FIELD OF THE INVENTION

The disclosure generally pertains to liquid compositions containing S-nitrosothiols and alkanediols or alkanediol polymers for controlled release of nitric oxide.

BACKGROUND

Nitric oxide (NO)-releasing solutions have a wide range of applications. They can be used as an in vitro source to generate NO for inhalation therapy of cardiopulmonary diseases. In the 1980s, the identification of endothelium-derived relaxing factor as NO paved the way for inhaled NO (iNO) therapy for cardiopulmonary and pulmonary diseases such as pulmonary hypertension, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. In these conditions, iNO acts as a selective pulmonary vasodilator that diffuses freely into vascular- smooth muscle, resulting in the expansion of pulmonary vasculature. This, in turn, decreases local vascular resistance and improves oxygenation in patients with respiratory disorders. The FDA officially approved iNO therapy in 1999 for treating neonates with hypoxic respiratory failure, and iNO’s therapeutic function has since been elucidated and expanded. It is an effective treatment against chronic infections happening in the lower respiratory tract due to its toxicity to bacterial pathogens. Supplemental iNO is also used as a preventive measure for organ transplants and cardiac surgeries during perioperative care by reducing reperfusion injury.

Nitric oxide-releasing solutions can be used as nasal sprays to be administered to nostrils or liquid drugs to be applied onto the skin. One example function of exogenous NO in nostrils is to prevent infections such as Coronavirus. NO may also enhance wound healing when applied on the skin. NO-releasing solutions can also be used as the lock solution of intravascular catheters to prevent thrombotic and infectious complications and as the inflation solution of urinary catheters to prevent catheter-associated urinary tract infections. NO-releasing solutions may also be mixed with insulin formulations to be infused via insulin infusion sets. In addition to the anti-bacterial functions, the released NO may also reduce inflammation caused by the insulin infusion cannula and the insulin formulation.

Since NO itself is highly unstable, NO donors that can decompose under suitable conditions such as light and heat can be dissolved in a liquid to form NO-releasing solutions. To attain the best efficacy and safety, NO needs to be released from solutions with appropriate concentrations and durations. A high concentration of NO donors is usually beneficial since it allows for a large loading of NO in a small volume. The small volume is desirable in in vitro devices such as iNO generators to make the device light and small. The small volume is necessary for many in vivo applications. For example, the volume of the lumen in intravascular and urinary catheters is limited. However, a high concentration of NO donors may cause a high burst release of NO in the beginning since most NO donors in solutions decompose via first or second order kinetics. The burst release could be toxic to patients and also waste a portion of the NO donors. On the other hand, the NO release rapidly decays to a level that is no longer effective. The low solubility of NO donors and their typical NO release profile with a high initial burst release and rapid decay is a major obstacle to the successful medical application of NO-releasing solutions. If a NO donor solution can release NO in a more steady and sustainable manner (hours to weeks), it may enable these applications. Aqueous media such as buffers have been widely used as the solvent for NO donors. The aqueous solvent usually has a pH that makes NO donors ionic to increase its solubility. However, steady NO release is hard to obtain from these solutions.

Novel liquid compositions containing NO donors that overcome the aforementioned obstacles are needed.

SUMMARY OF THE INVENTION

The disclosure provides NO donors dissolved in alkanediols and methods of using such liquid compositions.

One aspect of the disclosure provides a liquid composition comprising at least one S- nitrosothiol (RSNO) and at least one alkanediol or alkanediol polymer. In some embodiments, the at least one S-nitrosothiol includes S-nitroso-N-acetylpenicillamine (SNAP), S -nitrosoglutathione (GSNO), S-nitrosocysteine, S-nitroso-N-acetylcysteine, S-nitroso-penicillamine, and S- nitrosotriphenylmethanethiol. RSNOs may be fully dissolved or oversaturated. In some embodiments, the at least one alkanediol or alkanediol polymer is selected from the group consisting of ethylene glycol, propylene glycol, 1,3-propanediol, 1,4-butanediol; 1,3-butanediol; 1,2-butanediol; 2,3-butanediol; 1,2-pentanediol, 1,4-pentanediol, 1,5-pentanediol, 2,4- pentanediol, ethohexadiol, 2-methyl- 1,3 -propanediol, 2-methylpropane- 1 ,2-diol, 2- methylpropane-l,2-diol, 2-methyl-2-propyl- 1,3-propanediol, 2,2-dimethylpropane-l,3-diol, 2- butyl-2-ethyl-propanediol, 3-methyl- 1,3-butanediol, 2-methyl-2,4-pentanediol, 2,4-dimethyl-2,4- pentanediol, 3-methyl-1 ,5 -pentanediol, diethylene glycol, triethylene glycol, tetraethylene glycol, pcntacthylcnc glycol, hcxacthylcnc glycol, hcptacthylcnc glycol, octacthylcnc glycol, polyethylene glycol with an average molecular weight of 200 to 1000 Da (e.g., polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000), dipropylene glycol, tripropylene glycol, tetrapropylene glycol, pentapropylene glycol, hexapropylene glycol and polypropylene glycol with an average molecule weight of 200- 1000 Da (e.g., polypropylene glycol 425).

