HK40115333A - Aqueous solution comprising a glutathione salt - Google Patents

Description

Aqueous solutions containing glutathione salt

Cross-references to related applications

This application claims the benefit of U.S. Provisional Application No. 63/296,405, filed January 4, 2022, the entire contents of which are incorporated herein by reference.

background

Technical Field

This disclosure relates to the general field of chemical and pharmaceutical formulations. In some aspects, this disclosure relates to pharmaceutical formulations (e.g., pharmaceutical formulations containing glutathione suitable for pulmonary administration) and methods of use thereof. In some aspects, this disclosure provides pharmaceutical formulations and products related to ARINA-1 for the treatment of patients with chronic inflammatory lung diseases characterized by excessive mucus and inflammation, such as lung transplant recipients, patients with cystic fibrosis (CF), non-CF bronchiectasis, chronic obstructive pulmonary disease (COPD), and other inflammatory lung diseases.

Background Technology

Excessive mucus and inflammation are hallmarks of chronic inflammatory lung disease. Ion imbalances lead to excessive mucus production and chronic dysregulation of inflammation, both of which can cause lung damage and scarring, ultimately leading to lung failure. Ramsey et al., American Journal of Respiratory and Critical Care Medicine 201(6):661–670 (2020); Borok et al., The Lancet 338:215–216 (1991); Behr et al., Transplantation 69:1856–1860 (2000). Glutathione is an important antioxidant ion that contributes to the integrity of mucus structure (Meldrum et al., Scientific Reports 8(1): Article No. 5802(2018) https://doi.org/10.1038/s41598-018-24223-3) and regulates inflammatory responses. Elferink, Immunobiology 184(1):25-36(1991). Chronic inflammation also leads to increased airway acidity, causing lung damage. Glutathione dysregulation and increased airway acidity directly lead to thickened mucus, accompanied by delayed or absent mucociliary clearance, which in turn leads to obstruction of small and medium airways. This creates an environment conducive to bacterial colonization, chronic infection, and inflammation.

Current treatments for chronic inflammatory lung disease (CIL) include alfa-chain enzymes and hypertonic saline. Alfa-chain enzymes (Pulmozyme; Genentech, Inc.) are inhaled recombinant human deoxyribonucleases that cleave extracellular DNA accumulated in CF mucus (Yang et al., Cochrane Database Syst Rev. 4:CD001127 (2016)). Hypertonic saline is thought to hydrate the mucus layer via osmotic pressure-driven fluid transfer (Donaldson et al., N Engl J Med. 354:241-250 (2006)). However, these agents have limited and variable efficacy in improving lung function, disease progression, and quality of life (Yang et al., Cochrane Database Syst Rev. 4:CD001127 (2016); Henke et al., Paediatr Respir Rev. 8:24-29 (2007)).

ARINA-1 is a compound pharmaceutical formulation for the treatment of CF and other chronic inflammatory lung diseases. ARINA-1 is prepared by dissolving bicarbonate, L-glutathione, and ascorbic acid in water. The resulting aqueous solution is first nebulized and then applied to the patient's lungs. See, for example, Adewale et al., Am J Respir Cell Mol Biol. 63:362-373 (2020); U.S. Patent Nos. 9,308,234 and 11,058,743; and U.S. Patent Application Nos. 2019/0351005 and 2020/0397849. There is a need in the art for stable pharmaceutical formulations and products for the treatment of lung diseases.

Summary of the Invention

This disclosure provides pharmaceutical formulations and products related to ARINA-1 for the treatment of patients with chronic inflammatory lung diseases characterized by excessive mucus and inflammation, such as lung transplant recipients, patients with cystic fibrosis (CF), non-CF bronchiectasis, chronic obstructive pulmonary disease (COPD), and other inflammatory lung diseases.

The formulation of ARINA-1 and related pharmaceutical preparations involves dissolving bicarbonates (such as sodium bicarbonate), L-glutathione, and ascorbic acid in water. The applicant discovered that this aqueous composition exhibits surprising stability when prepared, packaged, and stored under a carbon dioxide atmosphere.

In one aspect, this disclosure provides a formulation comprising an aqueous solution in a closed container having a headspace, wherein:

(i) The aqueous solution contains a salt having formula I:

(ii) The atmosphere of the headspace contains 90% or more carbon dioxide by volume; and

(iii) M ⁺ is Na ⁺ , Li ⁺ , K ⁺ or Cs ⁺ .

