Nitric oxide generating formulations and kits

Abstract

Examples of nitric oxide generating formulations include S-nitrosothiol (RSNO) powders, salts, and additives. The additive will control or accelerate the rate at which nitric oxide (NO) is released from the RSNO after the RSNO powder, salt, and additive are dissolved in the liquid carrier. The nitric oxide generating formulation may be part of a kit comprising a container for dissolving a component in a liquid carrier. One kit can be used to prevent and / or treat paranasal sinus infections and another kit can be used to produce an antimicrobial blocking solution.

Description

[0001] This application is a divisional application of the invention patent application with application date of February 23, 2017, application number 201780013356.0 (PCT / US2017 / 019136), and invention name “Nitric Oxide Generating Preparations and Kits”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 299,850, filed February 25, 2016, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] The present application relates to nitric oxide generating preparations and kits. Background Art

[0005] In humans, nitric oxide (NO) can be produced by any of several isoforms of the enzyme nitric oxide synthase (NOS). NO is important for mammalian immune response or defense, and is a cytotoxic agent in the mechanism used by macrophages to kill Leishmania major (L. major), Mycobacterium bovis (M. bovis), and Mycobacterium tuberculosis (M. tuberculosis), as well as many other types of bacteria. NO is also an effective antiviral agent, active against rhinoviruses that cause the common cold. NO is produced in the airways (e.g., upper respiratory tract) from L-arginine by immune cells (macrophages, neutrophils, lymphocytes, etc.) and airway epithelial cells (e.g., conducting and respiratory epithelial cells) primarily through inducible nitric oxide synthase (iNOS). Defects in NO production can lead to reduced immune response and / or microbial biofilm formation. Defects in nasal NO levels have been associated with diseases such as primary ciliary dyskinesia and chronic sinusitis (CRS), and may be associated with an inability to combat viral agents that cause the common cold. Summary of the invention

[0006] Examples of nitric oxide generating formulations include S-nitrosothiol (RSNO) powders, salts, and additives. The additives will control or accelerate the rate of release of nitric oxide (NO) from the RSNO after the RSNO powder, salts, and additives are dissolved in a liquid carrier. The nitric oxide generating formulation can be part of a kit that includes a container for dissolving the components into a liquid carrier. The kit can be used to prepare an antimicrobial sinus treatment solution or a catheter sealing solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Features of examples of the present disclosure will become apparent by reference to the following detailed description and accompanying drawings. Figure 3-5, 7, 9, 11, 13, 24 and 25), data show nitric oxide (NO) release profiles or kinetics (e.g., ppb or PPB / mL; Y-axis) as a function of time (X-axis). In these figures / graphs, the data show broadband values ​​due to the high level of noise associated with chemiluminescent assays. The noise may be due to aerosol droplets from nitrogen-containing solutions, which scatter light and increase the noise of the gas phase levels detected by the optical photomultiplier tube detector within the chemiluminescent instrument.

[0008] Figure 1 Schematic diagram of an example of an antimicrobial sinus treatment solution (formed from an example of an antimicrobial sinus treatment formulation disclosed herein) for introduction into the upper airways via the nasal cavity, with an enlarged view showing the generation of nitric oxide gas (NO) from S-nitrosoglutathione (GSNO). (g) );

[0009] Figure 2 is a schematic diagram of an example of a catheter lock solution formed from an example of a catheter lock solution formulation disclosed herein and introduced into a catheter inserted into a blood vessel (e.g., an artery or a vein), wherein an enlarged view shows the generation of nitric oxide gas (NO) from an NO donor of the lock solution;

[0010] Figure 3 Graph depicting the distribution of nitric oxide (NO) release from solutions including 10 μM GSNO and 25 μM GSNO in phosphate buffered saline (pH 7.4) over a period of 7 hours;

[0011] Figure 4 Graph depicting the nitric oxide (NO) release kinetics of GSNO in saline (0.16 M NaCl + 0.03 M NaHCO3 in Millipore purified water) without ascorbic acid (GSNO) and with ascorbic acid (GSNO:Asc, 1:1 molar ratio; 100 μM each);

[0012] Figure 5 A diagram depicting the NO release kinetics of different concentrations of GSNO in saline (0.16 M NaCl + 0.065 M NaHCO 3 in Millipore water) at 37° C. and at room temperature (e.g., 20° C. to 25° C.);

[0013] Figure 6 To be a bar graph depicting the stability of dry powder GSNO formulations as % of initial GSNO versus time, as determined by UV / Vis spectrophotometry, when freshly prepared and after storage as a dry powder at different temperatures for various periods of time;

[0014] Figure 7To illustrate the NO release kinetics of GSNO:glutathione (GSH) (1:1 molar ratio; 100 μM each) in saline (0.16 M NaCl + 0.065 M NaHCO3 in Millipore water) at 37°C, at room temperature (e.g., 20°C to 25°C), and at 37°C in the presence of oxygen (air);

[0015] Figure 8 Bar graph depicting the stability of GSNO:GSH (1:1 molar ratio) formulations as % of initial GSNO versus time, as determined by UV / Vis spectrophotometry, when freshly prepared and after storage as dry powder at different temperatures for various periods of time;

[0016] Fig. 9 To illustrate the NO release kinetics of GSNO:GSH:ethylenediaminetetraacetic acid (EDTA) (1:1:0.1 molar ratio; 100 μM GSNO) in saline (0.16 M NaCl + 0.065 M NaHCO3 in Millipore water) at 37°C and at room temperature (e.g., 20°C to 25°C);

[0017] Fig.10 Bar graph depicting the stability of GSNO:GSH:EDTA (1:1:0.1 molar ratio) formulations when freshly prepared and after storage as dry powder at different temperatures for various periods of time as determined by UV / Vis spectrophotometry as % of initial GSNO versus time;

[0018] Fig.11 A diagram depicting the NO release kinetics of different molar ratios of GSNO:ascorbic acid (Asc) in saline at 37° C. and GSNO:Asc (1:1 molar ratio; 100 μM each) in saline at room temperature (e.g., 20° C. to 25° C.);

[0019] Fig.12 Bar graph depicting the stability of GSNO:Asc (1:1 molar ratio) formulations as % of initial GSNO versus time, as measured by UV / Vis, when freshly prepared and after storage as dry powder at different temperatures for various times;

[0020] Fig.13 To characterize the new preparation of different molar ratios in saline at 37°C

[0021] GSNO:Asc:EDTA and in saline at room temperature (e.g., 20°C to 25°C)

[0022] Graphical illustration of NO release kinetics from GSNO:Asc:EDTA (1:1:0.1 molar ratio);

[0023] Fig.14 Bar graph depicting the stability of GSNO:Asc:EDTA (1:1:0.1 molar ratio) formulations when freshly prepared and after storage as dry powder at different temperatures for various periods of time as determined by UV / Vis spectrophotometry;

[0024] Fig.15 A graphical illustration depicting the effect of different concentrations of GSNO:Asc (always at a 1:1 molar ratio) on the disruption of S. aureus biofilm;

[0025] Fig.16 A graphical illustration depicting the effect of different concentrations of GSNO:Asc (always at a 1:1 molar ratio) on the disruption of P. aeruginosa biofilm;

[0026] Fig.17A To illustrate the effect of different concentrations of a GSNO:Asc (1:1 molar ratio) mixture on killing of S. aureus biofilms grown on the apical surface of mucociliary differentiated airway epithelial cell cultures established using cells obtained from cystic fibrosis (CF) patients (these cells are deficient in endogenous production of NO, therefore any detected nitrite is due to the added GSNO:Asc);

[0027] Fig. 17B is a bar graph showing the levels of nitrite present on the apical surface of CF cell cultures colonized with S. aureus biofilms after treatment with different concentrations of GSNO:Asc;

[0028] Fig. 17C Bar graph showing the release of interleukin-8 (IL-8), a pro-inflammatory cytokine, from CF airway epithelial cell cultures colonized with S. aureus and treated with varying concentrations of GSNO:Asc;

[0029] Fig.18A and 18B Confocal images (black and white) to illustrate that treatment with GSNO:Asc reduces the density of bacterial biofilms grown on the apical surface of mucociliary differentiated cell cultures compared to mucociliary differentiated cell cultures treated with PBS;

