WO2008153762A2 - S-nitrosothiol formulations and storage systems - Google Patents

Abstract

The invention provides stable S-nitrosothiol, such as S-nitrosoglutathione, formulations for long term storage of S-nitrosothiols. The invention provides stable formulations comprising S-nitrosothiol, such as S-nitrosoglutathione, and methods of treating patients in need of S-nitrosothiol, such as S-nitrosoglutathione, and/or nitric oxide treatment.

Description

S-NITROSOTHIOL FORMULATIONS AND STORAGE SYSTEMS

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 60/931,730, filed May 25, 2007, the contents of which are herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to compositions and formulations comprising stabilized S-nitrosoglutathione and methods of storing and using the same.

BACKGROUND OF THE INVENTION

Nitric oxide (NO) is a ubiquitous molecule that has several biological functions, including decreasing blood pressure and inhibiting platelet function. To deliver NO bioactivity under physiological conditions, NO must be stabilized because it is too reactive by itself to reach a desired treatment location within the body. Current delivery methods typically involve polymers and small molecules, such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosocysteine (CysNO) that release NO in the body. These methods are flawed, however, because they release NO rapidly under physiological conditions and/or have a very short shelf life. Such methods are not able to deliver sufficient quantities of NO to a desired location for extended periods of time or in a controlled manner. Naturally occurring NO donor S-nitrosothiols (SNOs), such as S-nitrosoglutathione (GSNO) and S- nitrosocysteine, are particularly unstable. While both of these endogenous primary SNOs are more stable than tertiary SNOs thermodynamically, they are highly unstable kinetically at ambient temperatures and above. These issues make alternate technologies for delivery of S- nitrosothiols attractive, in particular as related to identifying methods for stabilizing S- nitrosothiols prior to delivery to patients while still allowing for spontaneous production of NO bioactivity under physiological conditions. Of particular value is identification of methods for kinetic stabilization (i.e., protection from redox and other reactions) of SNOs. GSNO, a key endogenous source of NO bioactivity, has several biological functions that have generated clinical interest, particularly in cardiovascular and bronchopulmonary diseases and disorders. For example, GSNO has an inhibitory effect on platelet activation. GSNO also inhibits nuclear factor kappa-B (NF-κB) activation and smooth muscle cell benefit patients following balloon angioplasty, as well as patients with acute myocardial infarction and unstable angina. GSNO can reduce the rate of cerebral embolization and has also been shown to induce apoptosis in T cells. In addition to providing benefits related to the cardiovascular system, GSNO is a powerful bronchodilator. GSNO has been demonstrated in vitro and ex vivo to reverse the airway epithelial molecular defect in cystic fibrosis, increasing the expression and function of the ΔF508 cystic fibrosis transmembrane regulator on epithelial cell surfaces. Also, endogenous GSNO levels are increased in the airway of patients having pneumonia and reduced in patients having cystic fibrosis or severe asthma. While GSNO is an attractive compound for treating a variety of diseases, formulations of the compound itself are unstable, as described above, and GSNO suffers from significant, temperature dependent aqueous instability. Solution formulations typically decompose quantitatively in hours. Therefore, there is a need for stable compositions and formulations of GSNO that can be stored for an adequate time and that are useful for delivery to patients in need of GSNO treatment and delivery of NO bioactivity to tissues.

SUMMARY OF THE INVENTION

The invention provides novel formulations that stabilize S-nitrosothiols (SNOs), such as S-nitrosoglutathione (GSNO), and storage systems that enable the long term storage of such formulations. The invention thus provides an effective means for delivering SNOs to a patient in need thereof.

The present invention provides pharmaceutical formulations including, for example, iS-nitrosoglutathione or a derivative or salt thereof, in a pharmaceutically acceptable fluid, wherein the liquid formulation is stable during storage at temperatures up to about 37° C. The pH of the formulation is about 5.0 to about 8.0, about 5.5 to about 7.5 or about 6.0 to about 7.0.

The pharmaceutically acceptable fluid can include a buffer. The buffer can be a non- thiol buffer with minimal nucleophilicity. The concentration of the buffer can be about 0.005 M to about 2 M, about 0.05 M to about 2 M, about 0.01 M to about 1.5 M, about 0.1 M to about 1.5 M or about 0.5 M to about 1 M. The pharmaceutically acceptable fluid may also include a component, such as pharmaceutically acceptable salt, to add a specific ionic strength or "tonicity" to the fluid. Appropriate salts may be monovalent or polyvalent. Specific examples include, but are not limited to, sodium and/or potassium salts of chlorides, sulfates and phosphates. Such salts may be present from 0.0001 M to 1 M to control the ionic strength of the fluid for both modulating the stability of the pharmaceutical formulation and/or controlling the need to have a biological tonicity for effective physiological delivery and purpose. It is also anticipated that the pharmaceutically acceptable fluid may also include a plurality of buffers and salts. The appropriate combinations of these components are known to those skilled in the art.

The stability of aqueous formulations of SNO vary, depending on the temperature at which the solution is stored. At pH = 7.4, the rate of degradation of SNO in the aqueous formulations provided herein is approximately 0.25% loss/hr at 4° C (97% of the initial amount of SNO left at 12 hrs), 0.95% loss/r at 25° C (89% of the initial amount of SNO left at 12 hrs), and 2.7% loss/hr at 37° C (68% of the initial amount of SNO left at 12 hrs). Thus, the formulations of the present invention provide long term storage such that at least about 85% of the initial S-nitrosoglutathione is present after at least 8-12 hours following storage at 4° C to 25° C, and at least 6-8 hours following storage at 25° C to 37° C. The formulations can be stored under anaerobic conditions such that the formulation includes less than about 200 μM pθ₂, less than about 100 μM pθ₂ or less than about 50 μM pθ₂. The use of inert gases is also contemplated.

The present invention also provides pharmaceutical SNO formulations including, for example, S-nitrosoglutathione or a derivative or salt thereof, in a solid dosage form, wherein the formulation is stable during long term storage. For example, iS-nitrosoglutathione can be milled, sieved or micronized into particles of about 1.5 μm to about 6.0 μm. Larger particle sizes or amorphous solid are also contemplated. The S-nitrosoglutathione can be stored in a unit dosage of about 0.01 mg/ml to about 20 mg/ml, about 1 mg/ml to about 15 mg/ml or about 5 mg/ml to about 10 mg/ml. The milled unit dosage powder contains limited moisture content in the range of 0% to about 10% H₂O, preferably about 0% to about 2% H₂O, more preferably about 1% to about 1.5% H₂O, even more preferably about 0% to about 1% H₂O (or any specific percentage within these ranges).

The invention further provides storage systems for the long term storage of such solid SNO formulations, including GSNO. In one embodiment, the solid dosage units are stored in oxygen impermeable containers or overwraps, for example sealed aluminum bags. In a further embodiment, suitable dessicants and/or oxygen scavengers are also included as components of the storage system. In yet another embodiment, the components of the storage system can be treated with at least one inert gas, for example nitrogen, prior to sealing the oxygen impermeable container or overwrap. The storage system of the present invention can provide an extended shelf life to the solid formulation as measured by purity of an S-nitrosothiol, such as GSNO, or the amount of degradation products, such as glutathione, glutathione disulfides, nitrates or nitrites. For example, GSNO can have a purity greater than 95.0% as determined by HPLC, direct UV absorption or other methods know to those skilled in the art. The formulation preferably contains less than 5.0% reduced and oxidized L-glutathione, less than 2.5% glutathione, less than 2.5% glutathione disulfide and/or less than 2.5% NO₂ ^" and NO₃ ^". The storage systems provides long term storage of SNOs for more than about 1 month, 3 months, 6 months, 9 month, 12 months, and preferably more than about 24 months (or any time within these time periods) at a temperature between about -20° C to about 37° C, between about 5° C to about 25° C or any temperature within these ranges.