In another aspect, this disclosure provides a method for preparing an aqueous solution containing a salt of formula I, the method comprising:

(i) L-glutathione, ascorbic acid and M ⁺ _HCO3- are ^dissolved in water for injection under a carbon dioxide atmosphere to obtain an aqueous solution, wherein M ⁺ is Na ⁺ , Li ⁺ , K ⁺ or Cs ⁺ ;

(ii) Transfer a portion of the aqueous solution to a container;

(iii) Cover the aqueous solution with carbon dioxide; and

(iv) Seal the container with a stopper.

In another aspect, this disclosure provides an aqueous solution containing a salt of formula I, which ^is prepared by dissolving L-glutathione, ascorbic acid and M ⁺ _HCO3- in water for injection under a carbon dioxide atmosphere, wherein M ⁺ is Na ⁺ , Li ⁺ , K ⁺ or Cs ⁺ .

Brief description of the attached figures

Figure 1 is a line graph showing the pH versus time of an aqueous solution containing sodium bicarbonate, L-glutathione, and ascorbic acid prepared under a nitrogen atmosphere.

Figure 2 is a line graph showing the pH versus time of an aqueous solution containing sodium bicarbonate, L-glutathione, and ascorbic acid prepared under a carbon dioxide atmosphere.

Figure 3 shows a partial NMR spectrum of an aqueous solution containing sodium bicarbonate, L-glutathione, and ascorbic acid prepared under a carbon dioxide atmosphere.

Figure 4 is a flowchart showing the compounding steps for preparing ARINA-1 under a carbon dioxide atmosphere.

Detailed Implementation

I. Formulations disclosed herein

In one aspect, referred to as "Embodiment I", this disclosure provides a formulation comprising an aqueous solution in a closed container having a headspace, wherein:

(i) The aqueous solution contains a salt having formula I:

(ii) The atmosphere of the headspace contains 90% or more carbon dioxide by volume; and

(iii) M ⁺ is Na ⁺ , Li ⁺ , K ⁺ or Cs ⁺ .

On the other hand, the aqueous solution of Embodiment I has a pH of 6.0 ± 0.4 for 24 hours or longer at about 5°C.

On the other hand, the aqueous solution of Embodiment I has a pH of 6.0 ± 0.3 for 24 hours or longer at about 5°C.

On the other hand, the aqueous solution of Embodiment I has a pH of 6.0 ± 0.2 for 24 hours or longer at about 5°C.

On the other hand, the aqueous solution of Embodiment I has a pH of 6.0 ± 0.1 for 24 hours or longer at about 5°C.

On the other hand, the aqueous solution of embodiment I also contains a salt having formula II:

On the other hand, the aqueous solution of embodiment I also contains a salt having formula III:

On the other hand, the atmosphere of the top space in Embodiment I contains 80% or more carbon dioxide by volume.

On the other hand, the atmosphere of the headspace in Embodiment I contains 85% by volume or more carbon dioxide.

On the other hand, the atmosphere of the top space in Embodiment I contains 90% by volume or more carbon dioxide.

On the other hand, the atmosphere of the headspace in Embodiment I contains 95% by volume or more carbon dioxide.

In another aspect, the aqueous solution of Embodiment I contains about 10% to about 20% by weight of a salt of Formula I.

In another aspect, the aqueous solution of Embodiment I contains about 13% to about 17% by weight of a salt of Formula I.

In another aspect, the aqueous solution of Embodiment I contains about 14.7% by weight of a salt of Formula I.

In another aspect, the aqueous solution of Embodiment I contains about 5% by weight to about 15% by weight of a salt of Formula II.

In another aspect, the aqueous solution of Embodiment I contains about 7% by weight to about 11% by weight of a salt of Formula II.

In another aspect, the aqueous solution of Embodiment I contains about 9.1% by weight of a salt of Formula II.

On the other hand, the aqueous solution of Embodiment I has a density of about 1.13 g/L.

On the other hand, the aqueous solution in Embodiment I is frozen.

On the other hand, in implementation I, M ⁺ is Na ⁺ .

On the other hand, in implementation I, M ⁺ is Li ⁺ .