[0030] Fig.19 Graph depicting the synergistic effect of GSNO:Asc (1:1 molar ratio, 0.625 mM GSNO:Asc) and gentamicin (50 μg / ml) on killing S. aureus biofilms established on the apical surface of CF airway epithelial cell cultures;

[0031] Fig. 20A graphical representation depicting the effects of GSH and ascorbic acid alone on killing S. aureus colonized on human CF epithelial cultures;

[0032] Fig.21 A graphical illustration depicting the effect of GSNO:Asc (1:1 molar ratio, 0.625 mM GSNO:Asc) on killing of S. aureus biofilms grown on the apical surface of sinonasal epithelial cell cultures established from chronic sinusitis patients;

[0033] Figures 22A to 22D Before treatment with GSNO:Asc ( Fig.22A and 22B ) or 3 h after treatment with GSNO:Asc (1:1 molar ratio, 0.625 mM GSNO:Asc) ( Fig. 22C and 22D ), scanning electron microscopy image of S. aureus biofilm growing on the apical surface of CF airway epithelial cell cultures;

[0034] Fig.23 Bar graph showing the ciliary beat frequency of uninfected CF cultures and CF cultures infected with S. aureus and then treated with PBS or GSNO:Asc (1:1 molar ratio, 0.625 mM GSNO:Asc);

[0035] Fig.24 A graphical representation of NO flux versus time on the outer surface of a silicone rubber catheter filled with a GSNO:GSH blocking solution (1:1 molar ratio, 2 mM concentration, respectively); and

[0036] Fig.25 Figure 2 is a graph illustrating NO flux over time on the outer surface of a polyurethane catheter filled with GSNO:GSH blocking solution (1:1 molar ratio, 10 mM concentration, respectively). DETAILED DESCRIPTION

[0037] Nitric oxide (NO) is a potent antibacterial and antiviral agent. NO deficiency in vivo may be genetic or polymorphism-related, or induced by pathogens that utilize upstream regulation of NO production. Defective NO production may reduce mucociliary function (one of the major innate immune defense mechanisms in the airway epithelium, also directly related to ciliary beat frequency), increase susceptibility to microbial infection, and / or promote the persistence of antibiotic-resistant bacterial biofilms.

[0038] In some examples disclosed herein, the nitric oxide generating preparation is specifically formulated as an antimicrobial sinus treatment preparation, which can be used to prepare an antimicrobial sinus treatment solution. The antimicrobial sinus treatment solution allows for the spontaneous and slow delivery of NO during the presence of residual sinus preparations in the sinus cavity. The antimicrobial sinus treatment solution disclosed herein increases the NO level inside and outside epithelial cells and immune cells, which can also help control the frequency of ciliary beating. Therefore, the antimicrobial sinus treatment solution including the example of the nitric oxide generating preparation described herein can help restore / enhance mucociliary function (as mentioned above, it is directly related to the frequency of ciliary beating). The restored / enhanced mucociliary function can improve the defense against long-term colonized pathogens and reduce or even prevent the perpetuation of the disease. In addition, the nitric oxide released from the treatment solution will have direct bactericidal and antiviral activity against most types of bacteria and viruses that can infect the sinus cavity. Therefore, this sinus treatment solution can be beneficial for treating or even preventing upper airway infections, including CRS.

[0039] In other examples disclosed herein, the nitric oxide generating formulation is specifically formulated as a catheter lock solution formulation, which can be used as a catheter lock solution. The catheter lock solution can be injected into the lumen of the catheter, wherein the solution spontaneously and slowly releases NO over a period of many hours to many days. NO can penetrate the catheter wall and, therefore, can help prevent the adhesion and growth of biofilm on the lumen wall and outer surface of the catheter. Therefore, the catheter lock solution can be an antimicrobial catheter lock solution. In some examples disclosed herein, the catheter lock solution also includes an additive that can act as a local anticoagulant or can enhance the antithrombotic activity at the distal end of the catheter.

[0040] Examples of nitric oxide generating formulations disclosed herein include S-nitrosothiol (RSNO) powder, salts, and additives. As further described below, the components of the nitric oxide generating formulation can be part of a single composition (i.e., all components are mixed together in a single tablet, granule, or powder mixture), or can be separate compositions to be mixed together in a liquid carrier. As examples of separate compositions, the RSNO powder can be in one container or formulated as one granule or tablet, the salt and additive can be together in another container or formulated as separate granules or tablets, or the RSNO powder, salt, and additive can be in separate containers or formulated as separate granules or tablets.

[0041] The RSNO selected for use in the nitric oxide generating formulation is a substance that occurs naturally in the human body, or is capable of being decomposed into a substance that occurs naturally in the human body, or is a drug that is suitable for human use (e.g., ingestion, consumption, etc.). In any example disclosed herein, the RSNO or RSNO powder is selected from S-nitrosoglutathione (GSNO, occurs naturally in the human body), S-nitroso-cysteine ​​(CYSNO, occurs naturally in the human body), S-nitroso-N-acetyl-penicillamine (SNAP, decomposes into the drug penicillamine), S-nitroso-penicillamine, and S-nitroso-human serum albumin (occurs naturally in the human body).

[0042] S-nitrosoglutathione (GSNO) is an example of an S-nitrosothiol (RSNO) molecule that releases NO. As NO (produced by endothelial cells, macrophages, sinusoidal epithelial cells, etc.) reacts with oxygen to form N2O3, N2O3 can provide nitrosonium ions (NO + ) to react with the thiol group of glutathione to form GSNO, which is present in the human body. Therefore, the use of GSNO in the nitric oxide generating formulations disclosed herein does not introduce any foreign or toxic agents into the nasal cavity or into the blood vessels through a catheter.

[0043] GSNO can be prepared from glutathione (GSH) by acidifying a sodium nitrite / GSH mixture with hydrochloric acid and then isolating the GSNO species (as a solid crystallized). Alternatively, GSNO can be a commercially available sample.

[0044] In some examples, S-nitrosothiol (RSNO) molecules other than GSNO can be used in nitric oxide generating formulations. Examples of these other S-nitrosothiols include S-nitroso-cysteine ​​(CYSNO, naturally occurring in the human body), S-nitroso-N-acetyl-penicillamine (SNAP, broken down into the drug penicillamine), S-nitroso-penicillamine, and S-nitroso-human serum albumin (naturally occurring in the human body).

[0045] When the nitric oxide generating formulation is an antimicrobial sinus treatment formulation, any salt that provides a physiological molar osmotic pressure concentration can be used. Examples of suitable salts for antimicrobial sinus treatment formulations are selected from sodium chloride, sodium bicarbonate, calcium chloride, sodium phosphate buffer, potassium phosphate buffer, and combinations thereof. In the example of a sinus treatment formulation, the salt includes a combination of sodium chloride and sodium bicarbonate.

[0046] When the nitric oxide generating formulation is a catheter seal formulation, any salt that provides physiological osmolarity and does not promote coagulation can be used. Examples of suitable salts for catheter seal formulations are selected from sodium chloride, sodium bicarbonate, sodium phosphate buffer, potassium phosphate buffer, and combinations thereof.

[0047] The additive is selected to control or accelerate the rate of release of nitric oxide from the RSNO material after the RSNO powder, salt and additive are dissolved in a liquid carrier (e.g., for a sinus rinse or catheter sealing solution) or incorporated into a spray (e.g., for a nasal spray). The inclusion of an additive in an antimicrobial sinus treatment formulation enables control of the temporal profile of NO release within the sinus cavity, which can enhance the therapeutic benefit. The inclusion of an additive in a catheter sealing solution formulation enables control of the temporal profile of NO release within and out of the catheter, which can enhance antimicrobial activity. Examples of additives include reduced glutathione (GSH), cysteine, ascorbic acid or ascorbate, copper ions, zinc ions, zinc oxide particles, an organic selenium material, or a combination thereof. An example of an organic selenium material is selected from selenocysteine ​​and ebeselen. An example of a combination of additives is reduced glutathione and ascorbate.