Unless otherwise defined, 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. Unless otherwise required by context, singular terms as used herein shall include pluralities and plural terms shall include the singular. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a scheme for the synthesis of GSNO.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and formulations that stabilize S- nitrosothiols (SNOs), such as S-nitrosoglutathione. The compositions and formulations enable long term storage and provide an effective means for delivering SNOs to a patient in need thereof. As used herein, the terms "S-nitrosothiols" or "SNOs" include, but are not limited to S-nitroso-beta-mercaptosuccinic acid, 1-S-nitrosothio-beta-D-galactopyranose, S- nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC), S-nitrosothioglycerol, S- nitroso-N-acetylpenicillamine (SNAP), S-nitrosohomocysteine, S-nitrosocysteine (CysNO), and S-nitrosocysteinylglycine. SNOs are commercially available or can be synthesized by those skilled in the art.

In an embodiment, the SNO is GSNO. GSNO can be synthesized according to the synthesis scheme shown in Figure 1 and by the methods described in Example 1. The GSNO synthesized by the recited methods and other methods known in the art provide GSNO in the form of a strong acid salt.

The present invention is based on the surprising discovery that the stability of SNOs in solution can be greatly increased by modulating pH, temperature and/or pθ₂. The formulations of the present invention increase the stability on SNOs in solution, which can extend in vitro shelf life and storage. More specifically, the present invention is based on the discovery that SNOs are greatly unstable at lower pH (acidic conditions) and slightly more unstable at higher temperatures and higher oxygen content. Further, the present invention is based on the discovery that the presence of various degradation products in SNO solutions decreases the stability of the SNO and induces further degradation which liberates protons thereby further increasing the acidic conditions in solution. The compositions and formulations of the present invention address these short comings and provide for a more stable, therapeutically effective, SNO formulation.

Aqueous Dosage Form Stability

The present invention provides a formulation comprising SNO or a derivative or salt thereof and a pharmaceutically acceptable fluid such that said formulations are stable for an extended shelf life and storage, as compared to prior art formulations. The formulations are suitable for administration to a subject in need thereof. Preferably, the formulations are aqueous formulations.

As used herein, a pharmaceutically acceptable fluid is a solvent suitable for pharmaceutical use which is not a liquified propellant gas. Exemplary pharmaceutically suitable fluids include polar fluids, including protic fluids such as water. As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.

The formulations of the present invention can comprise SNO or a derivative or salt thereof and a stabilizing buffer. A stabilizing buffer suitable for use with the compositions and formulations of the present invention include those non-thiol buffers displaying minimal nucleophilicity and providing a pKa between about 5 and about 8 at 25° C. The stabilizing buffer can be present in a concentration ranging from about 0.005 M to about 2 M, about 0.05 M to about 2 M, about 0.01 M to about 1.5 M, about 0.1 M to about 1.5 M, about 0.5 M to about 1 M (or any specific value within said range). Buffers suitable for stabilizing SNO include, but are not limited to, cacodylate, succinate (pk2), MES (2-(N-morpholino)ethanesulfonic acid), citrate (pk3), maleate (pk2), histidine, bis-tris, phosphate (pk2), ethanolamine, ADA (N-(2-acetamido)-2-iminodiacetic acid), carbonate (pkl), ACES (N-(carbamoylmethyl)-2-aminoethanesulfonaic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO (3-(N-morpholino)-2- hydroxypropanesulfonic acid), imidazole, BIS-TRIS (bis(2- hydroxyethyl)iminotris(hydroxyrnethyl)methane), BIS-TRIS PROPANE (1,3- bis(tris(hydroxymethyl)methylamino)propane), BES (N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonaic acid), MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (N-(2- hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2- aminoethanesulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid), DIPSO (3-(N,N- bis(2-hydroxyethyl)amino)-2-hydroxy-propanesulfonic acid), TAPSO (3-(N- tris(hydroxymethyl)methylamino)-2-hydroxy-propanesulfonic acid), TEA (triethanolamine), pyrophosphate, HEPPSO (N-(2-hydroxyethyl)piperazine-N'-(2 -hydroxypropanesulfonic acid), POPSO (piperazine-N,N'-bis(2-hydroxypropanesulfonic acid)), TRICINE (N- tris(hydroxymethyl)methylglycine), hydrazine, GLY-GLY (glycylglycine) (pk2), tris(hydroxymethylaminomethane), EPPS or HEPPS (N-(2-hydroxyethyl)piperazine-N'-(3- propane-sulfonic acid), BICINE (N,N-bis(2-hydroxyethyl)glycine), HEPBS (N-(2- hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)), TAPS (N-tris(hydroxy-methyl)methyl- 3-aminopropanesulfonic acid), or AMPD (2-amino-2-methyl-l,3-propanediol). More preferably, the buffer can be: cacodylate, MES, citrate, maleate, BIS-TRIS, phosphate, carbonate, BIS-TRIS propane, pyrophosphate, tricine, glycylglycine, Tris, EPPS or BICINE. Zwitterionic buffers or any other buffers displaying minimal nucleophilicity may also be utilized.

The formulations can be at a pH of about 5.0 to about 8.0, about 5.5 to about 7.5, about 6.0 to about 7.0 (or any specific value within said range). The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

Additionally, in embodiments where the SNO is GSNO and the GSNO is synthesized as an acid salt, the GSNO synthesis can include a salt neutralization step prior to the addition of the stabilizing buffer. Common salts resulting from GSNO neutralization known to those skilled in the art may include, but are not limited, to ions arising from Group Ia and Ha alkali earth metals and non redox active transition metal ions.

The storage temperature of the liquid formulations has been found to affect the stability of the composition. Specifically, liquid formulations stored at a lower temperature exhibit improved stability over liquid formulations stored at higher temperatures.

The liquid formulations provided herein remain stable for a relatively long period of time. For example, the compositions provided herein are stored between 25° C and 37° C, or between 4° C and 25° C (or any specific value within said ranges), and remain stable for the desired time. In one embodiment, the formulations are stored at ambient room temperature. Stabilized frozen solutions of GSNO that may be thawed prior to use are also anticipated.

The compositions and formulations of the present invention can be aerobic or anaerobic. Under aerobic conditions, the pH of the formulation can decrease as SNO is degraded. Specifically, as SNO degrades there is a liberation of protons and degradation products which increase the acidity of the formulation (decreased pH) and which can result in further SNO degradation. Thus in some embodiments, anaerobic conditions may be favored to maintain the pH of the formulations at less acidic pH (e.g., from about 5.0 to about 8.0). As such, the formulations of the present invention can be treated with one or more inert gases (e.g., nitrogen or argon gas) to decrease the oxygen content of the formulation. Preferably, the formulation comprises less than about 200 μM pθ₂, less than about 100 μM pθ₂ or less than about 50 μM pθ₂.

The estimated shelf-life of SNO in the aqueous formulations provided herein is significantly greater than that reported for known SNO formulations. The stability of aqueous formulations of SNO vary, depending on the temperature at which the solution is stored. The rate of degradation of SNO in the aqueous formulations provided herein is less than about 0.3% per hour to about 1 % per hour at 4° C to 25° C, and about 1 % per hour to about 3% per hour at 25° C to 37° C (or any specific value within said range). Preferably, the aqueous formulation is stable wherein at least 80%, preferably at least 85%, more preferably at least 90% even more preferably at least 95% of the initial amount of active ingredient, e.g., SNO or derivative or analog thereof, is present in the formulation as determined by HPLC or direct UV adsorption (A335nm). Thus, for example, an aqueous formulation of SNO that is stable would have greater than 50%, 80%, 85%, 90% , 95%, 96%, 97%, 98%, or 99% (or any specific value within said range) of the initial amount of active ingredient present in the composition approximately 8-12 hours following storage at 4⁰C to 25⁰C, and 50%, 80%, 85%, 90% or 95% (or any specific value within said range) of the initial amount of active ingredient present in the composition approximately 6-8 hours following storage at 25° C to 37° C.