On the other hand, in implementation I, M ⁺ is K ⁺ .

On the other hand, in implementation I, M ⁺ is Cs ⁺ .

In another embodiment, the formulation of Embodiment I is packaged as a single unit dose. Alternatively, the single unit dose is in a sealed vial.

On the other hand, the formulation of Implementation Method I is sold, distributed or administered as part of a pharmaceutical product.

In some respects, the formulation of embodiment I may also include any one or more of the other aspects disclosed herein.

II. Preparation method of the formulation

In another aspect, referred to as "Embodiment II," this disclosure provides a method for preparing an formulation of Embodiment I (or including one or more other aspects of the above disclosure of Embodiment I), the method comprising:

(i) L-glutathione, ascorbic acid and M ⁺ _HCO3- are ^dissolved in water for injection under carbon dioxide to obtain an aqueous solution, wherein M ⁺ is Na ⁺ , Li ⁺ , K ⁺ or Cs ⁺ ;

(ii) Transfer a portion of the aqueous solution to a container;

(iii) Cover the aqueous solution with carbon dioxide; and

(iv) Seal the container with a stopper.

In another embodiment, about 8% to about 18% by weight of L-glutathione, about 3% to about 13% by weight of ascorbic acid and about 3% to about 13% by weight ^of M ⁺ _HCO3- are dissolved in about 62% to about 82% by weight of water for injection to obtain an aqueous solution of embodiment II.

On the other hand, the aqueous solution of Embodiment II has a pH of 6.0 ± 0.4 over a period of 24 hours or longer at approximately 5°C.

On the other hand, the aqueous solution of Embodiment II has a pH of 6.0 ± 0.3 for 24 hours or longer at about 5°C.

On the other hand, the aqueous solution of Embodiment II has a pH of 6.0 ± 0.2 for 24 hours or longer at about 5°C.

On the other hand, the aqueous solution of Embodiment II has a pH value of 6.0 ± 0.1 for 24 hours or longer at about 5°C.

On the other hand, in embodiment II, M ⁺ is Na ⁺ , that is, M ⁺ _HCO3- is sodium ^bicarbonate .

On the other hand, in embodiment II, M ⁺ is Li ⁺ , that is, M ⁺ _HCO3- is lithium ^bicarbonate .

On the other hand, in embodiment II, M ⁺ is K ⁺ , that is, M ⁺ _HCO3- is potassium ^bicarbonate .

On the other hand, in embodiment II, M ⁺ is Cs ⁺ , that is, M ⁺ _HCO3- is cesium ^bicarbonate .

In another aspect, about 11% to about 15% by weight of L-glutathione, about 5% to about 9% by weight of ascorbic acid and about 5% to about 9% by weight of sodium bicarbonate are dissolved in about 68% to about 76% by weight of water to obtain an aqueous solution of embodiment II, wherein M ⁺ is Na ⁺ .

On the other hand, about 13.0% by weight of L-glutathione, about 7.6% by weight of ascorbic acid and about 7.3% by weight of sodium bicarbonate are dissolved in about 72.0% by weight of water to obtain an aqueous solution of embodiment II, wherein M ⁺ is Na ⁺ .

In some respects, the method of Implementation II may also include any one or more of the other aspects disclosed herein.

III. Products with Method-Specific Requirements

In another aspect, referred to as "Embodiment III", this disclosure provides an aqueous solution comprising a salt having Formula I:

It is prepared by dissolving L- ^glutathione , ascorbic acid and M ⁺ _HCO3- in water for injection under a carbon dioxide atmosphere, wherein M ⁺ is Na ⁺ , Li ⁺ , K ⁺ or Cs ⁺ .

In another aspect, about 8% to about 18% by weight of L ^- glutathione, about 3% to about 13% by weight of ascorbic acid and about 3% to about 13% by weight of M ⁺ _HCO3- are dissolved in about 62% to about 82% by weight of water for injection to obtain an aqueous solution of embodiment III.

On the other hand, the aqueous solution of Embodiment III has a pH of 6.0 ± 0.4 over a period of 24 hours or longer at approximately 5°C.

On the other hand, the aqueous solution of Embodiment III has a pH of 6.0 ± 0.3 for 24 hours or longer at about 5°C.