In some embodiments, the liquid composition further comprises water or aqueous solution, wherein the water or aqueous solution is present in an amount of 0.1-30 wt%. In some embodiments, the aqueous solution contains a strong base or a weak base. In some embodiments, at least one base is selected from the group consisting of carbonate salts, bicarbonate salts, phosphate salts, monohydrogen phosphate salts, dihydrogen phosphate salts, hydroxide salts, ammonia, amines, aniline, pyridine, and amino acids with basicity properties such as arginine, lysine, and histidine. The concentration of the base ranges from 1 micromole to 1 mole per liter of liquid formulation and the base does not have to be fully dissolved. Since the generation of NO reduces pH of the solution and a low pH may induce nitrogen oxide species other than NO, any chemicals that have the ability to take protons to suppress pH drop or increase the solution pH may be used for the purpose of reducing undesirable nitrogen oxide species such as NO2. Some bases may serve for this purpose based on mechanisms that are not only related their basicity. In some embodiments, the liquid composition further comprises a metal ion chelator to reduce the amount of metal ions that may catalyze the decomposition of RSNO. In some embodiments, the metal ion chelator is neocuproine or ethylenediaminetetraacetic acid. In some embodiments, the liquid composition further comprises one or more reducing agents, which may react with RSNO to modulate its decomposition rate. The reducing agent may also suppress the generation of undesirable nitrogen oxide species such as NO2. In some embodiments, the one or more reducing agent is ascorbic acid, ascorbate salts, iodide salts, sulfite salts, thiosulfate salts, thiols, metal hydrides, and activated carbon. In some embodiments, the liquid composition further comprises one or more metal ions. In some embodiments, the metal ion is copper ions, ferrous ions, lead ions, and selenium ions. In some embodiments, the liquid composition further comprises one or more surfactants to stabilize undissolved RSNO particles. The undissolved RSNO particles could be synthesized as particles that are small enough to be suspended in alkanediol and alkanediol oligomers/polymers. RSNO particles may also be ground to smaller particles to facilitate the suspension. In some embodiments, the surfactant is selected from a list including nonionic triblock copolymers or Poloxamers (F-127 as an example), polysorbates (Tween® 20 and 80 for instance), sorbitan esters (Span 80 and 85 for instance), quaternary ammonium salts (QAS) (CTAB- cetyltrimethylammonium bromide as an example), PEGs (Polyethylene glycols) and PEG derivatives like PEG ethers and PEG esters (PEG-2000, and Poly(ethylene glycol) monolaurate, for instance), alkyl sulfates-anionic surfactants (Sodium dodecyl sulphate (SDS) for instance), trioctylphosphine oxide [TOPO], fatty Acids (Oleic Acid, and Linoleic acid for instance), phosphatidylcholines (PC) or lecithins (Soybean lecithin for instance), linear alkylbenzene sulfonates (Sodium dodecylbenzene sulfonate (SDBS) for instance), alpha olefin sulfonates, Brij series-non-ionic surfactants, glycerol monostcaratc (GMS), Triton®-X-family surfactants (Triton® X-100, i.e. 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol for instance), and cetylpyridinium bromide. In some embodiments, the liquid composition further comprises a carrier gas infused therein. The carrier gas may be nitrogen or a mixture of nitrogen and oxygen (e.g., air).

Another aspect of the disclosure provides a method of delivering NO to a subject in need thereof, comprising providing to the subject, via inhalation, a liquid composition as described herein. In some embodiments, heat and/or light is applied to the liquid composition to generate NO prior to inhalation.

Another aspect of the disclosure provides an inhalation device comprising a liquid composition as described herein.

Another aspect of the disclosure provides a method of treating a respiratory disease or condition in a subject in need thereof, comprising administering to the subject, via inhalation, a liquid composition as described herein. In some embodiments, heat and/or light is applied to the liquid composition prior to inhalation.

Additional features and advantages of the present invention will be set forth in the description of disclosure that follows, and in part will be apparent from the description of may be learned by practice of the disclosure. The disclosure will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Decomposition curves of 0.1 M SNAP in alkanediols at 37°C in the absence of light. All solutions contain 1 mM neocuproine as a Cu+ chelator.

Figure 2. Decomposition curves of 0.1 M SNAP in ethylene glycol oligomers and polymers at 37°C in the absence of light. All solutions contain 1 mM neocuproine as a Cu+ chelator.

Figure 3. Components of an exemplary NO generator using a SNAP solution in alkanediol or alkanediol oligomers/polymers.