The formulations of the present invention also permit at least two freeze thaw cycles. The rate of degradation of SNO in the aqueous formulations provided herein is less than about 2-5% per freeze thaw cycle. As such, for example, following two freeze thaw cycles, about 90-95% SNO remains in the formulation. The aqueous formulations of the present invention permit as many freeze thaw cycles possible as long as at least 50% of the SNO remains in the formulation. The ionic strength of the formulations provided herein also has been found herein to affect the stability of the SNO. Ionic strengths of the formulations provided herein are from about 0 M to about 0.4 M, or from about 0.05 M to about 0.16 M (or any specific value within said ranges). The particular ionic strength of a given composition for long term storage provided herein may be determined empirically using standard stability assays well known to those of skill in the art.

In embodiments where the pharmaceutically suitable fluid is a saline solution, tonicity adjusting agents may be added to provide the desired ionic strength. Tonicity adjusting agents for use herein include those which display no or only negligible pharmacological activity after administration. Both inorganic and organic tonicity adjusting agents may be used in the compositions provided herein. Tonicity adjusting agents include, but are not limited to, ammonium carbonate, ammonium chloride, ammonium lactate, ammonium nitrate, ammonium phosphate, ammonium sulfate, ascorbic acid, bismuth sodium tartrate, boric acid, calcium chloride, calcium disodium edetate, calcium gluconate, calcium lactate, citric acid, dextrose, diethanolamine, dimethylsulfoxide, edetate disodium, edetate trisodium monohydrate, fluorescein sodium, fructose, galactose, glycerin, lactic acid, lactose, magnesium chloride, magnesium sulfate, mannitol, polyethylene glycol, potassium acetate, potassium chlorate, potassium chloride, potassium iodide, potassium nitrate, potassium phosphate, potassium sulfate, propylene glycol, silver nitrate, sodium acetate, sodium bicarbonate, sodium biphosphate, sodium bisulfite, sodium borate, sodium bromide, sodium cacodylate, sodium carbonate, sodium chloride, sodium citrate, sodium iodide, sodium lactate, sodium metabisulfite, sodium nitrate, sodium nitrite, sodium phosphate, sodium propionate, sodium succinate, sodium sulfate, sodium sulfite, sodium tartrate, sodium thiosulfate, sorbitol, sucrose, tartaric acid, triethanolamine, urea, urethan, uridine and zinc sulfate. Such agents may be present from 0.0001 M to 1 M (or any specific value within said range). In certain embodiments, the tonicity adjusting agent is sodium chloride, which is present at a concentration of from about 0 mg/mL to about 10, 15 or 20 mg/mL. In further embodiments, the compositions contain sodium chloride at a concentration of from about 0 mg/mL to about 7.5 mg/mL. In these embodiments, the pharmacologically suitable fluid is aqueous saline. For certain therapeutic treatments such as asthma or cystic fibrosis, an isotonic or hypertonic formulation is preferred.

The amount of SNO present in the stabilized formulations of the present invention can range from about 0.01 mg/ml to about 20 mg/ml, about 1 mg/ml to about 15 mg/ml or about 5 mg/ml to about 10 mg/ml (or any specific value within said range). The formulations can include a pharmaceutically acceptable derivative or salt of SNO. Pharmaceutically acceptable salts include, but are not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. As used herein, pharmaceutically acceptable derivatives of a compound include salts, esters, enol ethers, enol esters, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives may be readily prepared by those of skill in this art using known methods. The compounds produced may be administered to animals or humans without substantial toxic effects and are either pharmaceutically active or are prodrugs. Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N'- dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N- benzylphenethylamine, 1 -para-chloroben2yl-2-pyrrolidin- 1 '-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also include, but are not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids. Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of formula C=C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of formula C=C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl. Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecule, preferably 1 to about 100, more preferably 1 to about 10, most preferably one to about 2, 3 or 4, solvent or water molecules.

Solid Dosage Form Stability The present invention provides solid dosage forms of a pharmaceutical composition comprising SNO, including GSNO, such that said solid dosage forms are stable for an extended shelf life and storage. Preferably, the solid dosage form is a powder formulation. It has been found that moisture content plays a key role in the stability of solid dosage forms of SNO. For long term storage, the moisture content of solid dosage forms of SNO must be limited to avoid degradation. As such, the present invention provides powder formulations of a pharmaceutical composition comprising SNO wherein the moisture content of said powder formulations is about 0% to about 10% H₂O, preferably about 0% to about 2% H₂O, more preferably about 1% to about 1.5% H₂O, even more preferably about 0% to about 1% H₂O (or any specific percentage within these ranges). In one embodiment, the powder formulation of SNO comprises lyophilization and treatment with at least one inert gas, including but not limited to nitrogen, argon, helium, neon, krypton, and xenon.

Because SNO is sensitive to moisture and can be unstable in an aqueous environment, said solid dosage forms must be reconstituted in an appropriate aqueous solvent for administration to a patient in need thereof. Reconstitution of solid dosage forms of SNO and aqueous formulations thereof are described below.

Particles

The present invention provides compositions and formulations in which the SNO is processed prior to inclusion in the compositions or formulations, in order to produce particles in the desired size range. For example, the SNO can be milled or micronized using suitable equipment for example an air jet mill, hammer mill, ball mill or using a microfluidizer. Alternatively, particles in the desired particle range may be obtained by, for example, spray drying or controlled crystallization methods, for example, crystallization using supercritical fluids or via an emulsion method, such as microfiuidization or homogenization. When the compositions or formulations of the invention are delivered to a subject or patient in need there of, so as to produce a pharmacodynamic effect, the SNO particles can be about 0.5 μm to about 10 μm, about 1 μm to about 8 μm, or about 1 μm to about 5 μm (or any specific value within said range). In an embodiment, an SNO particle in a composition or formulation of the invention is about 1.5 μm to about 6 μm (or any specific value within said range). For some compositions and formulations about 90% of SNO particles in an SNO stabilizing formulation of the invention are less than about 6 μm, and about 50% are less than about 3 μm. In an embodiment, the SNO is GSNO.

The surfaces of the particles can also be modified prior to dispersion, for example, by spray drying a solution of drug and surfactant or by adsorption of surfactant onto SNO particles. Further techniques for modification of the surfaces of the particles can also be used, for example freeze drying, microfluidizing, and milling.

Storage Systems The invention further provides storage systems for the long term storage of solid forms of SNOs. The components of the storage systems include solid particles of SNOs packaged in oxygen impermeable containers. The solid form of SNOs in suitable particle sizes, including GSNO in powder form, can be packaged in convenient sizes, particularly unit dosage sizes as described in more detail below. Those skilled in the art can readily determine appropriate dosage sizes depending on the intended use and mode of administration.

Powdered GSNO can be placed in appropriate sterile containers by filling methods known to those skilled in the art or as described in the Examples below. Such containers can be vials, rubes, syringes, ampules and the like for holding powdered formulations, made of any inert material, such as plastics, polymers, glass and the like. The storage systems preferably include other components to aid in the long term stability of GSNO, including dessicants to maintain low moisture and oxygen scavengers. Suitable dessicants and oxygen scavengers for use in the storage systems are well known to those skilled in the art.

Because SNOs, and particularly GSNO, are light sensitive, the components of the storage system can be stored in suitable overwraps or containers that are impermeable to oxygen and light, for example aluminum pouches. Those skilled in the art can readily determine suitable containers, overwraps and the like for storing SNOs and other components useful for maintaining the stability of SNOs. Prior to sealing, the storage system is preferably treated with at least one inert gas, for example, nitrogen, argon, helium, neon, krypton and xenon, to reduce the oxygen content within the system. The storage system further stabilizes the solid formulations by retaining the purity level of SNOs over an extended period of time and at higher temperatures than previously regarded as acceptable.

Impurities

The present invention provides compositions and formulations which contain limited impurities. The compounds and formulations of the present invention have a purity greater than or equal to about 95.0% as determined by known methods in the art, for example, HPLC or direct UV adsorption (A335nm). In an embodiment, the compounds and formulations of the present invention have a purity ranging from about 95.0% to about 100% (or any specific value within said range) at initial synthesis.