On the other hand, the aqueous solution of Embodiment III has a pH of 6.0 ± 0.2 for 24 hours or longer at about 5°C.

On the other hand, the aqueous solution of Embodiment III has a pH of 6.0 ± 0.1 for 24 hours or longer at about 5°C.

On the other hand, the aqueous solution of embodiment III also contains a salt having formula II:

On the other hand, the aqueous solution of embodiment III also contains a salt having formula III:

On the other hand, in embodiment III, M ⁺ is Na ⁺ , that is, M ⁺ _HCO3- is sodium ^bicarbonate .

On the other hand, in embodiment III, M ⁺ is Li ⁺ , that is, M ⁺ _HCO3- is lithium ^bicarbonate .

On the other hand, in embodiment III, M ⁺ is K ⁺ , that is, M ⁺ _HCO3- is potassium ^bicarbonate .

On the other hand, in embodiment III, M ⁺ is Cs ⁺ , that is, M ⁺ _HCO3- is cesium ^bicarbonate .

In another aspect, about 11% to about 15% by weight of L-glutathione, about 5% to about 9% by weight of ascorbic acid and about 5% to about 9% by weight of sodium bicarbonate are dissolved in about 68% to about 76% by weight of water to obtain an aqueous solution of embodiment III.

On the other hand, about 13.0% by weight of L-glutathione, about 7.6% by weight of ascorbic acid and about 7.3% by weight of sodium bicarbonate are dissolved in about 72.0% by weight of water to obtain an aqueous solution of embodiment III.

In some respects, the aqueous solution of Embodiment III may also include any one or more of the other aspects disclosed herein.

IV. Instructions for Use

The formulations or aqueous solutions disclosed herein, including those described in Sections I-II above (e.g., Embodiments I and III and other aspects thereof), can be used to treat, prevent, or alleviate various diseases, conditions, or disorders. In particular, these formulations and aqueous solutions can be used as treatments for diseases, conditions, or disorders, or to alleviate their symptoms, wherein the application of L-glutathione or its salts and/or ascorbic acid or its salts provides benefit.

In one aspect, a method of treating or preventing lung or airway obstruction in a subject of need comprises applying a therapeutically effective amount of the preparation described in Section I or the aqueous solution described in Section III to the subject, for example by inhalation.

In one aspect, the lung or airway disorder is selected from the group consisting of: chronic inflammatory lung disease, pulmonary fibrosis, pulmonary vasculitis, pulmonary sarcoidosis, lung transplant-related inflammation and/or infection, acute or chronic lung rejection, chronic lung transplant dysfunction (CLAD), pulmonary hypertension, bronchitis, sinusitis, asthma, cystic fibrosis, bacterial infection, fungal infection, parasitic infection, viral infection, chronic obstructive pulmonary disease (COPD), bronchiolitis obliterans syndrome (BOS), and primary ciliary dyskinesia (PCD). Pulmonary alveolar proteinosis, idiopathic pulmonary fibrosis, eosinophilic pneumonia, eosinophilic bronchitis, acute respiratory distress syndrome (ARDS), inflammation and/or infection associated with mechanical ventilation, ventilator-associated pneumonia, complications associated with acute or chronic tracheostomy placement, airway obstruction or disease associated with asbestos, airway obstruction or disease associated with dust, silicosis, and airway disease or obstruction associated with radiation or chemical agents, and any combination thereof.

On the other hand, the lung or airway obstruction is selected from the group consisting of: chronic inflammatory lung disease, inflammation and/or infection associated with lung transplantation, acute or chronic lung rejection, chronic graft dysfunction (CLAD), asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), complications associated with acute or chronic tracheostomy placement, and any combination thereof. In another embodiment, the method further includes administering additional therapeutic agents to the subject.

On the other hand, the lung or airway obstruction is incidental to the patient's radiotherapy and/or chemotherapy.

In another aspect, this disclosure provides a method for treating or preventing airway infections in a subject, the method comprising administering a therapeutically effective amount of the preparation described in Section I to the subject. In another embodiment, the preparation described in Section I is administered to the subject in combination with one or more antibiotics. The antibiotics may be administered topically to the lungs and/or systemically.

On the other hand, this disclosure provides a method for treating or preventing airway inflammation in a subject in need, the method comprising administering the preparation described in Section I to the subject.