Figure 4. NO release at a therapeutic dose (approximately 20 ppm) from a 3 mL solution of 0.4 M SNAP in PEG 200 when exposed to a 9000K white LED with a brightness of 4000 lx and stirred at 2500 rpm.

Figure 5. NO release at a high therapeutic dose (approximately 38 ppm) from a 3 mL solution of 0.4 M SNAP in PEG 200 when exposed to a 490 nm blue LED array and stirred at 2500 rpm.

Figure 6. Rapid control of NO release by modulating light intensity.

Figure 7. Accelerated NO release from a SNAP solution through modulation of stirring rate.

Figure 8. Real-time NO and NO2 release from 4 mL of 0.4 M SNAP in a PEG 200 solution when exposed to a blue LED.

Figure 9. Real-time NO and NO2 release from 4 mL of 0.4 M SNAP in a PEG 200 solution mixed with a 0.5 mL saturated sodium carbonate solution when exposed to a blue LED.

Figure 10. Suspension of GSNO particles in 1,3-propanediol prepared by wet milling with trioctylphosphine oxide as a surfactant.

DETAILED DESCRIPTION

S-nitrosothiols (RSNOs) are nitric oxide (NO) donors with biomedical applications, which depend on stability and low toxicity. However, dissolving RSNOs in liquid solvents is problematic due to low stability, which prevents sustained release of NO, for example, for inhalation therapy. It is demonstrated herein that alkanediols are excellent liquid solvents/media for RSNOs in that: 1) RSNOs can be dissolved or suspended therein at high concentrations; 2) RSNOs display high stability in alkanediols; 3) the RSNO decomposition rate (and hence NO release profile) can be “tuned” using various combinations of alkanediols, as well as by adding other components such as water (generally less than 30%), saline, buffer, bases, reducing agents, surfactants, etc. and exposure to light and/or heat; 4) the generation of undesirable nitrogen oxide species such as NO2 can be suppressed by adding components such as hydroxide salts, carbonate salts, and reducing agents into the liquid formulation to ensure the purity of the NO gas.

RSNOs, also known as thionitrites, are organic compounds or functional groups containing a nitroso group attached to the sulfur atom of a thiol. RSNOs have the general formula R-S-N=O, where R denotes an organic group. Example RSNOs include, but are not limited to, S-nitroso-N- acetylpenicillamine (SNAP), S-nitrosocysteine, S-nitroso-acetylcysteine, S-nitroso-penicillamine, S -nitrosoglutathione (GSNO), and S-nitrosotriphenylmethanethiol.

As demonstrated herein, RSNOs such as SNAP are soluble in alkanediols at an extremely high concentration in the range of about 200 to 500 mM. In contrast, SNAP’s precursor, N-acetyl- DL-penicillamine, has a much lower solubility (less than 50 mM), suggesting that the high solubility is related to the S-NO group and such a high concentration of SNAP cannot be formed in situ in the alkanediols or alkanediol oligomer/polymers. Similarly, other RSNOs also cannot be synthesized in the solvent from the corresponding thiol precursors since thiols generally have very low solubility in alkanediol and its oligomers/polymers (much lower than 50 mM).

In addition to the soluble form of RSNOs, they may also exist in the solvent as solid particles. For example, GSNO has a low solubility in alkanediol and alkanediol oligomers/polymers. In order to prepare a relatively homogenous liquid formulation with a high concentration of GSNO (generally no less than 50 mM), GSNO powders are synthesized as particles with an average size of less than 100 pm or ground into particles with an average size of less than 100 pm. Preferably, the average particle size is 10 nm to 10 pm. These particles are then suspended in the solvent by themselves or in the presence of one or more surfactants. The amount of RSNO in the liquid formulation ranges from 1 mmol to 2 mol per liter. Preferably, it ranges from 10 mmol to 1 mol per liter. The amount of surfactant ranges from 1 pmol to 1 mol per liter. Preferably, it ranges from 10 pmol to 0.1 mol per liter. The surfactant is selected from a list including nonionic triblock copolymers or Poloxamers (F-127 as an example), polysorbates (Tween® 20 and 80 for instance), sorbitan esters (Span 80 and 85 for instance), quaternary ammonium salts (QAS) (CTAB -cetyltrimethylammonium bromide as an example), PEGs (Polyethylene glycols) and PEG derivatives like PEG ethers and PEG esters (PEG-2000, and Poly(ethylene glycol) monolaurate, for instance), alkyl sulfates-anionic surfactants (Sodium dodecyl sulphate (SDS) for instance), trioctylphosphine oxide [TOPO], fatty Acids (Oleic Acid, and Linoleic acid for instance), phosphatidylcholines (PC) or lecithins (Soybean lecithin for instance), linear alkylbcnzcnc sulfonates (Sodium dodccylbcnzcnc sulfonate (SDBS) for instance), alpha olefin sulfonates, Brij series-non-ionic surfactants, glycerol monostearate (GMS), Triton®- X-family surfactants (Triton® X-100 i.e. 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol for instance), and cetylpyridinium bromide. In some embodiments, RSNO exists in both dissolved and undissolved forms. Although stable suspension of particles in the liquid formulation is preferred, some particles may settle down in the formulation, which may not significantly affect the utility. The amount of RSNO in the suspension is generally greater than 47 mM, e.g., 50-500 mM.

