional idea of ​​NO as a freely diffusing gas that transmits signals without the need for membrane receptors, conceptually, NANOS mediates the controlled mobilization of NO bioactivity upon extracellular stimuli. In addition, the NANOS model may also shed new light on the understanding of the signaling of different NO store forms. The NANOS model provides a framework for the crosstalk between different NO species, such as the crosstalk between nitrite and SNO in vasodilation. This model not only provides a possible explanation for the mechanism by which nitrite and SNO cause vasodilation, but also provides a conceptual bridge between the findings on the endocrine vasodilatory functions of nitrite and SNO. Furthermore, the synergistic action of intracellular and extracellular NO species in vasodilation, i.e., NANOS and circulating nitrovasodilators in the vessel wall, adds new temporal and spatial dimensions to the signaling of NO and its store forms.

[0056] SNO, DNIC, heme-NO, nitroprusside, NTG, and other nitrovasodilators dilate blood vessels via activation of soluble guanylyl cyclase (sGC) in vascular smooth muscle cells. These nitrovasodilators have a strong NO-mimetic vasoactivity, even though they do not release the necessary amount of free NO. These nitrovasodilators activate sGC via preformed NANOS found in vascular smooth muscle cells. Other examples of nitrovasodilators include, but are not limited to, isosorbide dinitrate, isosorbide mononitrate, and nitrosyl complexes with metals or metalloproteins with metals other than iron. The effect of nitrite on the restoration of coronary NTG resistance has been confirmed both in vivo and in vitro. The enhancing effect of nitro-containing chemicals on vasodilation by nitrovasodilators, including NTG, has been confirmed both in vivo and in vitro.

[0057] Both exogenous and endogenous NO are easily metabolized to nitrovasodilators such as SNO, DNIC, and heme-NO. For example, it has been demonstrated that intravascularly applied NO generates SNO in the circulation. Endothelium-derived relaxing factor (EDRF) contains not only NO itself but also NO metabolites as its components. Even as minor components, these NO metabolites, many of which are nitrovasodilators, are of great importance for cardiovascular health. Thus, the nitrovasodilator-NANOS signaling axis also plays an important role in the action of both exogenous and endogenous NO. In addition to treatments that rely on the delivery of exogenous NO or the production of endogenous NO, perturbations that contribute to NANOS can be applied to enhance treatment. In one embodiment, inhalation of NO leads to the production of SNO, which activates NANOS. Through its contribution to NANOS, nitrite can enhance NANOS activation mediated by SNO generated during NO inhalation. Similarly, perturbations that facilitate the production of nitrovasodilators by NO treatment can also enhance treatment. In one embodiment, inhaling NO along with an nebulized thiol, such as L-cysteine, results in the production of L-cysNO, which signals via activation of NANOS.

[0058] In one embodiment, the present invention includes a method for treating a vascular or respiratory condition in a human subject by administering a therapeutically effective amount of a nitric oxide compound that contributes to NANOS to the subject together with a source of exogenous NO. In one particular embodiment, the nitric oxide compound is nitrite. In one particular embodiment, the exogenous NO is inhaled NO. Normal plasma nitrite is less than about 0.5 μM. Administration of a nitric oxide compound together with a source of exogenous NO results in an increase in plasma nitrite to about 1 μM to about 10 μM. Circulating nitrite levels are usually measured by one of several techniques known in the art, such as ozone-based chemiluminescence.

[0059] In some embodiments, the present invention includes a method for treating a vascular or respiratory condition in a human subject by administering to the subject a therapeutically effective amount of a thiol that promotes the production of SNOs together with a source of exogenous NO to activate NANO. In certain embodiments, the exogenous NO is inhalation of NO. In certain embodiments, the thiol is cysteine ​​or glutathione. In certain embodiments, the delivery of the thiol is inhalation of a nebulized thiol. In some embodiments, the present invention includes a method for treating a vascular or respiratory condition in a human subject by administering a therapeutically effective amount of a nitric oxide compound to the subject together with a source of endogenous NO. Administering the nitric oxide compound together with a source of endogenous NO results in an increase in plasma nitrite to about 1 μM to about 10 μM. Circulating nitrite levels are typically measured by one of several techniques known in the art, such as ozone-based chemiluminescence. In certain embodiments, endogenous NO is administered as a precursor for NO production, for example, as the amino acids L-arginine or L-citrulline, or a combination thereof.

