Method for treating respiratory bacterial infections

Abstract

A method of treating a subject for a respiratory bacterial infection, particularly tuberculosis, comprising administering to the subject by inhalation an effective amount of a hypertonic saline solution comprising an ibuprofenate salt, preferably further comprising arginine.

Description

[0001] METHOD FOR TREATING RESPIRATORY BACTERIAL INFECTIONS

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 668,055, filed July 5, 2024, the entire teachings of which are incorporated herein by reference.

[0004] FIELD OF INVENTION

[0005] The present invention relates to the treatment of lung disease such as bacterial infections, pneumonia, tuberculosis and bronchiolitis.

[0006] STATE OF THE ART

[0007] Non-steroidal anti-inflammatory drugs (NSAIDs) are a heterogeneous group of drugs that share their therapeutic action (analgesic, anti-inflammatory, and antipyretic effect), but differ in their relative toxicity and efficacy. The ibuprofen molecule with a molecular weight of 206.3 g / mol, as well as other derivatives of 2-arylpropionate, including ketoprofen, flurbiprofen, naproxen, etc., contains a chiral carbon in the alpha position of the propionate.

[0008] Ibuprofen is used as an antipyretic and for the symptomatic relief of headache (cluster), dental pain, muscle pain or myalgia, menstrual discomfort, mild neurological pain, and post-surgical pain. It is also used to treat inflammatory conditions such as those present in arthritis, rheumatoid arthritis, and gouty arthritis.

[0009] L-arginine is a semi-essential endogenous amino acid that plays an important role in cell division, wound healing, removal of ammonia from the body, immune function, hormone release, and is also the only biological precursor of nitric oxide (NO).

[0010] Nitric oxide (NO), which is produced from L-arginine by a family of isoenzymes called nitric oxide synthases (NOSs), plays an essential role in a variety of biological processes in the lung including host defense against pathogens, smooth muscle relaxation, bronchodilation, and inflammation [Ricciardolo, F. L. M.et al.. (2004). Nitric Oxide in Health and Disease of the Respiratory System. Physiological Reviews, 84(3), 731-765. https: / / doi.org / 10-1 152 / physrev.00034.2003]. In the vascular endothelium, NO has shown antithrombotic and antimicrobial properties. In this regard, NO release from endothelium and platelets plays a crucial role in maintaining fluidity and preventing coagulation. NO-induced vasodilation helps to eliminate "microaggregates" and inhibits platelet adhesion and aggregation, preventing vascular occlusion [Izzo J. L. (2008). Hypertension primer: [the essentials of high blood pressure; basic science population science and clinical management] (4. ed.). Lippincott Williams & Wilkins]. On the other hand, virucidal and bactericidal effects have also been described in the literature. The antibacterial effect of NO has been demonstrated against infectioncausing pathogens such as Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumanii, Listeria monocytogenes, and Enterococcus faecalis. The antimicrobial mechanisms of NO include nitrosation of amines and thiols in the extracellular matrix, lipid peroxidation and tyrosine nitration in the cell wall, and DNA cleavage in the cell matrix [Pant, J. et al. (2017). Tunable Nitric Oxide Release from S-Nitroso-N-acetylpenicillamine via Catalytic Copper Nanoparticles for Biomedical Applications. ACS Applied Materials and Interfaces, 9(18), 15254-15264. https: / / doi.org / 10.1021 / acsami.7b01408]. In addition, the ability of NO to suppress the replication of a respiratory coronavirus, which is unique to NO among other vasodilators, has been reported. [De Mel, A. (2020). Potential roles of nitric oxide in COVID- 19: A perspective. Integrative Molecular Medicine, 7(3). https: / / doi.org / 10.15761 / imm.1000403].