Alkanediols and their oligomers and polymers as solvents generally have boiling points greater than 150 °C. As used herein, the term “polymer(s)” will include “oligomer(s)”. Most solvents in this group have low toxicities and may be used in biomedical applications. Alkanediols comprise linear or branched hydrocarbon chains containing two hydroxy groups at different positions. Examples include, but are not limited to, ethylene glycol, propylene glycol (1,2- Propanediol), propanediol (1,3 -propanediol), 1,4-butanediol, 1,3-butanediol, 1,2-butanediol, 2,3- butanediol, 1 ,2-pentanediol, 1,3-pentanediol, 1 ,4-pentanediol, 1,5-pentanediol, 2,4-pentanediol, methylpropanediol (2-methyl-l,3-propanediol), 3 -methyl- 1,3-butanediol (isopentyldiol; Isoprene glycol), 2-butyl-2-ethyl-propanediol, 2-methylpentane-2,4-diol, 3-methyl- 1,5-pentanediol, polyethylene glycol, and polypropylene glycol. Suitable polyethylene glycols include low- molecular-weight polyethylene glycols (PEGs) that arc liquid at room temperature (i.e. 20-28°C), such as PEGs having a molecular weight of 100 to 1000 Da, e.g., 200-600 Da.

The term “alkanediols” as used herein may also include their oligomers and polymers that could be liquid at room temperature. Examples are dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, undecamers, dodecamers, tridecamers, and tetradecamers of any alkanediol. The solvent may be composed of a single alkanediol or a plurality of alkanediols.

The solvent portion of a liquid composition or solution as described herein may contain no more than 0-30 wt%, e.g. 1-30 wt%, 1-20 wt%, 1-15 wt%, 1-10 wt%, or 1-5 wt% of an aqueous solvent such as DI water, saline, or a buffer. In some embodiments, the solvent is 0-10 wt% water, saline, or buffer and 90-100 wt% alkanediol.

In some embodiments, the solvent may contain thickening agents to enhance the viscosity of the RSNO solution. Examples of thickening agents include, but are not limited to, glycerol, scratch, and fumed silica particles. The weight percentage of the thickening agent in the solvent docs not exceed 0-30 wt% c.g. 1-30 wt%, 1-20 wt%, 1-15 wt%, 1-10 wt%, or 1-5 wt%.

Further modulation of the rate of release of NO can be attained by adding metal ion chelators to reduce RSNO reactivity and adding chemical catalysts of RSNO decomposition to increase RSNO reactivity.

In some embodiments, the liquid composition or solution may contain one or more metal ion chelators such as neocuproine or ethylenediaminetetraacetic acid to remove metal ions such as copper ions that may catalyze the decomposition of RSNOs. As a result, the RSNO solution is more stable and the NO release is more sustainable. The metal ion chelator may be present in the solvent in an amount of 0.001-1 wt%. It should be appreciated that any chelators that can form complexes with metal ion catalysts may be used to slow down the RSNO decomposition.

In some embodiments, the liquid composition or solution may contain chemical catalysts such as ascorbic acid, thiols, metal ions (e.g. copper ions), copper or selenium-based particles, zinc oxide particles, active or noble metal particles such as palladium, platinum, tin, silver particles and/or nanoparticles, tin salts, and copper salts. These catalysts may enhance RSNO decomposition to generate NO at a faster rate when a fast NO release is desired. The catalyst may be present in the solvent in an amount of 0.001-1 wt %.

The dissolution of RSNO into a solvent or a solvent mixture may generate an acidic pH due to a carboxylate group and the release of NO may further reduce the solution pH due to mechanisms such as formation of nitrous acid. The pH of the solution may be controlled by adding acid or base to achieve a more acidic pH or a more basic pH. The pH of the final solution may range from 2 to 14, e.g. from 5 to 10. This pH range may be achieved by the addition of acids, bases, or buffers to the composition. It should be appreciated that the compositions of the present disclosure may be buffered by any common buffer system such as phosphate, borate, acetate, citrate, carbonate and borate-polyol complexes, with the pH and osmolality adjusted in accordance with well-known techniques to proper physiological values.