[0060] In certain embodiments, the combined application of nitrovasodilators and nitric oxide compounds is affected by the perturbation of the sGC-cGMP pathway. Vasodilators mediated by nitrovasodilators and / or nitric oxide compounds can be further enhanced by agents that enhance the sGC-cGMP pathway. Such agents include sGC stimulators and activators, cGMP-phosphodiesterase inhibitors, and the like. sGC stimulators directly activate reduced heme-containing sGC to generate cGMP, causing vasodilator. sGC activators activate sGC when heme is oxidized or the heme moiety is missing. sGC stimulators can be riociguat or veruiciguat. sGC activators can be lancaciguat, cinaciguat, ataciguat, or kynurenine. cGMP-phosphodiesterase inhibitors, such as sildenafil, vardenafil, and tadalafil, stabilize cGMP by preventing the degradation of cGMP by phosphodiesterase-5. In one embodiment, riociguat, sildenafil, or a combination of both are administered in addition to a nitrovasodilator, such as nitric oxide and / or nitrite, nitroglycerin, or both, thereby maximizing the vasodilatory effect of NANOS activation. Certain embodiments encompass a method of treating a vascular or respiratory condition in a human subject by administering a therapeutically effective amount of a nitric oxide compound and a cGMP-phosphodiesterase inhibitor. For example, a method of treating erectile dysfunction in a subject may encompass a step of administering a therapeutically effective amount of a nitric oxide compound (e.g., a nitrate-containing compound or a nitrite compound) and a cGMP-phosphodiesterase inhibitor (e.g., sildenafil, vardenafil, or tadalafil). The enhancing effect of riociguat and sildenafil on vasodilator induced vasodilator including GSNO has been confirmed in vitro.

[0061] In certain embodiments, the present invention includes a method for treating a vascular or respiratory condition in a human subject by administering a therapeutically effective amount of a first nitrovasodilator in a first formulation and a second nitrovasodilator in a second formulation. The nitrovasodilator acts extracellularly to activate NANOS, thereby causing vasodilation. The liposome-encapsulated nitrovasodilator cannot activate NANOS by extracellular stimuli. However, this formulation of the nitrovasodilator can efficiently load nitric oxide moieties into the cell and thus contribute to NANOS. In certain embodiments, the nitrovasodilator is encapsulated in a liposome. In certain embodiments, the first formulation is a sublingual tablet or translingual spray of nitroglycerin, and the second formulation is a liposomal formulation of DNIC, GSNO, or heme-NO. In certain embodiments, the present invention includes a method for treating a vascular or respiratory condition in a human subject by administering a therapeutically effective amount of a nitrovasodilator in a first formulation and the same nitrovasodilator in a second formulation. In certain embodiments, the first formulation is a sublingual tablet or spray of nitroglycerin and the second formulation is a liposomal formulation of nitroglycerin.

[0062] A number of embodiments have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the description. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. A method of treating a vascular or respiratory condition in a subject, comprising: administering to the subject a therapeutically effective amount of a nitric oxide compound and a nitrovasodilator. The method includes:

2. 10. The method of claim 1, wherein the vascular condition is one or more of coronary artery disease, pulmonary disease, or peripheral artery disease.

3. 2. The method of claim 1, wherein the nitric oxide compound is one or more of a nitro group-containing compound, a nitrate group-containing compound, a nitric oxide (NO) donor, and an endogenous NO-generating amino acid.

4. 4. The method of claim 3, wherein the nitro group-containing compound is one or more of nitrite, D-NG-nitroarginine methyl ester, and a low molecular weight molecule containing a nitro group, or a protein, or a lipid.

5. 4. The method of claim 3, wherein the nitrate group-containing compound is one or more of a nitrate salt, or a low molecular weight molecule containing a nitrate group, or a protein, or a lipid.