[0048] The following are some examples of how additives can control or accelerate the rate of release of nitric oxide from RSNO (specifically, from GSNO). By forming an initial N-hydroxysulfenamide species (e.g., GS-N(OH)-SG), which is then converted to the radical GS - , Group GS - Glutathione can react with another GSNO molecule to release NO and form a GSSG disulfide species, and glutathione can increase the rate of NO release from GSNO. Cysteine ​​can transfer the nitrosyl group with GSNO to form CysNO, which releases NO much faster than GSNO. Ascorbic acid or ascorbate can be easily oxidized to form a smaller threose structure (3-carbon sugar). Spontaneous oxidation of ascorbate can be combined with GSNO reduction to release NO and GSH. In addition, the oxidation product of ascorbate (i.e., the smaller threose structure) is also a reducing agent that can provide electrons to GSNO, and therefore, GSNO can also be directly reduced to NO. In one example, ascorbic acid or ascorbate can be oxidized in solution for up to 5 days, dried, and then incorporated into the nitric oxide generating preparation. Organic selenium substances can catalyze the production of NO from GSNO. The copper or zinc ions can be reduced to their +1 oxidation state by any trace free thiols present in the GSH preparation, and the Cu(I) or Zn(I) ions can then reduce GSNO to NO and GSH.

[0049] In the nitric oxide generating formulation, the molar ratio of RSNO powder to additive ranges from 1:10 (0.1) to 10:1 (10). In one example, the molar ratio of RSNO powder to additive is about 1:1.

[0050] The antimicrobial sinus treatment formulation (including RSNO powder, salts selected from sodium chloride, sodium bicarbonate, calcium chloride, sodium phosphate buffer, potassium phosphate buffer, and combinations thereof, and additives) can also be prepared so that when a predetermined amount of water or other liquid carrier is added to dissolve the components and form a sinus rinse / sinus treatment solution, the concentration of RSNO in the sinus treatment solution ranges from about 25 μM to about 10 mM. In one example, the concentration of RSNO in the sinus rinse / sinus treatment solution is about 100 μM.

[0051] The catheter lock solution formulation (including RSNO powder, salts selected from sodium chloride, sodium bicarbonate, sodium phosphate buffer, potassium phosphate buffer, and combinations thereof, and additives) can be prepared so that when a predetermined amount of water or other liquid carrier is added to dissolve the components and form the catheter lock solution, the concentration of RSNO in the catheter lock solution ranges from about 500 μM to about 50 mM.

[0052] The antimicrobial sinus treatment formulation (including RSNO powder, salts and additives selected from sodium chloride, sodium bicarbonate, calcium chloride, sodium phosphate buffer, potassium phosphate buffer and combinations thereof) may also include a metal ion chelator. Metal ion chelators can be used to control the level of free copper ions and / or zinc ions present in different chemical substances or reconstitution fluids / solutions. As an example, Cu(II) ions can act as a catalyst for the decomposition of RSNO (wherein Cu(II) is reduced to Cu(I) by free thiols, and Cu(I) reduces RSNO to NO and RSH). Metal ion chelators can be used to eliminate these effects if they are undesirable. Examples of metal ion chelators include ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), sodium citrate and combinations thereof. The antimicrobial sinus treatment formulation can be prepared so that when water or other liquid carriers are added to form a sinus rinse, the concentration of the metal ion chelator ranges from about 1 μM to about 1 mM.

[0053] The catheter lock solution formulation (including RSNO powder, salts selected from sodium chloride, sodium bicarbonate, sodium phosphate buffer, potassium phosphate buffer, and combinations thereof, and additives) may also include an anticoagulant. The anticoagulant may provide local anticoagulant activity at the catheter tip when the anticoagulant diffuses out of the catheter. Examples of suitable anticoagulants include heparin, EDTA, sodium citrate, and combinations thereof. Inclusion of heparin may also enhance antithrombotic activity at the catheter tip. The catheter lock solution formulation may be prepared so that when water or other liquid carrier is added to form the catheter lock solution, an effective amount of the anticoagulant is present at a concentration. An example range of an effective amount of heparin is about 2 units.

[0054] / ml (U / ml) to about 100 U / ml. An example range of an effective amount of EDTA or sodium citrate is about 0.5 mM to about 25 mM.

[0055] In any example disclosed herein, RSNO powder, salts, and additives may be formulated together in a single powder composition (which may be contained in a single bag, container, etc.) or as a single granule, tablet, etc. In some cases, other additives, such as metal ion chelators for antimicrobial sinus treatment formulations or anticoagulants for catheter sealing solution formulations, may also be part of a single powder composition or a single granule, tablet, etc. Alternatively, in any example disclosed herein, RSNO powder may be contained in a first container, and salts and additives may be contained in a second container separate from the first container. The contents of each container may be added to a liquid carrier to form an example of a solution disclosed herein. Further, in any example disclosed herein, RSNO powder, salts, and additives may be contained in separate first, second, and third containers, respectively. In this example, the contents of each container may be added to a liquid carrier to form an example of a solution disclosed herein.

[0056] Further, in any example disclosed herein, the RSNO powder, salts, and additives may be formulated together in a liquid carrier. Thus, in some examples, the sinus treatment formulation (including RSNO powder, salts and additives selected from sodium chloride, sodium bicarbonate, calcium chloride, sodium phosphate buffer, potassium phosphate buffer, and combinations thereof) also includes a liquid carrier. In these examples, the liquid carrier may be selected from water, saline solution, saline solution with sodium bicarbonate (NaHCO3), phosphate buffered saline solution (PBS), an aqueous solution with calcium chloride (CaCl2), and an aqueous solution with sodium phosphate buffer or with potassium phosphate buffer. In other examples, the catheter sealing solution formulation (including RSNO powder, salts and additives selected from sodium chloride, sodium bicarbonate, sodium phosphate buffer, potassium phosphate buffer, and combinations thereof) also includes a liquid carrier. In these examples, the liquid carrier may be selected from water, saline solution, saline solution with sodium bicarbonate (NaHCO3), phosphate buffered saline solution (PBS), and an aqueous solution with sodium phosphate buffer or with potassium phosphate buffer.

[0057] The nitric oxide generating formulation may be included in a nitric oxide generating kit. The NO generating kit may be an antimicrobial sinus treatment kit or a catheter sealing solution kit. In the NO generating kit, the RSNO may be part of the first powder composition, the additive may be part of the second powder composition, and the salt may be part of the first or second powder composition, or may be in its own separate (third) powder composition. The NO generating kit also includes a container for dissolving the first and second powder compositions (one of which includes the salt) or the first, second and third powder compositions into a liquid carrier.

[0058] In some examples of the kit, all of the nitric oxide generating formulation components are included in a single powder composition (which is a dry mixture of the listed components or a granule, tablet, etc. containing the listed components). In other words, the aforementioned first and second or first, second and third powder compositions are combined together to form a single powder composition or a single granule, tablet, etc. Within the kit, the single powder composition may be contained in a capsule, ampoule, vial or other suitable container, or may be pressed into granules, tablets, etc., which are soluble in a given volume of a liquid phase / liquid carrier. In the example of a sinus treatment kit, RSNO powder, salts and additives selected from sodium chloride, sodium bicarbonate, calcium chloride, sodium phosphate buffer, potassium phosphate buffer and combinations thereof, with or without a metal ion chelator, are combined together into a single dry granule or powder. In the example of a catheter sealing solution kit, RSNO powder, salts and additives selected from sodium chloride, sodium bicarbonate, sodium phosphate buffer, potassium phosphate buffer and combinations thereof, with or without an anticoagulant, are combined together into a single dry granule or powder.

[0059] In other examples, the nitric oxide generating formulation includes a first powder composition and a second powder composition as separate compositions, one of which includes a salt. In other examples, the nitric oxide generating formulation includes a first powder composition, a second powder composition, and a third powder composition as separate compositions. In the kit, the separate powder compositions are contained in separate capsules, bags, or other suitable containers, or the separate powder compositions are respectively pressed into two or three different granules, tablets, etc.

[0060] The nitric oxide generating kit may also include a container (of any type) for dissolving the powder composition into a liquid carrier. Examples of containers for sinus treatment kits include nasal rinse bottles or neti-pots. Examples of containers for catheter treatment kits include ampoules or vials or syringes.