The generation of undesirable nitrogen oxide species such as NCh needs to be suppressed in some applications such as inhaled NO therapy. To this end, at least one base is added into the formulation at an amount of 1 mmol to 1 mol per liter. The base may be soluble or oversaturated. In some embodiments, the at least one base is selected from the group consisting of carbonate salts, bicarbonate salts, phosphate salts, monohydrogen phosphate salts, dihydrogen phosphate salts, hydroxide salts, ammonia, amines, aniline, pyridine, and amino acids with basicity properties such as arginine, lysine, and histidine. It should be appreciated that any chemicals with the ability to accept protons may be used to suppress the pH drop or increase the pH of the formulation to reduce NO2 generation. Another method to reduce the generation of undesirable nitrogen oxide species is using reducing agents such as ascorbic acid, ascorbate salts, iodide salts, sulfite salts, thiosulfate salts, and thiols. Some classical examples of agents that reduce NO2 generation or scavenge NO2 are sodium carbonate, calcium hydroxide, ascorbic acid, sodium sulfite. The amount of the reducing agent ranges from 1 |imol to 1 mol per liter, e.g., 1 mmol to 100 mmol per liter. The mechanism may be based on its basicity, reducing property, or other direct chemical reactions with NO ₂ .

RSNO solutions/suspensions as described herein may have a half-life of more than 6 months at 4 °C with a concentration of tens of millimolar or above. The lifetime of RSNO may range from 1 day to 1 year at room temperature or elevated temperature such as 37 °C. This is substantially significantly better than other NO-releasing solution formulations.

Embodiments of the disclosure further include methods of preparing a liquid composition as described herein using chemical and biological techniques as known in the art and described herein.

Further embodiments provide a method of delivering nitric oxide to a subject in need thereof comprising administering an effective amount of a composition as described herein to the subject. The compositions of the disclosure may be useful for the treatment of any disease or disorder that would benefit from the administration of nitric oxide. Exemplary diseases/disorders include, but are not limited to, respiratory diseases or conditions, pulmonary hypertension, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, viral or bacterial infection, cancer, stroke, asthma, embolization, cystic fibrosis, diabetes, inflammation, and blood vessel stenosis.

A patient or subject to be treated by any of the compositions or methods of the present disclosure can mean either a human or a non-human animal including, but not limited to dogs, horses, cats, rabbits, gerbils, hamsters, rodents, birds, aquatic mammals, cattle, pigs, camclids, and other zoological animals.

In some embodiments, the active agent (e.g. RSNO or NO generated from RSNO) is administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of active agent to treat the disease or disorder at a reasonable bcncfit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific active agent employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels or frequencies lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage or frequency until the desired effect is achieved. However, the daily dosage of the active agent may be varied over a wide range from 0.01 to 1,000 mg per adult per day. In particular', the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, in particular from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. The NO concentration may be varied over a range from 0.1 to 10000 ppm in the gas phase. In particular, the NO concentration may be 5 to 500 ppm.

The active agent may be combined with pharmaceutically acceptable excipients. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi- solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The compositions of the present disclosure may also contain other components such as, but not limited to, antioxidants, additives, adjuvants, buffers, tonicity agents, bioadhesive polymers, and preservatives. An additive such as a sugar, a glycerol, and other sugar alcohols, can be included in the compositions of the present disclosure. Pharmaceutical additives can be added to increase the efficacy or potency of other ingredients in the composition. For example, a pharmaceutical additive can be added to a composition of the present disclosure to improve the stability of the bioactive agent, to adjust the osmolality of the composition, to adjust the viscosity of the composition, or for another reason, such as effecting drug delivery. Non- limiting examples of pharmaceutical additives of the present disclosure include sugars, such as, trehalose, mannose, D-galactose, and lactose.

In an embodiment, if a preservative is desired, the compositions may optionally be preserved with any well-known system such as benzyl alcohol with/without EDTA, benzalkonium chloride, chlorhexidine, Cosmocil® CQ, or Dowicil 200.

One exemplary application of the RSNO solution in alkanediols and their oligomers/polymers is inhalation NO (iNO) therapy. The dose of iNO used in clinical practice ranges from 1 to 80 ppm depending on the medical condition and must be gradually decreased before cessation to prevent rebound pulmonary hypertension/hypoxemia. Thus, NO delivery systems are employed to ensure the safety and efficacy of iNO therapy, which traditionally include a NO source, a breathing air source, flow regulators, a blender, a ventilator, sensors (gas analyzers), and a humidifier. The most common source of NO for these systems is a pressurized cylinder containing gaseous NO buffered in N2. A high concentration of NO in the cylinder (e.g., 800 ppm for INO max®) is blended with air and/or O2 to achieve the desired concentration and flow rate for delivery to patients. Although cylinder-based delivery technologies are mature, their storage and handling requirements limit accessibility to patients outside of hospitals and in low-resource settings. The cylinder-based iNO therapy is also quite expensive, with a cost of up to $3000 per day per patient in hospitals. Therefore, there has been a significant need for alternative NO generators that do not rely on pressurized cylinders to overcome these limitations.