In order to elicit the maximum pharmacodynamic and therapeutic effect of the compositions and formulations of the present invention, it is beneficial to limit the levels of reduced and oxidized L-glutathione impurities. These impurities can result in undesirable stability. The compounds and formulations of the present invention contain less than about 5.0% reduced and oxidized L-glutathione. In an embodiment, the compounds and formulations of the present invention contain reduced and oxidized L-glutathione in a range from about 0.0% to about 5.0% (or any specific value within said range). It is also beneficial to limit the levels of glutathione (GSH) and glutathione disulfide (GSSG) present in the compositions and formulations. Thus, the compounds and formulations of the present invention contain less than about 2.0% - 2.5% glutathione and less than about 2.0% - 2.5% glutathione disulfide. In an embodiment, the compounds and formulations of the present invention contain glutathione and glutathione disulfide in a range from about 0.0% to about 2.5% (or any specific value within said range), respectively. It is also beneficial to limit the amount of nitrogen oxide products (NO_x) present within the composition or formulation.

Thus, the compounds and formulations of the present invention contain less than about 2.5% nitrite (NO₂ ^") and less than about 2.5% nitrate (NO₃ ^"). In an embodiment, the compounds and formulations of the present invention contain NO₂ ^" and NO₃ ^" in a range from about 0.0% to about 2.5%, each (or any specific value within said range). The limited purity content of the compounds and formulations of the present invention achieved when synthesized and stored according to the methods and storage systems described herein enhances their stability. Storage time and/or variations in temperature have a minimal effect on the purity content of the compounds and formulations described herein. In an embodiment, the compounds and formulations of the present invention have a purity ranging from about 95.0% to about 100% (or any specific value within said range), preferably at least about 96%, more preferably at least about 97%, and even more preferably at least about 98% or 99% , over an extended period of time up to at least one month, three months, six months, preferably up to at least nine months, more preferably up to at least 12 months, even more preferably up to at least 24 months. In still another embodiment, the compositions and formulations of the present invention have a purity ranging from about 95.0% to about 100% (or any specific value within said range) over a temperature range of about -20° C to about 37° C, and preferably about 5° C to about 25° C (or any temperature within these ranges). In still another embodiment, the compounds and formulations of the present invention have a purity ranging from about 95.0% to about 100% (or any specific value within said range) over a temperature range of about -20° C to about 37° C and over an extended period of time up to at least one month, three months, six months, more preferably up to at least nine months, even more preferably up to at least 12 months, and even more preferably up to at least 24 months.

Pharmaceutical Compositions/Formulations

A pharmaceutical composition is a formulation containing the disclosed compounds in a form suitable for administration to a subject. A pharmaceutical composition of the invention is preferably formulated to be compatible with its intended route of administration. Examples of routes of administration include oral and parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, and rectal administration.

Solutions or suspensions can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The formulation of the present invention can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. As the compositions and formulations of the present invention are light sensitive, a light-blocking storage means. However, preferably the storage means should contain limited amounts of transition metals which can further accelerate the decomposition of SNO. Preferably, the storage means can be a clear plastic vial or clear glass ampoule, syringe or vial with a light-blocking over wrap. The present invention also provides pharmaceutical formulations comprising SNO in combination with at least one pharmaceutically acceptable excipient or carrier. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in "Remington: The Science and Practice of Pharmacy, Twentieth Edition," Lippincott Williams & Wilkins, Philadelphia, PA., which is incorporated herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Pharmaceutically acceptable carriers can also include solid carriers such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary liquid carriers include syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time-delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like. Other fillers, excipients, flavorants, and other additives such as are known in the art may also be included in a pharmaceutical composition according to this invention. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active reagent (e.g., polypeptide, peptide, antibody, or antibody fragment) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active reagents are formulated into ointments, salves, gels, or creams as generally known in the art. The reagents can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

The active reagents can be prepared with carriers that will protect against rapid elimination from the body. For example, a controlled release formulation can be used, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. The present invention provides a formulation comprising SNO and a stabilizing buffer and can further comprise a surfactant. The amount of surfactant that can be present in an SNO stabilizing formulation of the invention can range from about 0.1% w/w to about 10% w/w (or any specific value within said range) with respect to the SNO. In an embodiment, the amount of surfactant present is at least 1% w/w with respect to the SNO. In an embodiment, the amount of surfactant present is up to about 5% w/w with respect to the SNO.

Examples of suitable surfactants include, but are not limited to, fatty acid, fatty acid esters including fatty acid trigylcerides, fatty alcohols, salts of fatty acids, oleyl alcohol, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan mono-oleate, natural lecithin, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of oxyethylene and oxypropylene, oleic acid, salts of oleic acid, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, isopropyl palmitate, glyceryl mono-oleate, glyceryl monostearate, glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, cetyl pyridinium chloride, olive oil, glyceryl monolaurate, corn oil, cotton seed oil, sunflower seed oil, polyoxyethylenesorbitan monooleate, sorbitan trioleate, oligolactic acid, lecithin, (poly)alkoxy derivatives including polyalkoxy alcohols, in particular 2-(2-ethoxyethoxy) ethanol. Additional (poly)alkoxy derivatives include polyoxyalkyl ethers and esters, such as polyoxyethylene ethers and esters, including, but not limited to, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters and polyoxyethylene stearates.

A composition or formulation of the invention can optionally comprise additional ingredients, such as additives that serve as preservatives, antioxidants, radical quenchers, sweeteners, taste masking agents, pharmaceutically active agents, adjuvants, carriers, chemical stabilizers, and/or polymers. The amount of additional ingredients included in a formulation of the invention can be, for example, 0% to about 1% w/w (or any specific value within said range). The compositions and formulations of the instant invention can also comprise one or more desiccants. Suitable desiccants that can be used in the present invention are those that are pharmaceutically safe, and include, for example,, pharmaceutical grades of silica gel, crystalline sodium, potassium or calcium aluminosilicate, colloidal silica, anhydrous calcium sulphate and the like. The desiccant may be present in an amount from about 1.0% w/w to 20.0% w/w, or from about 2% w/w to 15% w/w (or any specific value within said range).

It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active reagent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active reagent and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active agent for the treatment of individuals. When the SNO comprised within the formulation is GSNO, the formulation comprises about 0.01 to about 20 mg/ml, about 1 mg/ml to about 15 mg/ml or about 5 mg/ml to about 10 mg/ml (or any specific value within said range).

Disorders

The present invention also provides methods of treating a subject afflicted with a disorder ameliorated by NO donor therapy (i.e., conditions or disorders where SNO treatment is desirable) where the method comprises administering to the subject a therapeutically effective amount of the compositions and formulations as defined above, or a pharmaceutically acceptable salt thereof, or a prodrug or metabolite thereof, in combination with a pharmaceutically acceptable carrier. The subject can be any mammal, e.g., a human, a primate, mouse, rat, dog, cat, cow, horse, pig. For example, the mammal is a human. The terms "subject" and "patient" are used interchangeably herein.

As used herein the term "therapeutically effective amount" means the amount necessary to alleviate at least one symptom of a disorder to be treated as described herein. In an embodiment, the therapeutically effective amount is any amount of SNO delivered to produce a pharmacodynamic effect.

As used herein, "treating" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition or disorder. More specifically, treating includes reversing, attenuating, alleviating, minimizing, suppressing or halting at least one deleterious symptom or effect of a disease (disorder) state, disease progression, disease causative agent (e.g., bacteria or viruses), or other abnormal condition. Treatment is continued as long as symptoms and/or pathology ameliorate. The disease, conditions or disorders can include, but are not limited to, cystic fibrosis, asthma, COPD and other pulmonary disorders involving diminished gas exchange or inflammation such as pulmonary fibrosis, and pneumonia; cardiovascular proliferative, inflammatory, contractile and hypertensive disorders, including hypertension, atherosclerosis, restenosis, ischemia and heart failure; preconditioning related disorders of the heart and brain; motility and smooth muscle disorders of the GI tract, including esophageal spasm, biliary spasm, and colic; erectile dysfunction stroke; infectious disease (viral, bacterial and other); and disorders of red blood cells characterized by SNO deficiency, abnormal rheology or impaired vasodilation, such as sickle cell disease and stored blood-related diathesis, and thrombotic disorders. The following examples, including the experiments conducted and results achieved are provided for illustrative purposes only and are not to be construed as limiting the invention.