[0061] Some examples of nitric oxide generating formulations disclosed herein can be added to a predetermined amount of a liquid carrier to form a sinus rinse or sinus treatment solution. The resulting sinus rinse / sinus treatment solution has a pH of 4 to 10. The sinus rinse / sinus treatment solution can be prepared by the user and used immediately.

[0062] In the example of a sinus rinse / sinus treatment solution, the liquid carrier can be any suitable liquid that can be introduced into the upper airway of a person through the nasal cavity. The liquid carrier can be water, a saline solution, a saline solution with sodium bicarbonate (NaHCO3), a phosphate buffered saline solution (PBS), an aqueous solution with calcium chloride (CaCl2), or an aqueous solution with a sodium phosphate buffer or a potassium phosphate buffer. It should be understood that the salt present in the liquid carrier can be introduced when the nitric oxide generating formulation (including salts) is dissolved therein. For example, sodium bicarbonate and sodium chloride can be included in the nitric oxide generating formulation and added to water to form the sinus rinse / sinus treatment solution, or can be a separate package that is added to the water in addition to the RSNO and additive components. In this specific example, when the salt is dissolved in water, the solution formed is a sodium bicarbonate / sodium chloride solution. In one example, this solution includes about 0.16M sodium chloride and about 0.065M sodium bicarbonate.

[0063] The examples of nitric oxide generating formulations disclosed herein can also be added to an aerosol to form a nasal spray. In this example, the spray container can be filled with water, and then the dry powder nasal formulation can be added to this reservoir to produce a solution containing RSNO, which can be administered upward into the nose and sinus cavities as a spray. The concentration of RSNO in the spray can be comparable to those used in the sinus rinse solution / sinus treatment solution method. In some examples, the concentration of RSNO in the spray can be higher than those used in the sinus rinse solution method.

[0064] In the examples disclosed herein, the nitric oxide generating preparation or kit can be used in a method of increasing nasal nitric oxide (NO) levels. Examples thereof are shown in Figure 1 The method includes introducing an S-nitrosothiol (eg, S-nitrosoglutathione (GSNO)) and an additive into the nasal cavity via a nasal / sinus rinse 10 or spray (contained in a suitable container 12).

[0065] When a nasal / sinus rinse is utilized, it may be necessary to freshly prepare rinse 10 in container 12 by adding the nitric oxide generating formulation to a liquid carrier and stirring or shaking to dissolve the powder, tablets, granules, etc., and then introduce the rinse into the nasal cavity. When a spray is utilized, the aerosol container may be configured to freshly prepare an aerosol solution by introducing a powder, tablet, granules, etc. into a liquid carrier and atomize the solution as it is dispensed into the nasal cavity.

[0066] When a sinus rinse / sinus treatment solution (including an example of a nitric oxide generating formulation in a saline / HCO3 solution) is introduced into the nasal cavity, RSNO and an additive (wherein Figure 1 When the sinus wash is introduced into the nostrils, a significant volume of the nitric oxide generating agent is present as a layer of sinus treatment solution (such as GSNO) in the capsule along the surface of the sinus / nasal mucosal tissue lining the inner wall of the sinus cavity. Figure 1 The RSNO in solution spontaneously decomposes at a controlled rate (which can be controlled by the additives used) to release NO. This increases NO levels in the airways and on the surface of the sinus / nasal mucosal tissues where biofilms may exist.

[0067] Increasing the level of NO produced will be therapeutic and sufficient to help kill bacteria and viruses, disrupt bacterial biofilm formation, disperse or prevent microbial biofilm formation (e.g., disperse antibiotic resistant biofilms), reduce inflammation, and increase the frequency of ciliary beats, thereby improving mucociliary clearance. Increased NO production can be observed almost immediately and for a long time (e.g., up to about 4-6 hours). The nasal preparations disclosed herein can be used to fight infection. In addition, daily use (e.g., once or twice a day) of the nasal preparations disclosed herein can help maintain NO levels equivalent to healthy individuals (i.e., can help maintain sinus health in a manner consistent with the human body's fight against infection), which in turn can prevent bacterial biofilm formation and CRS disease perpetuation.

[0068] The risk of producing too much NO (toxic levels) with the examples disclosed herein is unlikely. For example, a typical sinus cavity has a volume of about 60 ml. Therefore, if 10% of a 250 ml volume of a sinus rinse is retained in the sinuses (along the sinus cavity walls and in the crevices, etc.), and the rinse contains 10 μM GSNO, the maximum level of gas phase NO that is possible in the sinus cavity may be about 80 ppmv, if all the NO is released at once (which is unlikely given the slow release of NO from GSNO over hours, see e.g. Figure 3 and 6 ). In the examples disclosed herein, the gas phase NO levels within the sinus cavity resulting from the introduction of the nitric oxide generating formulation via a rinse or spray will be much lower and closer to the target normal 200-500 ppbv levels.

[0069] Other examples of nitric oxide generating formulations disclosed herein can be added to a predetermined amount of a liquid carrier to form a catheter lock solution. The resulting catheter lock solution has a pH ranging from 4 to 10. The catheter lock solution can be prepared by the user and used immediately.

[0070] The liquid carrier used to form the catheter lock solution can be any suitable liquid that can be introduced into the catheter without promoting clotting in the patient. In these examples, the liquid carrier can be water, a saline solution, a saline solution with sodium bicarbonate (NaHCO3), a phosphate buffered saline solution (PBS), or an aqueous solution with a sodium phosphate buffer or a potassium phosphate buffer. It should be understood that the salt present in the liquid carrier can be introduced when the catheter lock solution formulation (including salts) is dissolved therein. For example, sodium bicarbonate and sodium chloride can be included in the catheter lock solution formulation and added to the water to form the catheter lock solution, or can be a separate package that is added to the water in addition to the RSNO and additive components.

[0071] In the examples disclosed herein, the catheter lock solution formulation or kit can be used in a method of increasing the level of antimicrobial nitric oxide (NO) both inside and outside of a catheter infused into a patient. An example thereof is shown in Figure 2 middle.

[0072] The method includes preparing a catheter lock solution 10' by adding a catheter lock solution formulation 14 to a liquid carrier 16 in a container 12' and stirring or shaking to dissolve the powder, tablets, granules, etc. of the formulation 14 in the liquid carrier 16. In one example, about 2 mL to about 3 mL of purified water is added to the tablet of RSNO, salts, and additives to form the catheter lock solution 10'. In other examples, different powder compositions can be added to the liquid carrier 16 in the container 12' and stirred or otherwise mixed to dissolve the composition and form the catheter lock solution 10'.

[0073] The method then includes introducing catheter lock solution 10' into the lumen of catheter 20 (located in a patient's blood vessel 22). Syringe 18 (or other suitable device) may be used to draw catheter lock solution 10' (from container 12') and introduce catheter lock solution 10' into the lumen.

[0074] The reconstituted catheter lock solution 10' releases low levels of NO over a long period of time. At least the distal end of the catheter lumen releases the generated NO. The NO at the distal end inhibits local platelet activity, thrombosis, and distal blockage. When the lumen and the wall of the catheter 20 are permeable to nitric oxide (e.g., Figure 2 ), NO may also be advantageously distributed from the lumen and catheter wall. Examples of NO permeable materials that may be used for the lumen and catheter 20 include silicone rubber (SR), polyurethane (PU), copolymers of SR and PU, copolymers of PU and polycarbonate, and other NO permeable materials. When the lumen and walls of the catheter 20 are permeable to nitric oxide, NO may be discharged through the entire outer surface of the catheter 20, which at least substantially prevents bacterial adhesion, biofilm formation, and coagulation on the outer catheter surface. For example, increasing the level of NO produced will be therapeutic and will be sufficient to help kill bacteria and viruses, disrupt bacterial biofilm formation, and disperse or prevent microbial biofilm formation (e.g., disperse antibiotic resistant biofilm). At the same time, even higher levels of NO will be present in the sealing solution within the lumen of the catheter 20, which will prevent infection within the lumen and other lumens if the device 20 is a multi-lumen catheter. In addition, when an anticoagulant is included in the nitric oxide generating formulation 14 used to form the catheter sealing solution 10', the anti-thrombotic effect of the catheter sealing solution 10' at the distal end of the catheter 20 may be further improved.