On the other hand, this disclosure provides a method for treating or preventing mucosal tissue diseases or disorders in a subject, the method comprising administering a therapeutically effective amount of the preparation described in Section I to the subject. Non-limiting examples of mucosal tissues include the mouth, nose, eyes, ears, upper respiratory tract, lower respiratory tract, gastrointestinal tract, vagina, rectum, and urethra.

On the other hand, this disclosure provides a method for treating or preventing mucosal-related diseases or disorders in a subject in need, the method comprising applying a therapeutic amount of the preparation described in Section I to an appropriate mucosa of the subject. In one embodiment, the mucosa is the lung, such as the middle bronchus or deep lung (alveolar region); in other embodiments, the mucosa is one or more of the eye, mouth, nose, rectum, genitourinary tract, and vagina.

On the other hand, the preparations described in Section I are used to treat or prevent bronchiolitis obliteran and military-related lung injury, i.e., lung injury in military personnel whose airways have been damaged due to unknown exposure.

The treatment methods disclosed herein include administering a therapeutically effective amount of the preparation described in Section I to a subject in need, such as a human patient. The suitability of such treatment depends on the individual case and requires a medical evaluation (diagnosis) that takes into account the presence of signs, symptoms, and/or functional impairments, the risk of developing specific signs, symptoms, and/or functional impairments, and other factors.

In some respects, the formulations described in Section I are atomized before being applied to the subject.

V. Definition

The term "ARINA-1" refers to a pharmaceutical preparation obtained by mixing L-glutathione, ascorbic acid, and sodium bicarbonate in water to obtain an aqueous solution. When prepared and stored under a _CO2 atmosphere, the chemical entities present in ARINA-1 may include sodium L-ascorbate, sodium L-glutathione/bicarbonate, and sodium L-glutathione, as shown in Scheme 1.

Option 1

The stoichiometry of each component of ARINA-1 is shown in Table 1.

Table 1

The term "pharmaceutical product" refers to a product suitable for human use. In some respects, pharmaceutical products are suitable for human use and are regulated by the U.S. Food and Drug Administration (FDA) or its foreign equivalents (including, but not limited to, the European Medicines Agency (EMA), the China National Medical Products Administration (NMPA), and the Japanese Ministry of Health, Labour and Welfare (MHLW)).

The term “preparation” means one or more pharmaceutical products intended for human use, which are (i) finished dosage forms; or (ii) preparations, formulations and/or administrations of finished dosage forms.

The term "container" refers to a pharmaceutically acceptable container, including a cavity suitable for containing an aqueous solution comprising one or more pharmaceutical products. The "closed end" of a container refers to the end of the cavity without an opening. The "open end" of a container refers to the end of the cavity opposite the closed end. Exemplary containers include, but are not limited to, vials, syringes, cartridges, bags (such as polypropylene and polyurethane bags), and ampoules. In one embodiment, the container is a vial.

As used in this article, the term "stopper" refers to any product that prevents aqueous solutions and gases (such as carbon dioxide) from leaking out of the opening of a container.

The term "closed container" as used in this article refers to a container with a stopper.

The term “headspace” as used in this article refers to the region within the container cavity between the aqueous solution and the stopper when the open end of the container is away from the direction of gravity.

As used herein, the term "atmosphere" refers to a layer of one or more gases in the top space of a closed container. These gases include, but are not limited to, oxygen, nitrogen, argon, and carbon dioxide. In one embodiment, the atmosphere in the pharmaceutical product of this disclosure contains 75% by volume or more carbon dioxide, such as 80% by volume or more, 85% by volume or more, 90% by volume or more, 95% by volume or more, or 99% by volume or more.

As used herein, the term "% by weight" in relation to aqueous solutions refers to the mass of one component divided by the total mass of all components (including, for example, water for injection) multiplied by 100. For example, in an aqueous solution containing 161 g of sodium L-glutathione/bicarbonate (i.e., compound of formula I, where M ⁺ is Na ⁺ ), 99 g of sodium ascorbate (i.e., compound of formula II, where M ⁺ is Na ⁺ ), 1 g of sodium bicarbonate, and 829 g of water, the % by weight of sodium L-glutathione/bicarbonate is 14.8% (161 g/1090 g = 0.148 x 100 = 14.8%). Similarly, when 150 g of L-glutathione, 88 g of ascorbate, and 84 g of sodium bicarbonate are dissolved in 829 g of water to obtain an aqueous solution, the % by weight of L-glutathione is 13.0% (150 g/1151 g = 0.130 x 100 = 13.0%).