EXAMPLES Example 1. GSNO Synthesis When synthesizing GSNO, it is important to note that GSNO is air, UV-light, and heat sensitive. Thus additional measures are recommended to protect against unwanted exposure to such elements, e.g. , de-gassing all solvents (e.g. , a nitrogen sparge) prior to use, covering reaction flasks with foil to reduce light exposure, and storage of wet cakes and products at -20° C. In the absence of explicitly stated conditions, the following implied tolerance ranges apply: Reagent quantities +/- 2%, solvent quantities greater than IL +/-10%, solvent quantities less than IL +/- 50%, temperature +/- 10° C, and sampling time intervals +/- 60 min. Pre-Reaction Preparation:

In a clean, dry container, weigh out and charge 1 kg of USP water and 0.229 kg of sodium nitrite and mix until dissolved to create an aqueous sodium nitrite solution. Sparge the aqueous sodium nitrite solution for at least one hour. In a separate container, de-gas USP water for a minimum sparge time of 1 hour, or until needed. Reaction:

In a 50 L reaction flask, weigh out and charge 5.50 kg of the de-gassed USP water and begin stirring, taking caution to minimize splash on the walls of the reaction flask. Using nitrogen, sparge the water in the reaction flask vigorously for a minimum sparge time of 30 minutes. Next, weigh and charge 1.0 kg of reduced L-Glutathione into the 50 L reaction flask, creating a thin suspension. Measure and slowly charge 322 g of concentrated hydrochloric acid to the 5OL reactor while maintaining a solution temperature of 25° C or less during the addition of the hydrochloric acid. If necessary, place the reaction flask on ice to regulate the temperature of the solution.

Once the hydrochloric acid has been added, cool the reaction mixture to 0° C, and vigorously sparge with nitrogen as the solution cools (minimum sparge time of 1 hour). Once the solution has cooled, measure and charge all of the de-gassed aqueous sodium nitrite solution into the 50L reactor flask as quickly as possible, while maintaining a solution temperature of less than 2° C during the addition of the aqueous sodium nitrite. Once the aqueous sodium nitrite has been added, allow the mixture to stir for at least 2 hours at 0° C to -2° C while maintaining a positive flow of nitrogen to the flask. Monitor the progress of the reaction by HPLC. When the amount of starting material remaining is less than 1% by HPLC TAN, the reaction is complete. Once the reaction is complete, continue stirring the solution to avoid precipitation.

Next, using a filter flask fitted with a 1 micron filter and a nitrogen tent/bag to support a steady flow of nitrogen to the funnel, filter the reaction mixture (slurry) from the 5OL reactor flask via a nitrogen push to the filter. Collect the product on the filter (reaction filtrate) and store at 4° C. Transfer the wet solids collected in the funnel to a 5OL reaction flask, maintaining a stream of nitrogen in the reaction flask at all times. To wash the wet solids collected in the funnel, measure and charge 3.75 L of acetone into the 5OL reaction flask and start stir, taking caution to minimize splash on the walls of the reaction flask. Cool the solution below 5° C and stir for at least 15 minutes once the solution has reached below 5° C, making sure all solids are well suspended. Filter this solution, as previously described, and collect the product on the filter (wash filtrate #1). To ensure all solids from the 5OL reaction flask are removed and washed, rinse the reaction flask using 0.75 L of acetone and 0.75 L of water. Filter this rinse using the filter funnel as previously described, and collect the product collected on the filter (rinse filtrate). Wash the wet solids on the funnel using 2.5 L of acetone. Transfer the wet solids from the filter funnel to the 50L reaction flask while maintaining a stream of nitrogen in the flask at all times, and repeat the entire wash process. Drying and Processing:

Once the second wash process is complete, remove the wet solids from the filter funnel, place in a clean glass tray(s), and measure the net weight of the wet solids. Place the glass tray(s) in a vacuum oven with a house nitrogen sweep to dry, and dry at maximum pump speed for at least 15 minutes (no heat). Periodically weigh the glass trays, but not more frequently than every two hours, to monitor whether the solids are completely dried. When < 1% weight change of the product is measured between at least two weighings, the batch is considered dry and ready for micronization and packaging.

Example 2. Micronization and Packaging of GSNO

The GSNO solids synthesized according to the methods described in Example 1 are then micronized under N₂ gas into particle sizes of about 10 microns or less to form GSNO powder following standard protocols. The GSNO powder is ready for packaging into vials according to the following procedure.

Packaging components (e.g., depyrogenated glass vials, stoppers and seals) are sterilized via an autoclave prior to the filling process and protected with depyrogenated foil. The desired amount of GSNO powder is weighed into depyrogenated glass beakers (e.g., 400 mL beakers) and is kept protected from light and moisture using depyrogenated foil. The powder filler is calibrated to dispense at 100 + 5 mg GSNO powder per vial. The GNSO powder is then filled into the vials (10 mL, USP Type 1, glass, 20 mm mouth, depyrogenated) and an autoclaved 20 mm stopper is placed to the first stop in each filled vial upon addition.

The partially closed vials are then placed on a depyrogenated lyophilization tray, completely covered with depyrogenated foil and transferred to the lyophilization chamber for application of the nitrogen overlay and final sealing. The vacuum controller on the lyophilization chamber is set to 100 microns (100 m Torr). The following procedure is repeated three times: (a) the vacuum valve is opened; (b) the hand wheel on the lyophilizer is loosened and the vacuum break valve is closed; (c) the vacuum is slowly released using filtered N₂ gas through a 0.2 μm sterile filter; (d) the vacuum break valve is opened; and (e) the vacuum valve is closed between 10-20 psi. The vacuum is broken to atmospheric pressure (about 2 psi) and then closed. The vials are stoppered under vacuum and the vacuum is turned off. The lyophilization chamber is opened and the vials are unloaded.

Next, an autoclaved flip-off-tear-off aluminum (20 mm OD) seal is applied to each stoppered vial and crimped using a vial crimper. The vials are then stored between 2° C - 8° C. Dry vials are brought to room temperature prior to labeling. After labels are affixed, the vials are stored between -10° C to -25° C until ready for packaging.

A silica gel dessicant packet (MiniPax^®, Multisorb Technologies, Inc.), an O₂ scavenger packet (StabilOx^®, DF-100-H60, Multisorb Technologies, Inc.) and a labeled, filled vial (upright) are placed in a ziplock foil pouch. Each pouch is purged with N₂ for five (5) seconds and sealed with a heat sealer.

Example 3; Stability of GSNO

A number of packaged vials in Ziplock foil pouches described in Example 2 were then subjected to stability testing. The amounts of moisture, GSNO and GSNO degradation products (e.g., NO₂ ^', NO₃ ^", and glutathione disulfide (GSSG)) were measured at -20° C, 5° C and 25° C at the following time points: 0 (initial stability), 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, and 9 months.

Moisture Content in GSNO The following Karl Fischer method was used to determine the water content in the packaged GSNO.

Apparatus, Material, and Reagents:

Karl Fischer apparati suitable for coulometric titrations and volumetric titrations;

Analytical balance capable of weighing to 0.1 mg; Appropriate Karl Fischer vessel solution (commercially available); Deionized (DI) water.