The RSNO solutions in alkanediols (including their oligomers and polymers that are liquid) can be used to generate NO in an iNO generator. RSNO decomposition in these solvents may be slow under ambient conditions. To increase and control NO release from these RSNO solutions, external stimuli such as light and heat may be used because RSNO (e.g. SNAP) decomposition may be accelerated under light (photolytic activation) and/or heat (thermal activation). A carrier gas such as air or N2 is introduced into the chamber with the RSNO solution to sweep away the generated NO from the generation chamber. The carrier gas can be introduced into the RSNO solution or only to the headspace of the RSNO solution chamber. To enhance the efficiency in removing NO from the solution into the gas line, the solution can undergo further agitation. Examples of agitation methods include, but are not limited to, magnetic stirring (e.g., via a magnetic spin vane or bar), overhead stirrer, and sonication. The stirring rate can range from 100 ppm to 10000 ppm if a stirrer plate or an overhead stirrer is used. The flow of the carrier gas into the liquid formulation may bring NO from the solution/suspension into the gas line without other agitation methods.

The RSNO solution may have an initial concentration ranging from 0.1 to 2.0 M for the inhalation therapy. The solution can be prepared right before its use by mixing RSNO powders and the solvent. The solution can also be prepared by the manufacturer as a consumable and stored at room temperature or lower temperatures. The solvent may be one or more of alkanediols and alkanediol oligomer s/polymers. The volume of the solution may range from 0.2 mL to 2 L to generate NO at varying concentrations and for varying durations. In some embodiments, the rate of the delivering of the NO containing gas is about 0.1 to 2 liters per minute, e.g. about 1 liter per minute. The gas may be administered for 0.1 to 100 hours, e.g. 1-24 hours or 2-10 hours.

Figure 3 provides an example setup of an NO generator containing a SNAP solution. Any light with a wavelength between 200 to 1000 nm may be used as the light source (e.g. a desk lamp, LED light, etc.) although a range of 450 to 550 nm is generally highly effective. The light source can be at any side of the solution as long as the light can strike the solution. The light may also be introduced into the solution via an optical fiber. If heat is used in place of light or combined with light, the solution temperature may range from 30 to 130 °C. In general, a higher temperature leads to higher NO release. An air pump is used to introduce air into the chamber containing SNAP solution and function as the carrier gas. If NO2 or other undesirable nitrogen oxide species is generated from the formulation in a toxic level, it can be removed or converted back into NO via a separate scavenging or converting component. The scavenger/converter can be based on ascorbic acid, calcium hydroxide, sodium hydroxide, sulfite in a solution or solid form. The intensity of the light or heating unit may be pre-programmed during the experiment. It may also be controlled and continuously adjusted by a feedback loop with an NO sensor installed after the SNAP solution chamber. The feedback loop will allow for highly precise control of the NO concentration. Any RSNO solutions or suspensions may be used as the NO-generating component in the inhalation NO generator.

In a different approach, the illuminated and/or heated RSNO solution can be pumped to gas separation silicone fibers to have a large surface area of air exchange (Mol. Pharmaceutics 2017, 14, 11, 3762-3771). In this way, NO generated from the RSNO solution can be exchanged into the carrier gas, which could be air. In addition to the pumping process, no other agitation of the solution is needed.

Embodiments of the disclosure include inhalation devices containing a composition as described herein. Exemplary inhalation devices are known in the art and include, but are not limited to, inhalers (such as metered dose pressurized inhalers (MDIs) and dry powder inhalers (DPIs)), nebulizers, nasal sprays, or other devices for nasal or pulmonary delivery. Inhalation devices typically comprise a plurality of hardware components, which in the case of a MDI can include for example gasket seals; metered dose valves (including their individual components, such as ferrules, valve bodies, valve stems, tanks, springs retaining cups and seals); containers; and actuators.

In some embodiments, the RSNO solution/suspension in alkanediols and their oligomers/polymers is topically administered to patients (e.g., to nostrils or wounds). For example, the RSNO solution can be stored in a pressurized container and administered to nostrils as nasal sprays. In some embodiments, the RSNO solution may be mixed with water or saline-based solutions before it is administered to patients.