The terms “one” and “a kind” refer to one or more than one.

In this article, the term “about” includes the cited number ±10%. Therefore, “about 10” refers to 9-11.

Example

General methods

Material

L-Ascorbic acid (batch number 1200851004) and reduced L-glutathione (batch number B200465) used in the 2-L scale experiments were sourced from CSPC Weisheng Pharmaceutical (Shijiazhuang) Co., Ltd. and Shandong Jincheng Biopharmaceutical Co., Ltd., respectively. L-Ascorbic acid (product number A92902) and reduced L-glutathione (product number G4251) used in the 0.75-L scale experimental runs involving carbon dioxide coverage were purchased from Millipore Sigma. Sodium bicarbonate (product number S6014) used in all experiments was purchased from Millipore Sigma. Deionized water was supplied by Millipore Water Systems. All other raw materials and solvents used for the analysis were purchased from commercial suppliers and used as is.

NMR instruments

NMR was used to evaluate the titers of L-ascorbic acid, reduced L-glutathione, L-glutathione disulfide, and any unknown impurities during the demonstration run. ^1H NMR spectra were obtained at 25 °C using a Bruker Avance III 500 MHz NMR system equipped with a 5 mm triple-resonance cryoprobe. Heavy water was used as the NMR solvent, and the resonance from calcium formate was used as a reference to determine chemical shifts and integral values. NMR spectral data were processed using MNova software.

Example 1

ARINA-1 was compounded under N ₂ conditions.

Referring to Table 2, the 2-L scale preparation of ARINA-1 was carried out in a jacketed four-necked glass reactor equipped with a top stirrer, thermometer, gas inlet connector, solids feed port, and a second connector to limit the flow rate of nitrogen leaving the flask through the Claysen connector. Nitrogen was introduced into the flask through a flow meter capable of measuring a flow rate of 0.05–0.5 L/min. Temperature control of the reaction was achieved by recirculating the water/ethylene glycol mixture in the vessel jacket to a Haake EZ-Cool 80 circulating heater/cooler.

Table 2

pH was monitored at the end of L-glutathione neutralization, and at the temperatures (°C) shown in Table 3 and Figure 1, the pH was measured to rise by approximately +0.1 pH units per hour.

Table 3

Example 2

ARINA-1 was compounded under CO2 _conditions .

Referring to Table 4, the 750-mL scale preparation of ARINA-1 was carried out in a jacketed four-necked glass reactor equipped with a top stirrer, thermometer, gas inlet connector, and solids feed port. Carbon dioxide was introduced into the reactor via a gas flow meter with a measurement range of 0.05–0.5 L/min. Temperature control of the reaction was achieved by circulating a water/ethylene glycol mixture between the vessel jacket and a Haake EZ-Cool 80 circulating heater/cooler. The compounding process steps are shown in Figure 4.

Table 4

Process Description

1. Add 600g of deionized water to the reactor. This amount of water, together with the rinsing water introduced in step 6, yields a product density of 1.12 to 1.13 g/mL. Start stirring by setting the top stirrer speed to approximately 90-100 rpm. The water temperature in the demonstration experiment was 21°C.

2. Add 63.0g of sodium bicarbonate and stir continuously for about 10 minutes until most of the sodium bicarbonate dissolves.

3. Replace the air in the top space of the flask completely with gaseous carbon dioxide from a gas cylinder source equipped with a suitable gas regulator.

4. Add 66.0 g of L-ascorbic acid to the flask, ensuring the rate of addition is controlled to allow for the evolution of carbon dioxide gas. The reaction should be completed within 2-3 minutes after the solid addition, yielding a clear solution. Then, step 5 can begin.

5. Add 112.5 g of reduced L-glutathione to the flask, ensuring the rate of addition is controlled to prevent carbon dioxide gas generation. The reaction should be completed within 4-5 minutes after the final addition of the solid, yielding a clear solution.

6. Weigh out 21.75g of deionized water and use it to rinse any solids remaining in the solids feeding funnel and/or the upper wall of the container, where there may be adhering solids. Rapidly increase the stirring speed and maintain it for 10-15 seconds to splash off any solids or liquids adhering to the container walls, thereby obtaining a completely homogeneous mixture.