Preparation of Calibration and Check Standards:

At least one DI water check was made on the coulometric apparatus each analysis day by using a syringe to inject 2 μL of water into the vessel solution to ensure the titrated value was 2000 μg ± 10% (1800-2200 μg) each time. For a volumetric Karl Fischer apparatus, approximately 20 to 30 μL of DI water was injected into the vessel solution. The weight of the injected water was entered to the nearest 0.0001 g into the instrument or computer software. The titrant's water equivalence (mg H20/mL titrant) was automatically calculated by the instrument. A duplicate DI sample was injected to ensure that the water equivalents agreed within 5%. The average equivalent was into the instrument or computer software as a common variable. System Suitability:

The calibration factor for the volumetric Karl Fischer titrant was RSD < 5% for all calibration trials. Procedure:

Calibration of the analytical balance was checked on the day of the analysis, and te Karl Fischer instrument was set up, as previously described. Samples were either directly added to the sample to the vessel solution or dissolved, diluted, or extracted in an appropriate solvent which was then injected. If a solvent was used, a blank was also analyzed. For a coulometric moisturemeter, the sample weight was recorded to at least 0.0001 g, the result from the instrument's digital display was recorded, and/or the printout was affixed when the sample completed titrating. Sample weights were optimally chosen such that > 50 μg H2O was titrated.

For the volumetric autotitrator, at least 0.0001 g of the sample weight was entered into the instrument or computer software. The water content in the sample was then automatically titrated and calculated by the instrument. A sample weight was optimally chosen such that > 2 mg of water was titrated. Calculations and Reporting

The sample was analyzed duplicates. Samples were calculated in the following manner:

For the coulometric moisturemeter:

μg HbO = Ug H?O (from instrument display) g sample weight in g

To calculate wt % water (if applicable): Wt % H₂O = Ug H₂O : 10.000 ÷ 10,000 g

For the volumetric autotitrator, the wt % water was calculated by the instrument according to the following general equations: ppm water = WE x mL titrant x 1000 μg/lmg Sample Weight in g

Where: WE = water equivalence in mg H₂OAnL titrant Wt % Water = ppm Water ÷ 10,000 Nitrite (NO₇ ^') and Nitrate (NCV) content in GSNO

The following ion chromatography procedure used to determine NO₂ ^" and NO₃ ^" content in the packaged GSNO (down to 0.01 wt % NO₂V 0.05 wt % NO₃ ^"). The separation was performed at 30° C using an AS22 column (Dionex) and an isocratic mobile phase of 4.5 mM Na₂CO₃/ 1.4 mM NaHCO₃. Quantitation of nitrite and nitrate was performed by external standard calibration using a four-point calibration plot using standards prepared from stock solutions of nitrite and nitrate. A diluent of nitrogen-purged DI water was used. Apparatus:

Ion chromatography (IC) system; Chromatography data system; Dionex IonPac AS22, 4 x 250 mm column or equivalent; AG22 Guard, or equivalent; ASRS-ULTRA II suppressor, or other appropriate Dionex suppressor; Analytical balance capable of weighing to 0.0001 g; Volumetric flasks: 50 mL, 100 mL, 500 mL, and 2 L; Auto pipettes, 0.1-10 mL; Standard laboratory glassware {e.g., sample bottles, scintillation vials, pipets, autosampler vials, etc.). Materials and Reagents:

100 μg/mL nitrite (CPI, cat. #4400-IC4 or equivalent); 100 μg/mL nitrate (CPI, cat. #4400-IC3 or equivalent); Sodium carbonate (Fisher certified, or equivalent); Sodium bicarbonate (Fisher certified, or equivalent); Water (house deionized); Methanol (Fisher HPLC grade, or equivalent; House nitrogen. Preparation of Mobile Phase:

0.45 MNa₂CO₃ZO.14 MNaHCO₃ (JOOx eluent): 23.85 g Na₂CO₃ and 5.88 g NaHCO₃ was weighed (to within ± 0.05 g) into a small beaker or disposable cup then quantitatively transfer to a 500 mL volumetric which was partially filled with water and mixed well for at least one hour. The mixture was then diluted to volume with water, mixed by inversion and transferred to a sample bottle (note: this solution is stable for approximately three months). 4.5 TnMNa₂CO₃ZlA mM NaHCO₃: 20.0 mL of 10Ox eluent was transferred to a 2 L volumetric flask, diluted to mark with DI water, mixed well, vacuum degassed, then transferred to the IC reservoir. Preparation of Autosampler Flush (5% MeOH)

Using a graduated cylinder, 25 mL methanol and 475 mL DI water were each measured then poured into the autosampler flush reservoir and mixed well. Preparation of Calibration and Check Standards:

Working Calibration Standards: Aliquots of 100 μg/mL nitrite (NO₂ ^") and 100 μg/mL nitrate (NO₃ ^") stock solution were pipetted into separate volumetric flasks according to the following Tables 1 and 2, respectively, then diluted to volume with DI H₂O that was been nitrogen-purged for at least an hour, and mixed by inversion.

Table 1:

Table 2:

Note: Calibration standards may be prepared by using proportional equivalents to the stated amounts.

Preparation of Samples:

50 ± 5 mg of sample was weighed into a dry 50-mL volumetric flask in duplicate using a glove box, then dissolved and diluted to mark with DI water that was nitrogen purged for at least an hour. The solution was gently mixed by repeated inversion to dissolve sample. Due to GSNO instability in solution, the preparation was injected within 10 minutes of DI water addition. System Suitability:

A diluent blank (nitrogen-purged DI H2O) was injected in duplicate to ensure no peaks were present at the retention time of nitrite or nitrate. Peaks with an area response that was < 2% than that of the 5 μg/mL standard were acceptable if other system suitability criteria were met. If a significant impact existed, new mobile phase was prepared or the column was changed. All analyte peaks were on-scale to assure proper integration and area percentage calculations. For the calibration standards, concentration versus the peak area response was plotted and linear regression was performed. The coefficient of determination (R²) was 0.995 for nitrite and nitrate. Standards were checked to verify recovery of 90-110%. Procedure

The ion chromatographic system was set up follows: Column Temp: 30 ± 1° C

IC Column: Dionex Ionpac AS22

Guard Column: Dionex Ionpac AG22 Flow Rate: 1.2 mL/min Injection Volume: 20 μL Run Time: 9.5 minutes Detection Mode: Conductivity, 10 μS range Suppression: AutoSuppression with ASRS-ULTRA, 50 mA Mobile Phase: 4.5 mM Na₂CO₃/! .4 mM NaHCO₃

The estimated retention times were 5.2-6.2 minutes for nitrite and 7.5-8.5 minutes for nitrate.

The column with the mobile phase was equilibrated a minimum of one hour prior to commencing analysis. The autosampler and pump were flushed with the appropriate solutions on a daily basis prior to analysis. After use, the column was washed with eluent. (note: the mobile phase was recommended for long term storage of the column).

The data system and load autosampler were set up using the following example format (Table 3) for the sequence of injections: Table 3:

The chromatograms were reviewed for each injection of standard or sample: Full scale: X scale range = 0 minutes to 9.5 minutes; Y scale range was set to review entire nitrite or nitrate peak Calculations and Reporting:

Nitrite/Nitrate Content of Sample: The nitrite/nitrate content for each sample injection was determined using the appropriate calibration plot. Nitrite (or Nitrate) content (wt %) = fPA_NO -,T x DFSmp

I CF

WSmp X 10000

Where:

PANO = Peak area of nitrite/nitrate in sample solution W_Smp= Weight of sample (g)

DFS_mp = Dilution volume for sample (50 mL, times any subsequent dilution factor, as needed)

CF = Calibration factor of analyte or slope [Peak Area / Concentration (μg/mL)] I = Intercept from calibration plot

If average of duplicate sample preparations was above the minimum quantitation level, the two values were averaged to obtain the test result. The nitrite and nitrate content corrected for moisture and ash (determined as part of release testing) were determined as follows:

100- [water content (% w/w) + ash content (% w/w)] e.g. 0.25% x l00 = 0.26% 100-(3.0+0.1) The results for both nitrite and nitrate were reported to two decimal places (Note: If duplicate sample preparations revealed analyte content that was below the low standard when averaged, then the analyte content was reported as "< 0.01 wt % NO₂ ^"" and/or "< 0.05 wt % NO₃ ".