Alkanediols themselves have certain antimicrobial properties. Thus, the combination of RSNOs, which is antimicrobial via the NO release, and alkanediols may have enhanced antimicrobial properties. The composition may be bactericidal against various strains of bacteria, including both Gram-positive and Gram-negative organisms, fungi, mycobacteria, parasites, and viruses. Embodiments of the disclosure include methods of inhibiting microbial growth, e.g. bacterial or viral, on a surface, such as a medical device or implant, by contacting the surface with a composition as described herein. As used herein, a “medical device” is any device intended for medical purposes. Exemplary types of a medical device include an instrument, apparatus, constructed element or composition, machine, implement, or similar or related article that can be utilized to diagnose, prevent, treat or manage a disease or other conditions. The medical devices provided herein may, depending on the device and the embodiment, be implanted within a subject. utilized to deliver a device to a subject or utilized externally on a subject. The medical devices provided herein arc sterile and arc subject to regulatory requirements relating to their sale and use. Representative examples of medical devices and implants include, for example, cardiovascular devices and implants such as implantable cardioverter defibrillators, pacemakers, stents, stent grafts, bypass grafts, catheters and heart valves; orthopedic implants (e.g., total or partial arthroplastic joints such as hip and knee prosthesis); spinal implants and hardware (spinal cages, screws, plates, pins, rods and artificial discs); a wide variety of medical tubes, cosmetic and/or aesthetic implants (e.g., breast implants, fillers); a wide variety of polymers, bone cements, bone fillers, scaffolds, and naturally occurring materials (e.g., heart valves, and grafts from other naturally occurring sources); intrauterine devices; orthopedic hardware (e.g., casts, braces, tensor bandages, external fixation devices, tensors, slings and supports) and internal hardware (e.g., Jewries, pins, screws, plates, and intramedullary devices (e.g., rods and nails)); cochlear implants; dental implants; medical polymers, a wide variety of neurological devices; artificial intraocular eye lenses, skin dressings (e.g., wound care dressings), and wearable devices. In certain embodiments, the medical devices may also include a plurality of biomedical devices that are used in clinical and biomedical research settings (e.g., PCR machines or any other research instruments).

In some embodiments, the RSNO solution/suspension is co-administered with an antimicrobial agent. In some embodiments, a surface is contacted with the RSNO solution and an antimicrobial agent. In some embodiments, the RSNO solution is mixed with an additional antimicrobial agent. Non-limiting examples of antimicrobial agents include hexachlorophene, cationic biguanides (i.e. chlorhexidine, cyclohexidine) iodine and iodophores (i.e. povidone- iodine), para-chloro-meta-xylenol, triclosan, furan medical preparations (i.e. nitrofurantoin, nitrofurazone), methenamine, aldehydes (glutaraldehyde, formaldehyde), silver sulfadiazine and alcohols. Nonlimiting examples of classes of antibiotics that may be used include tetracyclines (e.g., minocycline), rifamycins (e.g., rifampin), macrolides (e.g., erythromycin), penicillins (e.g., nafcillin), cephalosporins (e.g.. cefazolin), other beta-lactam antibiotics (e.g., imipenem, aztreonam), aminoglycosides (e.g., gentamicin), chloramphenicol, sufonamides (e.g., sulfamethoxazole), glycopeptides (e.g., vancomycin), quinolones (e.g., ciprofloxacin), fusidic acid, trimethoprim, metronidazole, clindamycin, mupirocin, polyenes (e.g., amphotericin B), azoles (e.g., fluconazole) and beta-lactam inhibitors (e.g., sulbactam). Nonlimiting examples of specific antibiotics that may be used include those listed above, as well as minocycline, rifampin, erythromycin, nafcillin, ccfazolin, imipenem, aztreonam, gentamicin, sulfamethoxazole, vancomycin, ciprofloxacin, trimethoprim, metronidazole, clindamycin, teicoplanin, mupirocin, azithromycin, clarithromycin, ofloxacin, lomefloxacin, norfloxacin, nalidixic acid, sparfloxacin, pefloxacin, amifloxacin, enoxacin, fleroxacin, temafloxacin, tosufloxacin, clinafloxacin, sulbactam, clavulanic acid, amphotericin B, fluconazole, itraconazole, ketoconazole, and nystatin.

Additional exemplary uses of the RSNO solutions/suspensions include, but are not limited to: i. To be used as nasal spray to prevent infectious diseases including COVID. ii. To be used as lock solutions of IV catheters and inflation solutions of urinary catheters to prevent thrombosis and infections. iii. To be infused into insulin infusion cannula to increase its lifetime. The short longevity of insulin infusion cannulas has been a major challenge in diabetes management. iv. To be used as topical dressings to promote wound healing. v. To be used as injectable drugs through intravenous, intramuscular, subcutaneous, and other routes to aid in the therapy of diseases, such as stroke and cancer. vi. To be the source of NO inhalation to treat diseases such as pulmonary hypertension and airway infections. vii. To be used as coatings on medical implants to prevent complications such as infection and blood vessel stenosis.

It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that state range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

EXAMPLE 1: SNAP solubility experiments in alkanediols and their oligomers/polymers

The solubility of S-nitroso-N-acetylpenicillamine (SNAP) in various alkanediols is shown in Table 1. The solubility is overall very high with a range of 200 to 500 mM. If a higher amount of SNAP is needed, excess SNAP powders can exist in the liquid formulation. The SNAP powders can be suspended in the formulation or settled on the bottom of the container. The undissolved SNAP can release NO from its solid state. The undissolved SNAP may also get dissolved as the dissolved SNAP decomposes.