[0011] In the lungs, NO serves diverse purposes. For example, it functions as a selective pulmonary vasodilator to improve oxygenation and reduce pulmonary vascular resistance [Star, R. A. (1993). Southwestern internal medicine conference: Nitric oxide. American Journal of the Medical Sciences, 306(5), 348-358. https: / / doi.org / 10.1097 / 00000441 - 199311000-00015. - Tripathi, P. (2007). Nitric oxide and immune response. Indian Journal of Biochemistry & Biophysics, 44(5), 310-319.. - Susswein, A. J., Katzoff, A., Miller, N., & Hurwitz, I. (2004). Nitric Oxide and Memory. The Neuroscientist, 10(2), 153-162. https: / / doi.org / 10.1 177 / 1073858403261226. - Robbins, R. A., & Grisham, M. B. (1997). Nitric Oxide. Int. J. Biochem. Cell Bid, 29(6), 857-860. https: / / doi.org / 10.1016 / S1357- 2725(96)00167-7], As a bronchial / airway dilator, NO promotes oxygen inhalation, increasing blood flow in the capillaries which exchange gas with the alveoli and accelerating the oxygen circulation in the body [Fang, W. et al. (2021 ). The role of NO in COVID-19 and potential therapeutic strategies. Free Radical Biology and Medicine, 163, 153-162. https: / / dai.Org / 10.1016 / i.freeradbio ed.2020.12.Q08]. As a regulator of the immune system, it has been recognized that NO performs many functions where there are large numbers of cells in the system that produce and respond to NO [Tripathi, P., Tripathi, P., Kashyap, L., & Singh, V. (2007). The role of nitric oxide in inflammatory reactions. FEMS Immunology and Medical Microbiology, 51(3), 443-452. https: / / doi.org / 10-1 11 1 / j.1574- 695X.2007.00329.x], As a vascular anticoagulant, it inhibits blood coagulation and excessive platelet activation. And as an anti-inflammatory molecule, it prevents excessive inflammation through early non-specific immunity and regulates vascular inflammation and proliferation of immune cells [Fang, W. et al. (2021 ). The role of NO in COVID-19 and potential therapeutic strategies. Free Radical Biology and Medicine, 163, 153-162.

[0012] In patients with cystic fibrosis (CF), higher concentrations of exhaled NO are closely related to improvement in lung function [Grasemann, H. et al. (1997). Decreased Concentration of Exhaled Nitric Oxide (NO) in Patients With Cystic Fibrosis. Pediatric Pulmonology, 24(3), 173-177. https: / / doi.Org / 10.1002 / (sici) 1099-

[0013] 0496(199709)24:3<173::aid-ppul2>3.0.co;2-o - Ho, L. P. et al. (1998). Exhaled nitric oxide is not elevated in the inflammatory airways diseases of cystic fibrosis and bronchiectasis. European Respiratory Journal, 12(6), 1290-1294. https: / / doi.org / 10.1 183 / 09031936.98.12061290 - Keen, C. et al. (2010). Low levels of exhaled nitric oxide are associated with impaired lung function in cystic fibrosis. Pediatric Pulmonology, 45(3), 241-248. https: / / doi.org / 10.1002 / ppul.21 137]. Indeed, Ratjen and cols. [Grasemann, H. et al. (2006). Inhaled L-arginine improves exhaled nitric oxide and pulmonary function in patients with cystic fibrosis. American Journal of Respiratory and Critical Care Medicine, 174(2), 208-212. https: / / doi.org / 10.1164 / rccm.200509-14390C] found that the administration of L-arginine inhalation therapy resulted in a transient improvement in the pulmonary function of CF patients. In later studies [Grasemann, H. et al. (2013). A randomized controlled trial of inhaled l-Arginine in patients with cystic fibrosis. Journal of Cystic Fibrosis, 12(5), 468-474. https: / / doi.org / 10.1016 / jjcf.2012.12.008], they found that L-arginine inhalation was well tolerated and resulted in a significant increase in exhaled NO. FEV1 increased by an average of 56 ml compared to -8 ml after saline solution, but this difference did not reach statistical significance. Moreover, there was no change in inflammatory markers in sputum. They concluded that the repeated inhalation of L-arginine alone in patients with CF was safe and well tolerated. Inhaled L-arginine increased NO production but no evidence of changes in airway inflammation was found. It is interesting to note that they opted to use a twice-daily inhalation of 5 mL of a 100 mg / mL solution, resulting in a cumulative daily dose of 1 g L-arginine. They showed that, after 14 days of inhalation treatment with a high concentration of L-arginine, there was a measurable increase in the concentrations of the NOS inhibitor ADMA and the L-arginine / ADMA ratio (NOS substrate over inhibitor), which is decreased in CF patients and correlates with low airway NO. However, the study found that the potential effect of increased L-arginine concentrations is counteracted by an increase in both L-ornithine, which competes with L- arginine for transport into the cell, and ADMA, which acts as a competitive NOS inhibitor.