GSNO Identity. Assay and Related Substances by HPLC The following HPLC reversed-phase procedure was used to determine the purity/assay and impurity profile of GSNO. The separation was performed at 25 ⁰C using a Cl 8 column (Waters Atlantis dC18) and a binary mobile phase of water w/0.075% TFA and water: acetonitrile (80:20) w/0.072% TFA. The method began with an isocratic step for 25 minutes. The gradient program linearly increased the amount of organic from 0% (initial) to 25% (final) over 10 minutes. The pump was then held at 25% organic for 5 minutes prior to returning to the initial isocratic conditions. Detection for this method was by UV at 210 nm. Quantitation of GSNO and GSSG (a GSNO degradant) were performed via external standard calibration (down to 0.05% w/w and 0.1 w/w % impurities, based on GSNO response, respectively; down to 0.1% w/w LOD for this GSSG). Care was taken to minimize exposure to the elements (e.g. , careful sparging of diluent and mobile phase, limiting the amount of time that the samples were held at room temperature during preparation of the standards and samples, and covering of all samples with aluminum foil to protect from light). Once diluted in the acidic diluent, samples were relatively stable (48 hours) provided they were kept cold (5° C) and dark (no amber glass; cover with foil). Apparatus:

High performance liquid chromatography (HPLC) system; Chromatography data system; Waters Atlantis dC18, 150 x 3.0 mm, 3 lam, HPLC column or equivalent; Security Guard with Cl 8 Cartridge, or suitable alternate; Analytical balance capable of weighing to

0.00001 g; Volumetric flasks, 100 mL, 10 mL, and 50 mL; Standard laboratory glassware

(e.g., sample bottles, autosampler vials, etc.); pH meter.

Materials and Reagents: GSNO reference standard (lot #52698-9-4, Ricerca CACCI #CS 06143 or equivalent);

GSSG standard (Sigma-aldrich/Fluka lot #1245475 or suitable alternate); Acetonitrile (HPLC grade or suitable alternate); Water (HPLC grade or suitable alternate); Trifluoroacetic acid

(TFA) (HPLC grade, or suitable alternate).

Preparation of Mobile Phase: Aqueous Mobile Phase, 0.075% TFA in Water: 1 L of water was measured by graduated cylinder and added to a mobile phase reservoir. 0.75 mL of TFA was then added and the solution was stirred well to ensure thorough mixing.

Organic Mobile Phase: 0.072% TFA in Water.ACN (80:20): 800 mL of water and

200 mL of acetonitrile were separately measured using a graduated cylinder. Each solution was poured into mobile phase reservoir B. 0.72 mL of TFA was added and the solution was stirred well to ensure thorough mixing.

Preparation of the Diluent

0.075% TFA in water: Using a graduated cylinder, 500 mL of water was measured and pour into a sample bottle, then sparged with helium (or nitrogen) for 20 - 30 minutes to degas. 0.375 mL of TFA was then added and the solution was gently mixed then sparged with helium (or nitrogen) for an additional 5 - 10 minutes prior to use.

Preparation of Autosampler Flush

200 mL of water and 200 mL of acetonitrile were separately measured using a graduated cylinder, poured into an Erlenmeyer flask and mixed well. Preparation and Calibration of Check Standards:

GSNO Standard (0.22 mg/mL in diluent): Standards were prepared in duplicate at a target concentration of 0.22 mg/mL in a 100 mL volumetric flask by accurately weighing 22 mg (±4 mg) of GSNO reference standard. Each flask was partially filled with freshly sparged diluent and gently mixed to avoid introducing air into the solution and diluted to volume with diluent. The flask was protected from light with foil or stored in the dark.

GSNO LOQ Standard [0.1 w/w% (0.00022 mg/mL) in diluent]: 1.0 mL of the GSNO standard prepared above was transferred into a 100 mL volumetric flask, diluted to volume with diluent, and mixed gently. This intermediate LOQ solution contained approximately

0.0022 mg/mL GSNO. 5.0 mL of the intermediate LOQ solution was then transferred into a 50 mL volumetric flask, diluted to volume with diluent, and mixed gently. This LOQ solution contained approximately 0.1 w/w% (i.e., 0.22 μg/mL GSNO).

GSSG Standard 1.2 w/w% (0.0044 mg/mL) in diluent]: 11 mg (± 2 mg) of GSSG was weigned, in duplicate, into a 100 mL volumetric flask, then dissolved and diluted to volume with diluent. 2 mL of the stock GSSG solution was transferred to a 50 mL volumetric flask, then diluted to volume with diluent. Preparation of Samples:

Samples were prepared in duplicate at a target concentration of 0.22 mg/mL in a 100 mL volumetric flask by accurately weighing 22 mg (± 4 mg) of sample. Each flask was partially filled with freshly sparged diluent and gently mixed to dissolve, then diluted volume with diluent and mixed by hand swirling the flask. System Suitability

The GSNO LOQ solution was injected one time. The average calibration factor for the six injections of the GSNO standard was used to calculate the % recovery for the GSNO at the LOQ. Expected recovery was between 50 and 150% (corrected for purity). The peak area of the LOQ injection was used to set the area reject for reporting < LOQ values. All peaks were on-scale to assure proper integration.

The working GSNO Standards were injected six consecutive times. The following system suitability criteria for these six injections were obtained: a. % RSD API peak area: < 2.0% b. %RSD GSSG impurity peak area: < 5.0% c. % RSD API retention time: < 2.0% d. Average API tailing factor: < 3.0 e. Average column efficiency (N Tan Plates) using API: > 2000 f. Average capacity factor of API: > 2 g. Resolution between API and GSSG impurity: > 2.

The mean calibration factor from the first six injections of working standard was used to calculate the weight percent GSNO in the duplicate standard preparation. The result for the percent GSNO for the injection of the duplicate standard preparation was between 98% and 102% (values should be corrected for purity).

The %RSD of the peak area response was < 2% for all injections of GSNO standard made throughout the analytical sequence. Each of the GSSG standards were then injected. The response factor was calculated and, if within 5% absolute, the results were averaged. Procedure

The liquid chromatographic system was set up as follows:

HPLC Column: Waters Atlantis dC 18, 150 x 3.0 mm, 3 urn packing

Guard Column: Phenomenex Security Guard Cl 8 (or suitable alternate)

Column Temp: 25 ± 2° C

Sample Temp: 5° C (this is a critical parameter for solution stability)

Autosampler Flush: Wateπacetonitrile (1:1)

Flow Rate: 0.4 mL/min

Injection Volume: 10 μL

UVDetection: 210 nm Pump Run Time: 52 minutes Analysis Time: 50 minutes

Mobile Phase: Aq - 0.075% TFA in Water

Org - 0.072% TFA in WateπACN (80:20) The gradient pump program is shown in Table 4. Table 4: Gradient Pump Program*

*Developed on a Perkin Elmer Series 200 pump

The column was equilibrated with the mobile phase prior to commencing any analysis. For a gradient analysis, it wasnecessary to run the system through the gradient program at least one time prior to initiating an analysis. After use, the column was washed with water: acetonitrile (~40:60). These solvents were also recommended for long-term storage of the column.

The data system and load autosampler were set up using the following example format (Table 5) for the sequence of injections: Table 5:

The chromatograms for each injection of standard or sample were reviewed:

Full scale: X scale range = 0 minutes to 50 minutes.

Y scale range was set to review entire GSNO peak.

Expanded time scale: X scale range = GSNO retention time ± 4 min Y scale range was set to review the GSNO baseline/ resolution.

Expanded response scale: X scale range = 0 minutes to 20 minutes Y scale range was set to review impurities baseline. Calculations and Reporting:

Confirmation of Identity: The identity of GSNO and GSSG in the sample wasconfϊrmed if the principal peak in the chromatogram of the sample corresponded to the retention time of the peak produced by the reference material within ± 2.5%.