[0075] In the examples disclosed herein, a dry nitric oxide generating formulation is added to a liquid carrier to form an active solution. It may be desirable to reconstitute the nitric oxide generating formulation in a liquid carrier immediately prior to use in the nasal cavity or in a catheter because a freshly prepared solution is more stable than a solution in which the RSNO has been stored for a long time (e.g., several days).

[0076] In order to further illustrate the present disclosure, examples are given herein. It should be understood that these examples are provided for illustrative purposes and should not be construed as limiting the scope of the present disclosure.

[0077] Example 1

[0078] Two different solutions, one with 10 μM GSNO and one with 25 μM GSNO, were prepared in phosphate buffered saline at physiological pH 7.4. These solutions did not include the additives disclosed herein.

[0079] The NO release profiles of the two solutions were measured by chemiluminescence detection at 37 °C and the results are shown in Figure 3 As shown, both solutions continued to produce NO for more than 7 hours, and the solution containing the higher concentration of GSNO produced higher levels of NO. It should be noted that the NO levels observed for these solutions in this example, which were generally in the 5 ppbv to 40 ppbv range, were significantly diluted compared to the gas levels actually observed in the sinus cavity by continuously purging the test solutions with a nitrogen gas stream that swept the released NO into the chemiluminescent NO analyzer.

[0080] Two additional solutions were prepared in 0.16 M NaCl / 0.03 M NaHCO3, one with 100 μM GSNO and one with 100 μM GSNO and 100 μM ascorbate (ie, one example of an additive disclosed herein).

[0081] The NO release of the two additional solutions was measured by chemiluminescence and the results are shown in Figure 4 As shown, the inclusion of ascorbate enhances the NO release profile, with all NO released within a 2-3 hour window. It should be noted that the other solutions tested were purged with nitrogen to sweep the NO released from GSNO into the chemiluminescent NO gas analyzer, and therefore, the release rate was faster than that observed when the formulation was used as a sinus rinse or spray.

[0082] Example 2

[0083] preparation

[0084] In this example, several nasal formulations were prepared using previously synthesized S-nitrosoglutathione (GSNO) alone or in combination with reduced L-glutathione (GSH,>98%) or L-ascorbic acid (Asc). Some formulations also included ethylenediaminetetraacetic acid (EDTA) and / or saline salts (NaCl and NaHCO3). The components were mixed as dry powders and stored in 4 mL amber vials in a freezer (-18°C), at 4°C, at room temperature (RT, about 20°C to about 25°C), or at 37°C.

[0085] Different dry powder nasal formulations included GSNO; GSNO and GSH (1:1 molar ratio); GSNO, GSH, and EDTA (1:1:0.1 molar ratio); GSNO:Asc (1:1 molar ratio); and GSNO:Asc:EDTA (1:1:0.1 molar ratio).

[0086] A dry powder nasal formulation of GSNO and EDTA was prepared with saline salts, and a dry powder nasal formulation of GSNO and GSH was prepared with saline salts. Both formulations were prepared so that upon dilution to 200 mL (with water), the final concentration of GSNO was 100 μM.

[0087] NO release rate

[0088] The NO release rates of some solution formulations were measured by a chemiluminescent nitric oxide analyzer (NOA) at room temperature and at 37°C in an amber NOA cell while purging with 50 mL / min nitrogen through a glass pipette. Solution formulations were freshly prepared (initial data) (i.e., dry powder nasal formulations were dissolved in 10 mL of saline solution (NaCl and NaHCO3, in water) immediately before the assay) or prepared from stored dry powder aliquots (stored dry powder aliquots were freshly dissolved in 10 mL of saline solution (NaCl and NaHCO3, in water) immediately before the assay). NO release rates were measured for one or more of the following time periods: 0 (freshly prepared), 1 month, 2 months, 4 months, and 6 months.

[0089] NOA was Sievers NOA. Release kinetics were studied including analysis of the overall shape of the transient curve, half-life, and total NO released compared to theoretical GSNO content (%). Different machines produce different PPB / mL values ​​due to differential constants, therefore the shape of the graph is considered to be very qualitative in nature. If necessary, GSNO quantification in each formulation was tested by the copper / cysteine ​​method which releases all NO very quickly from the GSNO.

[0090] Stability measurement

[0091] Some dry powder formulations were tested for stability by UV / Vis analysis and / or by a Sievers Nitric Oxide (NO) Analyzer (as described previously).

[0092] For UV / Vis analysis, three samples (3-5 mg each) from the dry powder formulations (12 vials per formulation at each temperature) were placed in 4 mL amber vials at each time interval (0 (fresh), 1 month, 2 months, 4 months, and 6 months). The respective formulations were then diluted in 4 mL PBS+0.1 mM EDTA (PBSE) solution and mixed for 30 seconds, and 1 mL of the diluted formulation was injected into 3 mL PBSE in a disposable UV / VIS cuvette. After measuring the PBSE spectrum as a blank, the absorbance spectrum was generally measured between 300-600 nm.

[0093] GSNO was quantified by absorbance measured at 334 nm. These values ​​were then compared to the calculated percentage of initial or theoretical GSNO content for each formulation. As discussed below, GSH, ascorbate, and EDTA have no absorbance at 334 nm, therefore, this wavelength was used for the selective quantification of GSNO in each formulation.

[0094] GSH stability

[0095] To test GSH stability, the Ellman assay was used. This assay measures the amount of free thiol groups in a compound after the compound reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to produce a yellow product with absorbance at 412nm. This indicates that GSH does not interfere with the absorbance at 334nm.

[0096] Ascorbic acid stability

[0097] Ascorbate (i.e., vitamin C) is known to be stable for long periods of time under dry conditions. As discussed in more detail below, the UV / Vis analysis of GSNO mixed with ascorbic acid was similar for freshly prepared samples and samples prepared after the dry powder formulation had been stored for several months. These results indicate that ascorbic acid is relatively stable.

[0098] EDTA stability

[0099] It is also known that EDTA is stable over long periods of time under dry conditions. As will be discussed in more detail below, the NO release profiles of GSNO mixed with GSH or ascorbic acid and EDTA are similar for freshly prepared samples and samples prepared after the dry powder formulation has been stored for several months. These results indicate that EDTA is stable.

[0100] GSNO Results

[0101] Figure 5 The NO release kinetics of different GSNO formulations in saline at 37°C and at room temperature are depicted. Different dry powder mixtures were prepared and solutions were prepared just before the NO release assay. For the 37°C solutions, the GSNO concentrations were 1 μM, 5 μM, 10 μM, 25 μM, 50 μM and 100 μM. For the room temperature solutions, the GSNO concentration was 100 μM. Figure 5 The results in show that there is a considerable lag time for the release of NO from GSNO without any additive, reaching a peak value at 5-6 hours for all concentrations. In addition, as shown, the amount of NO released is positively correlated with the concentration of GSNO in the test solution.

[0102] Figure 6 Bar graph depicting the stability of GSNO formulations as % of initial GSNO versus time (determined by UV / Vis spectroscopy) when freshly prepared (ie, initial) and when stored as a dry powder at different temperatures for various times. Figure 6 Results shown in , all dry powder formulations contained 100 μM GSNO. As mentioned above, for UV / Vis, the corresponding dry powder formulations were diluted in 4 mL PBS+0.1 mM EDTA (PBSE) solution and mixed for 30 seconds, and 1 mL of the diluted formulation was injected into 3 mL PBSE in a disposable UV / VIS cuvette. Stability testing of the GSNO formulations was completed over a 6-month period. UV / VIS analysis showed no significant decrease in stability, although the 37°C aliquot showed the greatest decrease in stability, with 67% retained after 6 months (compared to 100% in the freshly prepared / initial sample). The headspace in the vials used to store the GSNO formulations was room air, which had some moisture. Therefore, the true shelf-life stability of the GSNO formulations in a completely dry headspace atmosphere may be much better.