7. Resume a slower stirring speed (but maintain a stirring speed sufficient to promote heat transfer) and immediately cool the solution to 2-6°C.

pH was monitored at the temperatures (°C) shown in Table 5 and Figure 2 at the end of L-glutathione neutralization.

Table 5

In Example 1, the problem with continuous purging or blowing of nitrogen was that the nitrogen flow removed residual _CO2 from the system, which in turn promoted the decomposition of carbonic acid formed by the neutralization reaction. This raised the pH value beyond the target range of 5.8–6.4. In experiments using slow nitrogen purging, the pH of the compound product increased from 6.0 to 7.3 after approximately 18 hours of inerting following the reaction (data not shown).

Henry's Law states that the amount of gas dissolved in a liquid is directly proportional to its partial pressure above the liquid. Under slow, continuous nitrogen purging, the partial pressure of _CO2 in the headspace above the product solution remains close to zero. However, if the headspace is inerted with gaseous _CO2 , the _CO2 pressure will become 1 atmosphere, which will provide the driving force to reverse the _CO2 loss and establish a stable pH equilibrium. This principle is demonstrated in this example.

Reduced L-glutathione is an acid neutralized by sodium bicarbonate, and gaseous _CO2 generation was not expected to cease until an equilibrium was established between dissolved _CO2 in the solution and _CO2 in the headspace. As with previous runs under, for example, nitrogen, the pH rose from approximately 5.95 to 6.10 after solid dissolution, and gas generation remained significant even when the solution was cooled to 2–6 °C. Equilibrium was established once the target pH range of 6.0–6.1 was reached. In the following 4 hours of experimentation, increasing and decreasing the temperature had no significant effect on the pH. A pH of 6.0 was again obtained after maintaining the solution under a slight positive pressure of _CO2 for 20+ hours. NMR analysis of solution samples yielded determinations of 99.7% for L-ascorbic acid and 99.0% for reduced L-glutathione. See Figure 3. The disulfide level was 0.25%, and no unexpected resonances were found, indicating the absence of unidentified degradation products at or above 0.2%. Therefore, using _CO2 covering unexpectedly stabilized the pH within its target range, keeping the product pH (and _CO2 content) consistent throughout the vial filling cycle as needed, while excluding oxygen and controlling disulfide formation.

All inputs in this embodiment were carefully measured by weight to allow analysis of the actual product _CO2 content corresponding to a pH of 6.1. As shown in the following stoichiometric calculations for 1 L of the ARINA-1 formulation, the expected product weight is 1107.6 g if all _CO2 has been expelled, and 1151.0 g if all _CO2 is unexpectedly retained in solution. A 500 mL portion of the formulation in this embodiment was weighed, yielding a result of 1125.8 g/L.

This corresponds to a carbonic acid content of 18.2 g/L (0.29 mol), or approximately 30% of the combined molar equivalent of the two APIs. Considering that _CO2 has a solubility of 0.034 g/L in water at 0°C and 1 atm, it can be inferred that the presence of the solute contributes to stabilizing carbonic acid in an unexpected way. Reduced L _- glutathione sodium salt is most likely to play a stabilizing role because it can form a carbonic acid complex:

Having provided a thorough description of the compounds, compositions, methods, formulations, and products described herein, those skilled in the art will understand that these can be practiced under a wide range of equivalent conditions, formulations, and other parameters without affecting the scope of the compounds, compositions, methods, formulations, and products or any embodiments thereof provided herein. All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

Claims (27)