6. 4. The method of claim 3, wherein the NO donor is a NONOate or a hydroxamic acid, such as deferoxamine, acetohydroxamic acid, and hydroxyurea.

7. 4. The method of claim 3, wherein the endogenous NO-generating amino acid is arginine or citrulline or a combination thereof.

8. 10. The method of claim 1, wherein the therapeutically effective amount of the nitric oxide compound ranges from about 1 mg to about 10 g.

9. 2. The method of claim 1, wherein the nitrovasodilator is nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, nitroprusside, amyl nitrite, S-nitrosothiol, heme-NO, or dinitrosyl iron complex.

10. 10. The method of claim 9, wherein the therapeutically effective amount of nitroglycerin is from about 0.1 mg to about 0.6 mg.

11. 10. The method of claim 9, wherein the therapeutically effective amount of isosorbide mononitrate is from about 10 mg to about 300 mg.

12. 2. The method of claim 1, wherein the nitric oxide compound is a nitrite compound.

13. 13. The method of claim 12, wherein the nitrovasodilator and the nitrite compound are administered to the subject in a ratio ranging from 1:1 to 1:3333.

14. 14. The method of claim 13, wherein the nitrovasodilator and the nitrite compound are administered to the subject as a fixed dose combination.

15. 2. The method of claim 1, wherein the nitric oxide compound is a nitrate compound.

16. 16. The method of claim 15, wherein the nitrovasodilator and the nitrate compound are administered to the subject in a ratio ranging from 1:1 to 1:33333.

17. 17. The method of claim 16, wherein the nitrovasodilator and the nitrate compound are administered to the subject as a fixed dose combination.

18. administering to the subject a therapeutically effective amount of a stimulator of soluble guanylyl cyclase. The method of claim 1 further comprising:

19. 19. The method of claim 18, wherein the stimulator of soluble guanylyl cyclase is riociguat or veruiciguat.

20. 10. The method of claim 1, further comprising the step of administering to the subject a therapeutically effective amount of an activator of soluble guanylyl cyclase.

21. 21. The method of claim 20, wherein the activator of soluble guanylyl cyclase is lancaciguat, cinaciguat, ataciguat, or kynurenine.

22. administering to the subject a therapeutically effective amount of a cGMP-phosphodiesterase inhibitor The method of claim 1 further comprising:

23. 23. The method of claim 22, wherein the cGMP-phosphodiesterase inhibitor is sildenafil, vardenafil, or tadalafil.

24. 10. The method of claim 1, wherein the nitrovasodilator is nitric oxide administered via inhalation.

25. 25. The method of claim 24, wherein nitric oxide is administered along with the nebulized thiol.

26. 26. The method of claim 25, wherein the thiol that is sprayed is L-cysteine ​​or glutathione.

27. 2. The method of claim 1, wherein the respiratory condition is apnea, and pulmonary hypertension, or chronic obstructive pulmonary disease.

28. 1. A method of treating a vascular condition in a subject, comprising: administering to the subject therapeutically effective amounts of a first nitrovasodilator in a first formulation for activating nitrovasodilator-activatable intracellular NO stores (NANOS) and a second nitrovasodilator in a second formulation for contributing to NANOS. The method includes:

29. 29. The method of claim 28, wherein the first nitrovasodilator is nitroglycerin and the first formulation is a sublingual tablet or a translingual spray.

30. 30. The method of claim 29, wherein the second nitrovasodilator is nitroglycerin and the second formulation is a nanoliposome.

31. 30. The method of claim 29, wherein the second nitrovasodilator is a dinitrosyl iron complex, S-nitrosoglutathione, or heme-NO, and the second formulation is a nanoliposome.

32. 1. A method of treating a vascular condition in a subject, comprising: administering to the subject a therapeutically effective amount of a nitric oxide compound and a cGMP-phosphodiesterase inhibitor. The method includes:

33. 33. The method of claim 32, wherein the nitric oxide compound is a nitrate group-containing compound or a nitrite compound.

34. 33. The method of claim 32, wherein the cGMP-phosphodiesterase inhibitor is sildenafil, vardenafil, or tadalafil.

35. 33. The method of claim 32, wherein the vascular condition is erectile dysfunction.

Images