[0103] GSNO:GSH (1:1) Results

[0104] Figure 7 To depict the NO release kinetics of GSNO: glutathione (GSH) (1:1) in saline at 37°C, at room temperature and at 37°C in the presence of oxygen (air). These solutions were freshly prepared from dry powder formulations that were not stored. For each solution, the GSNO and GSH concentrations were 100 μM, respectively. Figure 7 The results in show that at higher temperatures, the NO release rate increases. In addition, compared with the solution prepared in the absence of GSH (see Figure 5 ), the NO release rate was slightly increased, especially at the beginning.

[0105] Figure 8Bar graph depicting the stability of GSNO:GSH (1:1) formulations as % GSNO versus time (determined by UV / Vis spectroscopy) when freshly prepared (ie, initial) and when stored as dry powder at different temperatures for various times. Figure 8 Results shown in , all dry powder formulations contained 100 μM GSNO:100 μM GSH. As mentioned above, for UV / Vis, the corresponding dry powder formulations were diluted in 4 mL PBS+0.1 mM EDTA (PBSE) solution and mixed for 30 seconds, and 1 mL of the diluted formulation was injected into 3 mL PBSE in a disposable UV / VIS cuvette. Stability testing of the GSNO:GSH reagent was completed over a 6-month period. UV / VIS analysis showed that the stability did not significantly decrease over a 6-month period. The headspace in the vials used to store the GSNO formulations was room air, which had some moisture. Therefore, the true shelf life stability of the GSNO formulations in a completely dry headspace atmosphere may be much better.

[0106] GSNO:GSH:EDTA (1:1:0.1) results

[0107] Fig. 9 To illustrate the NO release kinetics of GSNO:GSH:ethylenediaminetetraacetic acid (EDTA) (1:1:0.1) in saline at 37°C and at room temperature. These solutions were freshly prepared from dry powder formulations that were not stored. For each solution, the GSNO and GSH concentrations were 100 μM, respectively. Fig. 9 The results showed that in the presence of EDTA, the rate of NO release was slightly slower due to EDTA chelating trace metal ion contaminants in the chemicals used to prepare the solution, and these trace metal ions, especially copper ions, can act as catalysts for the decomposition of GSNO.

[0108] Fig.10 Bar graph depicting the stability of GSNO:GSH:EDTA (1:1:0.1) formulations when freshly prepared (ie, initial) and when stored as dry powder at different temperatures for various times as % GSNO versus time (determined by UV / Vis spectroscopy). Fig.10Results shown in , all dry powder formulations contained 100 μM GSNO:100 μM GSH:10 μM EDTA. As mentioned above, for UV / Vis, the corresponding dry powder formulations were diluted in 4 mL PBS+0.1 mM EDTA (PBSE) solution and mixed for 30 seconds, and 1 mL of the diluted formulation was injected into 3 mL PBSE in a disposable UV / VIS cuvette. Stability testing of the GSNO:GSH reagent was completed over a 6-month period. UV / VIS analysis showed that the samples stored at 4°C and -18°C had a slight decrease in stability over a 2-month period, but showed total stability over a 6-month period. The headspace in the vials used to store the GSNO formulations was room air, which had some moisture. Therefore, the true shelf-life stability of the GSNO formulations in a completely dry headspace atmosphere may be much better.

[0109] GSNO:Asc results

[0110] Fig.11 A diagram depicting the NO release kinetics of different ratios of GSNO:ascorbic acid (Asc) in saline at 37°C and GSNO:Asc (1:1) in saline at room temperature. These solutions were freshly prepared from dry powder formulations that were not stored. For the 37°C solutions, the GSNO:Asc concentrations were 100 μM:1000 μM, 100 μM:10 μM, and 100 μM:100 μM. Fig.11 The results in Figure 2 show that at higher ascorbate concentrations, a large spike was seen before the 2 hour time, indicating that the presence of ascorbate greatly accelerated the release of NO from GSNO. 100 μM: 100 μM concentration in saline at 37°C still produced an initial spike, but took longer to reach baseline. The lowest ascorbate additive concentration, 10 μM, had little effect on the rate of NO release, with maximum production at the 5-6 hour time point, just like GSNO in saline without other additives.

[0111] Fig.12 Bar graph depicting the stability of GSNO:Asc (1:1) formulations as % GSNO versus time (determined by UV / Vis spectroscopy) when freshly prepared (ie, initial) and when stored as dry powder at different temperatures for various times. Fig.12Results shown in , all dry powder formulations contained 100 μM GSNO:100 μM Asc. As mentioned above, for UV / Vis, the corresponding dry powder formulations were diluted in 4 mL PBS+0.1 mM EDTA (PBSE) solution and mixed for 30 seconds, and 1 mL of the diluted formulation was injected into 3 mL PBSE in a disposable UV / VIS cuvette. Stability testing of the GSNO:Asc reagent was completed over a 6-month period. UV / VIS analysis showed excellent stability for all samples, although the stability of samples stored at room temperature was more significantly reduced at 4 months. The headspace in the vials used to store the GSNO formulations was room air, which had some moisture. Therefore, the true shelf-life stability of the GSNO formulations in a completely dry headspace atmosphere may be much better.

[0112] GSNO:Asc:EDTA (1:1:0.1) results

[0113] Fig.13 Graph depicting the NO release kinetics of different ratios of GSNO:Asc:EDTA in saline at 37°C and GSNO:Asc:EDTA (1:1:0.1) in saline at room temperature. These solutions were each freshly prepared from a dry powder formulation that had not been stored. As has been seen with the GSNO:Asc formulation, it is important to control the ascorbate concentration to avoid a large initial spike and a rapid decrease in NO release ppb / mL. The presence of EDTA reduces free trace copper ion levels (or other trace metal ions) and prolongs this decrease, allowing NO release to continue for nearly 5 hours, as opposed to 3-4 hours in the absence of EDTA.

[0114] Fig.14 Bar graph depicting the stability of GSNO:Asc:EDTA (1:1:0.1) formulations when freshly prepared and when stored as dry powder at different temperatures for various times as % GSNO versus time (determined by UV / Vis spectroscopy). Fig.14Results shown in , all dry powder formulations contain 100 μM GSNO: 100 μM Asc: 10 μM EDTA. As mentioned above, for UV / Vis, the corresponding dry powder formulations were diluted in 4 mL PBS + 0.1 mM EDTA (PBSE) solution and mixed for 30 seconds, and 1 mL of the diluted formulation was injected into 3 mL PBSE in a disposable UV / VIS cuvette. Stability testing of GSNO: Asc: EDTA reagents was completed over a 6-month period. UV / VIS analysis showed excellent stability for all samples. Data for room temperature samples showed a decrease in stability at 4 months. However, this decrease may be due to weighing errors when filling the vials and / or uneven dry powder formulations when divided into batches at different storage temperatures. In addition, the headspace in the vials used to store the GSNO formulations is room air, which has some moisture. Therefore, the true shelf life stability of the GSNO formulations may be much better in a completely dry headspace atmosphere.

[0115] Preparations combined with saline

[0116] Two reagents (GSNO:EDTA with saline and GSNO:GSH with saline) were formed to test the packaging of the sinus rinse. The 1 month period was passed and no decomposition was detected at this time.

[0117] Example 3

[0118] Effect of 3A–GSNO:Asc (1:1) on the biofilm destruction of Staphylococcus aureus

[0119] Three strains of Staphylococcus aureus (SA817, SA1691 and SA1681) were inoculated in 96-well microtiter plates (1×10 4 Colony forming units / 100 μl or CFU / 100 μl) and incubate overnight. Remove planktonic bacteria and wash once with saline. Dilute different concentrations of GSNO:ascorbic acid (1:1 or equimolar) in saline and add the saline solution (0.12 M, 100 μl) to the bacterial biofilm retained on the well surface. Incubate the bacteria at room temperature (RT) for 4 hours and then determine the biofilm density by staining with crystal violet. Perform the experiment three times with six replicates for each strain (i.e., for Fig.15 The experimental results are shown in Fig.15 Specifically, Fig.15 The effect of GSNO / Asc on three different Staphylococcus aureus isolates (SA817, SA1691 and SA 1681) is shown. Fig.15As depicted in Figure 1, a significant reduction in biofilm density was observed as GSNO / Asc concentration increased between 0 mM and 1 mM. A single exposure of bacterial biofilms to GSNO / Asc solution for 4 hours reduced the biofilm density of the S. aureus strain by nearly 50% at a concentration of approximately 0.3 mM.