Table 1

EXAMPLE 2: SNAP decomposition experiments

SNAP has a long lifetime of 30 to > 60 days in alkanediols at 37 °C (Figures 1 and 2). More importantly, the decomposition reaction has nearly constant rates except for the very last stage of the decomposition (the last 10-20%). The solutions contain 1 mM neocuproine.

At room temperature, SNAP is more stable. For example, approximately 90% SNAP is left when 0.1 M SNAP solution in 1,5-pentanediol is stored at room temperature for 4 weeks. Over 95% SNAP is left when 0.1 M SNAP solution in polyethylene glycol 200 is stored at room temperature for four weeks.

EXAMPLE 3: NO release from a SNAP solution in the presence of flowing air as the carrier gas.

Figure 4 shows NO release at a therapeutic dose (approximately 20 ppm) from a SNAP/PEG 200 solution when exposed to a 9000K white LED with a brightness of 4000 lx and stirred at 2500 rpm. The flow rate of air is 0.5 L/min. Air is introduced to the headspace of the container with the SNAP solution via an inlet of the sealed container. The container has an outlet that is connected to the NO analyzer for the NO measurement or to the patient for inhalation therapy. A schematic illustration of the setup is shown in Figure 3.

Figure 5 shows NO release at a high therapeutic dose (approximately 38 ppm) from a SNAP/PEG 200 solution when exposed to a 490 nm blue LED array and stirred at 2500 rpm.

Figure 6 shows rapid control of NO release by modulating light intensity.

Figure 7 shows accelerated NO release from a SNAP solution through modulation of stirring rate. Notably, a stirrer plate is not always necessary. For example, the flow of the carrier gas into the solution via a ceramic frit or a tube or a needle can induce agitation of the solution and also cany NO from the solution to the gas phase.

EXAMPLE 4: Reduction of NO2 generation to enhance the safety of inhalation NO therapy

Figures 8 and 9 show that adding sodium carbonate can reduce the formation of toxic NO2. Since SNAP is a weak acid, the dissolution of SNAP in a solvent results in an acidic pH. If the pH is not adjusted, NO2 will be formed quickly as shown in Figure 8. However, if the solvent is adjusted to a higher pH, for example, by adding a sodium bicarbonate solution in the PEG200, the NO2 formation is largely suppressed as shown in Figure 9. Including any strong or weak base in the formulation should have a similar effect. Any buffer for a basic pH may also be used to reduce the NO2 formation. Sodium carbonate may also directly react with NO2 via redox reactions, which reduces NO2 not or not only based on its basicity. Other bases such as calcium hydroxide can also reduce NO2 formation. Other reducing agents may reduce NO2 as well.

EXAMPLE 5: Suspensions of RSNO in an alkanediol prepared by wet milling

Figure 10 shows the suspension of GSNO particles in 1,3-propanediol. GSNO in such suspension has a lifetime of over a month at 37 °C, representing unprecedented durability. To prepare GSNO suspension, 0-8 mg of surfactant, 40 mg of GSNO, and 200 pl of 1,3-propanediol were added in a 3 ml glass vial. Then an adequate amount of ZrO2 beads with an average diameter of 0.3-0.4 mm were added on top of the mixture. The amount of the diol should be sufficient to minimize overheating while maintaining a high level of stress on particles by the milling beads. An overhead mixer (c.g., Overhead Mixer - 38C828IBDC250 - Grainger) was utilized to grind the GSNO particles. An ice bath was used to prevent the drug decomposition due to the heat production in the grinding process. To protect the light-sensitive drug, the entire setup was placed under a cover of aluminum foil. The original metal blade of the mixer was replaced by a handmade plastic one (made from a plunger of a disposable plastic syringe) to avoid the decomposition of GSNO in contact with metal surfaces. The GSNO particles are ground using the mixer at 1000 rpm for about 2 h. At the end, 600 pl of pure 1,3-propanediol was added to extract the final product following a vigorous vortexing for a few minutes, to blend the pure diol with the actual product. The dilution process is optional. The milling beads can be separated from the liquid using a sieve or other methods. This procedure is an example of preparing the GSNO suspensions. Any other procedures that can prepare GSNO particles with desirable sizes may be used to allow suspension of the particles. Usually, the particle size needs to be reduced since the synthesized GSNO powder often has a large particle size, which causes quick and severe settlement of particles from the liquid medium. An ideal size to prepare the suspension is 50 nm to 50 pm. Preferably, the size is 50 nm to 1 pm. For example, dry milling may be used to grind the GSNO particles into small particles and then get suspended in alkanediols or their oligomers/polymers. The GSNO particles can form stable suspension without using any surfactant although a surfactant may be added to modulate the stability of suspension or the decomposition rate of GSNO. This method is not limited to GSNO since other RSNOs above their solubility can be ground and suspended as well. For example, when SNAP exceeds its solubility, the liquid formulation may undergo wet milling to include both soluble and suspended SNAP.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.