1. A formulation comprising an aqueous solution in a closed container having a headspace, wherein: (i) The aqueous solution contains a salt having formula I: (ii) The atmosphere of the headspace contains 90% or more carbon dioxide by volume; and (iii) M ⁺ is Na ⁺ , Li ⁺ , K ⁺ or Cs ⁺ . 2. The formulation of claim 1, wherein the aqueous solution has a pH of 6.0 ± 0.4 for 24 hours or longer at about 5°C. 3. The formulation of claim 2, wherein the aqueous solution has a pH of 6.0 ± 0.1 for 24 hours or longer at about 5°C. 4. The formulation according to any one of claims 1-3, wherein the aqueous solution further comprises a salt having formula II: 5. The formulation according to any one of claims 1-4, wherein the aqueous solution further comprises a salt having formula III: 6. The formulation of any one of claims 1-5, wherein the atmosphere of the headspace comprises 95% by volume or more carbon dioxide. 7. The formulation according to any one of claims 1-6, wherein the aqueous solution comprises about 10% by weight to about 20% by weight of a salt having formula I. 8. The formulation of claim 7, wherein the aqueous solution comprises about 13% to about 17% by weight of a salt having formula I. 9. The formulation of any one of claims 8, wherein the aqueous solution comprises about 14.7% by weight of a salt having formula I. 10. The formulation according to any one of claims 3-9, wherein the aqueous solution comprises about 5% by weight to about 15% by weight of a salt having formula II. 11. The formulation of claim 10, wherein the aqueous solution comprises about 7% by weight to about 11% by weight of a salt having formula II. 12. The formulation of claim 11, wherein the aqueous solution comprises about 9.1% by weight of a salt having formula II. 13. The formulation according to any one of claims 1-12, wherein the density of the aqueous solution is about 1.13 g/L. 14. The formulation according to any one of claims 1-13, wherein the aqueous solution is frozen. 15. The formulation according to any one of claims 1-14, wherein M ⁺ is Na ⁺ . 16. The formulation according to any one of claims 1-15, wherein it is packaged as a single unit dose. 17. The formulation according to any one of claims 1-16, wherein the formulation is a pharmaceutical product. 18. A method for preparing the formulation of claim 15, the method comprising: (i) L-glutathione, ascorbic acid and sodium bicarbonate were dissolved in water for injection under carbon dioxide to obtain an aqueous solution; (ii) Transfer a portion of the aqueous solution to a container; (iii) Cover the aqueous solution with carbon dioxide; and (iv) Seal the container with a stopper. The aqueous solution is prepared by dissolving about 8% to about 18% L-glutathione, about 3% to about 13% ascorbic acid, and about 3% to about 13% sodium bicarbonate in about 62% to about 82% water for injection. 19. The method of preparing the formulation according to claim 18, wherein about 11% to about 15% by weight of L-glutathione, about 5% to about 9% by weight of ascorbic acid and about 5% to about 9% by weight of sodium bicarbonate are dissolved in about 68% to about 76% by weight of water to obtain the aqueous solution. 20. The method for preparing the formulation according to claim 19, wherein about 13.0% by weight of L-glutathione, about 7.6% by weight of ascorbic acid and about 7.3% by weight of sodium bicarbonate are dissolved in about 72.0% by weight of water to obtain the aqueous solution. 21. An aqueous solution comprising a salt having formula I: It is prepared by the following method: L-glutathione, ascorbic acid, and sodium bicarbonate were dissolved in water for injection under a carbon dioxide atmosphere, wherein: M ⁺ is Na ⁺ ; and The aqueous solution is obtained by dissolving about 8% to about 18% L-glutathione, about 3% to about 13% ascorbic acid and about 3% to about 13% sodium bicarbonate in about 62% to about 82% water. 22. The aqueous solution of claim 21, wherein the aqueous solution has a pH of 6.0 ± 0.4 over a period of 24 hours or longer at about 5°C. 23. The aqueous solution of claim 22, wherein the aqueous solution has a pH of 6.0 ± 0.1 over a period of 24 hours or longer at about 5°C. 24. The aqueous solution according to any one of claims 21-23, wherein the aqueous solution further comprises a salt having formula II: Where M ⁺ is Na ⁺ . 25. The aqueous solution according to any one of claims 21-24, wherein the aqueous solution further comprises a salt having formula III: Where M ⁺ is Na ⁺ . 26. The aqueous solution according to any one of claims 21-25, wherein about 11% to about 15% by weight of L-glutathione, about 5% to about 9% by weight of ascorbic acid and about 5% to about 9% by weight of sodium bicarbonate are dissolved in about 68% to about 76% by weight of water to obtain the aqueous solution. 27. The aqueous solution of claim 26, wherein about 13.0% by weight of L-glutathione, about 7.6% by weight of ascorbic acid and about 7.3% by weight of sodium bicarbonate are dissolved in about 72.0% by weight of water to obtain the aqueous solution.