[0120] 3B–Effect of GSNO:Asc (1:1) on killing P. aeruginosa biofilm growth on CF cultures

[0121] Pseudomonas aeruginosa (1×10 4 Colony forming unit

[0122] / 100 μl or CFU / 100 μl) and incubate overnight. Remove planktonic bacteria and wash once with saline. Dilute different concentrations of GSNO:ascorbic acid (1:1 or equimolar) in saline and add the saline solution (0.12 M, 30 μl) to the bacterial biofilm retained in the wells. Incubate the bacteria at room temperature (RT) for 4 hours and then determine the biofilm density by staining with crystal violet. Perform 6 replicates (i.e., for Fig.16 2 experiments were performed (with N = 6 data points, plus or minus standard deviation) Fig.16 The experimental results are shown in Fig.16 Specifically, Fig.16 The effect of GSNO / Asc on Pseudomonas aeruginosa isolates is shown. Fig.16 As depicted in FIG, a significant decrease in biofilm density was observed as GSNO / Asc concentration increased between 0 mM and 2.5 mM. Fig.16 These results are similar to Fig.15 The results in Figure 2 show that although the destruction of the preformed biofilm of Pseudomonas aeruginosa microorganisms reaches the maximum limit, a slightly higher dose of GSNO / Asc is required. This may be related to the fact that Pseudomonas aeruginosa is a nitrifying organism with some NO reductase activity in it. In addition, the plateau observed at high doses may be related to the faster loss of NO from the thin layer of GSNO / Asc solution applied to the biofilm surface to the atmosphere above the biofilm / pore. At higher concentrations, the NO production rate will increase, but this is offset by the faster loss of NO into the air above the film.

[0123] Effect of 3C–GSNO:Asc (1:1) on killing of S. aureus biofilm growth on CF cultures

[0124] Progenitor (basal) airway epithelial cells isolated from cystic fibrosis patients were cultured at an air / liquid interface to promote mucociliary differentiation, similar to the in vivo airway epithelium. These cultures are referred to herein as CF airway epithelial cells.

[0125] The apical surface of CF airway epithelial cells was washed with PBS and infected with 10 μl of Staphylococcus aureus (1×10 5CFU / well or at a multiplicity of infection of 0.01) and incubated for 24 hours. Control samples (for determination of IL-8 and nitrite analysis) were washed in a similar manner but were not infected with S. aureus. The basolateral medium was changed, the apical surface was washed once with saline, and then 30 μl PBS containing different concentrations of GSNO / Asc (i.e., 0 mM, 0.3 mM, 0.625 mM, 1.25 mM, 2.5 mM, and 5 mM) was added to the apical surface and incubated for 4 hours. The apical surface was washed once with 0.2 ml of 0.15% bicarbonate in PBS. The wash was used for nitrite analysis ( Fig. 17B Cells were lysed in 1% Triton X-100 and serial dilutions of the cell lysate were plated to determine bacterial density ( Fig.17A IL-8 protein in the basolateral culture medium was measured by ELISA ( Fig. 17C ). Fig.17A Data represent median values ​​and ranges from 3 to 6 experiments. Fig. 17B and 17C The data represent mean ± standard deviation (SD). Fig.17A *p<0.05, which is different from the cell cultures treated with saline alone. Figures 17A to 17C The results in shoW that 0.625 mM GSNO / Asc reduced bacterial density by no more than 1 log unit, Without inducing IL-8, a pro-inflammatory cytokine. In addition, GSNO / Asc treatment did not induce cytotoxic or pro-inflammatory effects (determined by IL-8 release) at doses up to 5 mM. Fig. 17B It was shown that nitrite levels at the apical surface increased as expected due to the use of increasing levels of GSNO solution.

[0126] To demonstrate that the reduction in viable counts correlated with biofilm disruption, confocal microscopy was performed. CF cell cultures with established S. aureus biofilms on the apical surface were treated with PBS or GSNO / Asc as described above. The cultures were briefly washed and fixed in paraformaldehyde, blocked with 5% normal donkey serum, and then incubated with antibodies to S. aureus overnight at 4°C. Unbound antibodies were washed away, bound antibodies were detected by a secondary antibody conjugated to Alexafluor-488, and cells were counterstained with DAPI to detect nuclei. The cultures were fixed and visualized under a confocal microscope. Compared to PBS-treated cultures, GSNO / Asc-treated cultures showed few bacteria and visually no bacterial biofilm, indicating that GSNO / Asc disrupts bacterial biofilms in addition to killing bacteria. The results, which were initially colored and depicted bacteria in green and nuclei in blue, are shown in Figure 2. Fig.18A and 18B The PBS-treated cultures are shown in black and white, with lighter dots depicting bacteria and darker dots depicting nuclei. Fig.18A In the initial image, the color was predominantly green. In contrast, the GSNO / Asc treated cultures were shown in Fig.18B In the figure, the initial image is predominantly blue. Treatment with GSNO / Asc significantly reduced the density of bacterial biofilm growth on the apical surface of mucociliary differentiated cell cultures.

[0127] 3D-GSNO:Asc(1:1) and gentamicin kill Staphylococcus aureus colonizing CF airway epithelial cells Synergy

[0128] The apical surface of CF airway epithelial cells was washed with PBS and inoculated with 10 μl of Staphylococcus aureus (2 × 10 6 CFU / well) and incubated for 24 hours. The basolateral medium was changed, the apical surface was washed once with saline and treated apically with 30 μl PBS or saline containing 1.25 mM GSNO / Asc or saline containing 50 μg gentamicin or saline containing a mixture of 1.25 mM GSNO / Asc and 50 μg gentamicin. All cells were incubated for 4 hours. The apical surface was washed once with 0.2 ml of 0.15% bicarbonate in PBS and then once with PBS. The cells were lysed in 1% Triton X-100 and serial dilutions of the cell lysate were plated to determine bacterial density. The results are shown in Fig.19 (N=3). Data represent median and range from 3 experiments. Fig.19 In the figure, *p<0.05, which is different from PBS-treated cultures; #p<0.05, which is different from GSNO / Asc-treated cultures. These results show that a single application of GSNO / Asc has a significant effect on the number of viable S. aureus cells retained on the surface (20-30-fold increase). These results also show that the combination of gentamicin and GSNO / Asc is significantly more effective in killing bacteria than GSNO / Asc alone.

[0129] Effects of 3E-GSH and ascorbic acid on killing of Staphylococcus aureus colonizing CF airway epithelial cells

[0130] The apical surface of CF airway epithelial cells was washed with PBS and inoculated with 10 μl of Staphylococcus aureus (2 × 10 6 CFU / well) and incubated for 24 hours. The basolateral medium was changed, the apical surface was washed once with saline and treated apically with 30 μl PBS or saline containing 1.25 mM GSH or saline containing 1.25 mM ascorbic acid. All cells were incubated for 4 hours. The apical surface was washed once with 0.2 ml of 0.15% bicarbonate in PBS and then once with PBS. The cells were lysed in 1% Triton X-100 and serial dilutions of the cell lysate were plated to determine bacterial density. The results are shown in Fig. 20 Data represent medians and ranges from 4 to 5 experiments. Fig. 20 No significant changes in bacterial density were observed in cultures treated with GSH or ascorbic acid. Fig.17A and 19 The effect seen in Figure 5 is due to the release of NO from GSNO.