GSNO Content: Calibration factor (CF) for Standard 1 (calculated using the first six injections of calibration standard)

Calibration Factor (CF) = Ag^

Concstd x Pstd

Where: *-std = Area of GSNO peak in standard solution Concstd = Concentration of GSNO in standard solution (mg/mL) Pstd = Purity of GSNO working standard (%)

8018318.43 ₇

0.2509*0.957

GSNO Content of Sample: TheGSNO content for each sample injection was determined as follows:

GSNO content (% w/w) = A <GSNO> x 100

ConcSmp x CF

Where: A<GSNO> = Area of GSNO peak in sample solution

ConcSmp = Cone of GSNO in sample solution (mg/mL) CF Calibration factor of GSNO (area/cone) e.g. 7170412.32 x 100 = 94.6% 0.2270 x 3.339x£7

If agreement of replicate sample preparations was acceptable (< 2% absolute), the two values were averaged to obtain the test result. Results were reported to one decimal place.

GSSG Content: Calibration factor (CF) for Standard 1 (calculated using the average of the two injections of GSSG calibration standard)

Calibration Factor (CF) = Astd

Concstd x Pstd Where: Astd = Area of GSSG peak in standard solution

ConcStd = Concentration of GSSG in standard solution (mg/mL) Pstd = Purity of GSSG working standard (%) e.g., 66984.67 = 1.3559 x IQ⁷ 0.00497 x 0.994

GSSG Content of Sample: GSSG content for each sample injection was determined as follows:

GSSG content (% w/w) = A <GSSG > x 100

ConcSmp x CF Where: A<GSSG> = Area of GSSG peak in sample solution

ConcSmp = Cone of GSSG in sample solution (mg/mL) CF = Calibration factor of GSSG (area/cone) e.g., 79521.87 x 100 = 2.63% 0.2233 x 1.3559x£7 If agreement of replicate sample preparations is acceptable (< 5% difference), average the two values to obtain the test result. Results were reported two decimal places.

Results of GSNO Stability Testing:

The stability results are reported in Tables 6 though 14 below. Note that in Tables 6 through 14 below, NO2^', NO3^" and GSNO Wt. % values are corrected for moisture. The stability data in Tables 6-14 show that GSNO remains highly stable over a wide range of temperatures (-20° C to 25° C) for time periods of up to at least nine months. Twelve month data (not shown) shows comparable results. Table 6: Initial GSNO stabili

Table 7: GSNO stabilit at 2 weeks

Table 8: GSNO stabilit at one month

Table 9: GSNO stability at two months

Table 10: GSNO stabilit at three months

Table 11: GSNO stabilit at four months

Table 12: GSNO stabilit at five months

'Average is rounded to 0.1

Table 13: GSNO stability at six months

Average is rounded to 0.1

Table 14: GSNO stabilit at nine months

Average is roun e to .1

It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

Claims

What is claimed is:

1. A pharmaceutical formulation comprising an S-nitrosothiol in a pharmaceutically acceptable fluid for stabilizing the S-nitrosothiol during long term storage.

2. The pharmaceutical formulation of claim 1, wherein said S-nitrosothiol is S- nitrosoglutathione (GSNO) or a derivative or salt thereof.

3. The pharmaceutical formulation of claims 1 or 2, wherein the pH of said formulation is about 5.0 to about 8.0.

4. The pharmaceutical formulation of claims 1 or 2, wherein the pH is about 5.5 to about 7.5.

5. The pharmaceutical formulation of claims 1 or 2, wherein the pH is about 6.0 to about 7.0.

6. The pharmaceutical formulation of claims 1 to 5, wherein said pharmaceutically acceptable fluid comprises a buffer having a concentration from about 0.005 M to about 2 M.

7. The pharmaceutical formulation of claim 6, wherein said buffer has a concentration from about 0.1 M to about 1.5 M.

8. The pharmaceutical formulation of claim 6, wherein said buffer has a concentration from about 0.5 M to about 1 M.

9. The pharmaceutical formulation of any of claims 6-8, wherein said buffer is a non- thiol buffer with minimal nucleophilicity.

10. The pharmaceutical formulation of any of claims 1 - 9, wherein at least about 80% of the initial S-nitrosothiol in said pharmaceutically acceptable fluid is present after storage at 4° C to 25° C.

11. The pharmaceutical formulation of any of claims 1-10, wherein the formulation is stored under anaerobic conditions having less than about 200 μM pθ₂.

12. The pharmaceutical formulation of claim 11 , wherein the formulation is stored under anerobic conditions having less than about 100 μM pθ₂.

13. The pharmaceutical formulation of claim 12, wherein the formulation is stored under anerobic conditions having less than about 50 μM pθ₂.

14. The pharmaceutical formulation of any of claims 1-9, wherein said formulation is subjected to a means for producing a solid form of said formulation.

15. The pharmaceutical formulation of claim 14, wherein said formulation contains up to 10% H₂O.

16. The pharmaceutical formulation of claim 15, wherein said formulation contains up to 2.0% H₂O.

17. The pharmaceutical formulation of claim 16, wherein said formulation contains up to 1.5% H₂O.

18. The pharmaceutical formulation of claim 17, wherein said formulation contains up to 1.0% H₂O.

19. The pharmaceutical formulation of claim 18, wherein said formulation contains up to 0.5% H₂O.

20. The pharmaceutical formulation of any of claims 14-19, wherein said S-nitrosothiol is present in particles of about 1.5 μm to about 6.0 μm.

21. The pharmaceutical formulation of any of claims 14-20, wherein GSNO is stored in a unit dosage of about 0.01 mg/ml to about 20 mg/ml.

22. The pharmaceutical formulation of claim 21 , wherein GSNO is stored in a unit dosage of about 1 mg/ml to about 15 mg/ml.

23. The pharmaceutical formulation of claim 22, wherein GSNO is stored in a unit dosage of about 5 mg/ml to about 10 mg/ml.

24. A storage system for stabilizing a S-nitrosothiol, or a derivative or salt thereof, for long term storage, said storage system comprising:

(a) the pharmaceutical formulation of any of claims 11 -23 in a storage container; and

(b) a light-blocking protective cover or container.

25. The storage system of claim 24, wherein said storage system further comprises a dessicant.

26. The storage system of claim 25, wherein said storage system further comprises an oxygen scavenger.

27. The storage system of any of claims 24- 26, wherein said storage system is treated with at least one inert gas prior to sealing said storage system.

28. The storage system of claim 27, wherein said inert gas is nitrogen, argon, helium, neon, krypton and xenon.

29. The storage system of claim 27, wherein said inert gas is nitrogen.

30. The storage system of any of claims 24-29, wherein the purity of the S-nitrosothiol is at least 95% as determined by HPLC after long term storage at a temperature in the range of about -20° C to about 37° C.

31. The storage system of claim 30, wherein said long term storage is at least 24 months.

32. The storage system of claim 31, wherein said long term storage is at least 12 months.

33. The storage system of claim 32, wherein said long term storage is at least 9 months.

34. The storage system of claim 33, wherein said long term storage is at least 6 months.

35. The storage system of claim 34, wherein said long term storage is at least 1 month.

36. The storage system of any of claims 30-35, wherein the S-nitrosothiol is S- nitrosoglutathione (GSNO) and the formulation contains less than 2.5% NO₂ after long term storage.

37. The storage system of any of claims 30-35, wherein the S-nitrosothiol is S- nitrosoglutathione (GSNO) and the formulation contains less than about 2.5% NO₃ after long term storage.

38. The storage system of any of claims 30-35, wherein the S-nitrosothiol is S- nitrosoglutathione (GSNO) and the formulation contains less than about 5% glutathione disulfide after long term storage.

39. The storage system of claim 30, wherein said storage system is stored at a temperature in the range of about -20° C to about 25° C.

40. A method of treating a patient in need of S-nitrosothiol therapy comprising:

(a) obtaining a storage system of any of claims 30-40;

(b) reconstituting said formulation with a pharmaceutically acceptable carrier; and

(c) administering said reconstituted formulation to said patient.

41. The method of claim 41, wherein said S-nitrosothiol is S-nitrosoglutathione.