[0014] Another article studied the supplementation of gaseous NO to treat antibioticresistant bacterial and fungal lung infections in patients with cystic fibrosis [Deppisch, C. et al. (2016). Gaseous nitric oxide to treat antibiotic resistant bacterial and fungal lung infections in patients with cystic fibrosis: a phase I clinical study. Infection, 44(4), 513-520. https: / / doi.org / 10.1007 / s15010-016-0879-x]. In this article, the authors reported large reductions in bacterial numbers that led to reduced pulmonary inflammation and increases in the lung function parameter FEV1 from baseline to a degree seldom observed after antibiotic therapy courses in CF patients. Nonetheless, the fact that treatment with gaseous NO for long periods of time would unequivocally lead to the formation of toxic NO2 levels, MetHb, and hypoxemia, represents a major disadvantage of this treatment alternative.

[0015] On the other hand, it has also been shown that NO can have detrimental effects on the organism of people suffering from pulmonary diseases. For example, asthmatic patients have higher concentrations of exhaled NO than healthy people and it is known that reactive nitrogen species are involved in the pathogenesis of asthma and the development of “nitrosative stress” [Kleniewska, P., & Pawliczak, R. (2017). The participation of oxidative stress in the pathogenesis of bronchial asthma. Biomedicine and Pharmacotherapy, 94, 100-108. https: / / doi.org / 10.1016 / j-biopha.2017.07.066]. In patients with pneumonia by COVID-19, high production of reactive species oxygen (ROS) and reactive nitrogen species (RNS, e g., nitric oxide (NO)) can lead to septic shock [Chavarria, A. P. et al. (2021 ). Antioxidants and pentoxifylline as coadjuvant measures to standard therapy to improve prognosis of patients with pneumonia by COVID-19. Computational and Structural Biotechnology Journal, 19, 1379-1390. https: / / doi.Org / 10.1016 / j.csbj.2021.02.009]. Moreover, cells that are damaged due to NO production express nitrotyrosine which, in turn, can have a pathogenic effect due to its ability to react with many different molecules [Adebayo, A., Varzideh, F., Wilson, S., Gambardella, J., Eacobacci, M., Jankauskas, S. S., Donkor, K., Kansakar, II., Trimarco, V., Mone, P., Lombardi, A., & Santulli, G. (2021 ). L- arginine and covid-19: An update. Nutrients, 13(11). https: / / doi.org / 10.3390 / nu131 13951].

[0016] In summary, NO has many beneficial effects that can be useful to treat lung diseases; however, an excess of NO can also lead to cytotoxic effects causing oxidative damage and cell death. Whether or not NO has a toxic or protective effect depends on many factors. Oxidative stress is caused by an excessive systemic manifestation of reactive oxygen species (ROS) compared to a reduced capacity of a biological system to rapidly neutralize the reactive intermediates or repair the resulting damage. Increased ROS concentrations are capable of reducing the amount of bioactive NO by chemical inactivation to form toxic peroxynitrite. Peroxynitrite, in turn, can "uncouple" endothelial NO synthase and become a dysfunctional superoxide-generating enzyme that further contributes to vascular oxidative stress [Fbrstermann, U. (2010). Nitric oxide and oxidative stress in vascular disease. Pflugers Archiv European Journal of Physiology, 459(6), 923-939. https: / / doi.org / 10.1007 / s00424-010-0808-2]. In this vein, a correlation between the presence of systemic or local oxidative stress and various pulmonary diseases, including all those treated in the examples herein, has been described in the literature [Ornatowski, W. et al. (2020). Complex interplay between autophagy and oxidative stress in the development of pulmonary disease. Redox Biology, 36. https: / / doi.org / 10.1016 / j-redox.2020.101679 - Zinellu, E. et al. (2021 ). Oxidative stress biomarkers in chronic obstructive pulmonary disease exacerbations: A systematic review. Antioxidants, 10(5), 710. https: / / doi.org / 10.3390 / antiox10050710 - Farouk, A. et al. (2016). Role of oxidative stress and outcome of various surgical approaches among patients with bullous lung disease candidate for surgical interference. Journal of Thoracic Disease, 8(10), 2936-2941. https: / / doi.org / 10.21037 / jtd.2016.10.41 - Bai, Y. et al. (2018). A Chinese herbal formula ameliorates pulmonary fibrosis by inhibiting oxidative stress via Upregulating Nrf2. Frontiers in Pharmacology, 9(JUN). https: / / doi.org / 10.3389 / fphar.2018.00628 - Horvath, I. et al. (1998). Increased levels of exhaled carbon monoxide in bronchiectasis: A new marker of oxidative stress. Thorax, 53(10), 867-870. https: / / doi.org / 10.1 136 / thx.53.10.867 - Jesenak, M. et al. (2017). Oxidative stress and bronchial asthma in children-causes or consequences? Frontiers in Pediatrics, 5. https: / / doi.org / 10.3389 / fped.2017.00162 - Nikolova, G. D. et al. (2018). Oxidative stress and related diseases. Part 1 : Bronchial asthma. Bulgarian Chemical Communications, 50, 30-35 - Fernandes, I. G. et al. (2020). SARS-CoV-2 and Other Respiratory Viruses: What Does Oxidative Stress Have to Do with It? Oxidative Medicine and Cellular Longevity, 2020. https: / / doi.org / 10-1155 / 2020 / 8844280 - Chavarria, A. P. et al. (2021 ). Antioxidants and pentoxifylline as coadjuvant measures to standard therapy to improve prognosis of patients with pneumonia by COVID-19. Computational and Structural Biotechnology Journal, 19, 1379-1390. https: / / doi.Org / 10.1016 / j.csbj.2021.02.009], and therefore disease treatments targeting ROS inhibition and restoration of the oxidant / antioxidant imbalance have also been proposed. A prior patent application by the same team of inventors (W02023100127) discloses a similar formulation comprising ibuprofen and arginine in hypertonic saline; however, it neither contemplates nor suggests its application for the treatment of tuberculosis, nor does it disclose or anticipate the surprising immunological effects observed in macrophages infected with Mycobacterium tuberculosis. In contrast, the present invention demonstrates for the first time that this composition restores the functional activity of infected alveolar macrophages, reprograms the host immune response, and significantly contributes to bacterial clearance, particularly in the context of pulmonary tuberculosis. These unexpected findings define a clear and inventive advance over the prior art.