[0131] Effect of 3F–GSNO:Asc (1:1) on killing of sinonasal epithelial cells in culture established from patients with CRS Role of Staphylococcus aureus biofilm growth

[0132] Sinus epithelial cell cultures were established from two patients (A and B) with CRS. Cell cultures were infected with S. aureus as described in Example 3C. The basolateral media was changed, the apical surface was washed once with saline, and apically treated with 30 μl of saline or saline containing 1.25 mM GSNO / Asc and incubated for 4 hours. The apical surface was washed once with 0.2 ml of 0.15% bicarbonate in PBS and then once with PBS. Cells were lysed in 1% Triton X-100, and serial dilutions of the cell lysate were plated to determine bacterial density. Results are shown in Fig.21 Data represent medians and ranges from 4 or 6 experiments. Fig.21 *p<0.05, which is different from the corresponding PBS-treated cultures. Fig.21 The results in Figure 2 show that GSNO / Asc treatment significantly reduced bacterial density on CRS cell cultures. More specifically, a single application of GSNO / Asc resulted in an approximately 10-fold reduction in viable bacterial counts on the cell surface.

[0133] Effect of 3G-ascorbic acid on the growth of Staphylococcus aureus biofilm on CF airway epithelial cells

[0134] 10 μl of Staphylococcus aureus (1×10 10 CFU / ml) were apically infected on the apical surface of CF airway epithelial cells and cultured for 24 hours. The basolateral medium was changed, the apical surface was washed with saline, and apically treated with 30 μl PBS or saline containing 1.25 mM GSNO / Asc and cultured for 3 hours. The cultures were washed once with PBS, fixed in glutaraldehyde, and processed for scanning electron microscopy. Fig.22A and 22B Bacterial biofilms on the surface of CF airway epithelial cells not treated with GSNO-Asc were shown. Fig. 22C and 22D Shown are bacterial biofilms on the surface of CF airway epithelial cells treated with GSNO-Asc. Fig.22A and 22B and Fig. 22C and 22D In comparison, samples treated with GSNO-Asc had considerably fewer bacteria and a smaller biofilm size.

[0135] Effect of 3H-ascorbic acid on ciliary beat frequency

[0136] Control CF airway epithelial cells were not infected. 10 μl of Staphylococcus aureus (2.5×10 3 CFU / ml) and cultured for 24 hours. The basolateral medium was changed, the apical surface was washed with saline, and the infected cultures were treated apically with 30 μl PBS or saline containing 1.25 mM GSNO / Asc and cultured for 3 hours. The cultures were washed with 0.2 ml 0.15% sodium bicarbonate and imaged using high-speed video microscopy. The liquid was aspirated from the apical surface and 10 μl of medium was added to prevent drying while the cells were imaged using high-speed video microscopy to quantify the ciliary beat frequency. Specifically, the ciliary beat frequency was measured in random fields using NIH image J software. The results are shown in Fig.23 Data represent median values ​​from 6 experiments and *p<0.05, which is different from the corresponding PBS-treated and infected cultures. Fig.23 Results for infected cultures treated with PBS showed that infection with S. aureus reduced the ciliary beat frequency of CF airway epithelial cells. Fig.23 The results for infected cultures treated with GSNO:Asc show a trend toward improved ciliary beat frequency (compared to infected cultures treated with PBS).

[0137] Example 4

[0138] Two catheter blocking solutions were prepared in PBS (i.e., vehicle and salt) with different GSNO and GSH concentrations. The first catheter blocking solution contained a 1:1 molar (equimolar) ratio of GSNO:GSH, wherein GSNO and GSH were each at a 2 mM concentration. The second catheter blocking solution contained a 1:1 molar (equimolar) ratio of GSNO:GSH, wherein GSNO and GSH were each at a 10 mM concentration.

[0139] A silicone rubber catheter tubing (ID = 1.58 mm, OD = 3.18 mm, volume 176 μL) was used and closed at the distal end with a plug. The tubing was filled with the first catheter closing solution. NO permeated the silicone rubber catheter tubing, therefore, NO release from the outer surface of the tubing was monitored by chemiluminescence. The results are shown in Fig.24 As depicted, NO release was observed for at least 4 hours with flux approaching physiologically relevant levels required for antiplatelet and antimicrobial activity.

[0140] A polyurethane (PU) catheter tube was also used and closed at the distal end with a plug. The PU tube was filled with a second catheter closing solution. NO permeated the PU tube, and therefore, NO release from the outer surface of the tube was monitored by chemiluminescence. The results are shown in Fig.25As depicted, NO release was observed for at least 15 hours. The flux was initially slightly above physiologically relevant levels (thus, high antimicrobial activity), and then the flux slowly decreased over time to more physiologically relevant levels required for antiplatelet and antimicrobial activity.

[0141] References throughout the specification to "one example," "another example," "an example," etc., mean that a particular element (e.g., feature, structure, and / or characteristic) described in conjunction with the example is included in at least one example described herein, and may or may not be present in other examples. It should also be understood that the elements described for any example may be combined in the various examples in any suitable manner, unless the context clearly dictates otherwise.

[0142] It should be understood that the ranges provided herein include the ranges and any value or sub-range within the ranges. For example, the range of about 25 μM to about 10 mM should be interpreted as not only including the clearly stated limits of about 25 μM to about 10 mM, but also including individual values ​​(such as 29 μM, 250 μM, 1000 μM (1 mM), 5.75 mM, etc.) and sub-ranges (such as about 50 μM to about 7 mM, about 550 μM to about 1.5 mM, etc.). In addition, when describing a numerical value with "about", this means including a smaller change (up to + / - 10%) from the value.

[0143] In describing and claiming the examples disclosed herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0144] While several examples have been described in detail, it should be understood that the disclosed examples may be modified. Therefore, the foregoing description should be considered non-limiting.

Claims

1. A nitric oxide (NO) generating formulation comprising: A single powder composition formed by mixing together the following ingredients: S-Nitrosothiol (RSNO) powder; Salt; and ascorbic acid or an ascorbate salt to accelerate the rate of release of NO from the RSNO after the single powder composition is dissolved in a liquid carrier; in: a) the NO producing formulation is an antimicrobial sinus treatment formulation and the salt is selected from the group consisting of sodium chloride, sodium bicarbonate, calcium chloride, sodium phosphate buffer, potassium phosphate buffer, and combinations thereof; or b) the NO generating preparation is a catheter lock solution preparation, and the salt is selected from the group consisting of sodium chloride, sodium bicarbonate, sodium phosphate buffer, potassium phosphate buffer, and combinations thereof.

2. The NO producing formulation of claim 1, wherein the NO producing formulation is an antimicrobial sinus treatment formulation and the amount of RSNO powder in the antimicrobial sinus treatment formulation corresponds to a RSNO concentration ranging from about 25 μM to about 10 mM in an antimicrobial sinus treatment solution formed when the RSNO powder, salt, and ascorbic acid or ascorbate are dissolved in a predetermined amount of a liquid carrier.

3. The NO generating formulation of claim 1, wherein the NO generating formulation is an antimicrobial sinus treatment formulation, and the antimicrobial sinus treatment formulation further comprises a metal ion chelator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), sodium citrate, and combinations thereof.

4. The NO producing formulation of claim 1, wherein the NO producing formulation is a catheter lock solution formulation, and the amount of RSNO powder in the catheter lock solution formulation corresponds to a RSNO concentration ranging from about 500 μM to about 50 mM in a catheter lock solution formed when RSNO powder, salt, and ascorbic acid or ascorbate are dissolved in a predetermined amount of a liquid carrier.

5. The NO generating preparation according to claim 1, wherein the NO generating preparation is a catheter lock solution preparation, and the catheter lock solution preparation further comprises an anticoagulant selected from the group consisting of heparin, ethylenediaminetetraacetic acid (EDTA), sodium citrate, and combinations thereof.

6. The NO generating formulation of claim 1, wherein the molar ratio of RSNO to the ascorbic acid or ascorbate ranges from 1:10 to 10:

1.

7. The NO generating preparation as claimed in claim 1, wherein the RSNO powder is selected from the group consisting of S-nitrosoglutathione (GSNO), S-nitroso-cysteine, S-nitroso-N-acetyl-penicillamine, S-nitroso-penicillamine and S-nitroso-human serum albumin.

8. The NO generating formulation of claim 1, wherein the single powder composition consists of the S-nitrosothiol (RSNO) powder, the salt, and the ascorbic acid or ascorbate.

9. The NO generating formulation of claim 3, wherein the single powder composition consists of the S-nitrosothiol (RSNO) powder, the salt, the ascorbic acid or ascorbate, and the metal ion chelator.

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