[0017] Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide (https: / / www.who.int / news-room / fact-sheets / detail / tuberculosis). Despite the availability of standardized multidrug regimens, the disease continues to present significant public health challenges, particularly in cases of multidrug-resistant tuberculosis (MDR-TB), where treatment requires prolonged administration of second-line agents with higher toxicity profiles and lower efficacy. Even in drug-sensitive TB (DS-TB), the extended duration of treatment and the risk of hepatotoxicity, treatment interruption, or poor adherence contribute to treatment failure and relapse.

[0018] In pulmonary TB, the inflammatory response and destruction of lung tissue result in cavitation, mucus obstruction, and compromised ventilation / perfusion ratios, which reduce the penetration and local efficacy of systemic antibiotics. Furthermore, a growing body of evidence implicates oxidative stress, reactive nitrogen species (including nitric oxide), and immune dysregulation in the pathogenesis and persistence of Mycobacterium tuberculosis infection. While L-arginine has been studied as a modulator of endogenous NO production, its therapeutic effects in monotherapy remain inconsistent, and the potential cytotoxicity of unregulated NO release raises safety concerns.

[0019] The therapeutic use of ibuprofen as an anti-inflammatory agent in TB has also been limited due to systemic side effects. Moreover, the need to balance localized antiinflammatory effects with immune modulation and pathogen control has not been successfully addressed by current treatments. Inhaled nitric oxide (gNO), while effective in reducing bacterial burden in some studies, carries the risk of N02formation, methemoglobinemia, and systemic oxidative damage, particularly in prolonged use. Thus, there exists a pressing need for novel, localized, lung-targeted therapeutic strategies that can enhance pathogen clearance, reduce inflammation, and improve pulmonary function without the systemic risks associated with current pharmacological options. In view of the limitations of the current treatment strategies for pulmonary tuberculosis, particularly in patients with cavitary disease, comorbidities, or multidrug resistance, there is a clear unmet medical need for a safe, efficient, and lung-targeted therapeutic alternative that enhances the host's ability to eliminate Mycobacterium tuberculosis while minimizing systemic toxicity and adverse effects.

[0020] The present invention relates to a method of treating a subject for tuberculosis, comprising administering to the subject by inhalation a composition comprising an ibuprofenate salt and arginine in a saline aqueous solution containing between 0.3 M and 2.0 M of salt. The combination of ibuprofen and arginine in inhalable form is designed to act synergistically at the site of infection by modulating the local immune response, reducing oxidative stress, promoting bronchodilation, and enhancing mucociliary clearance.

[0021] Surprisingly and unexpectedly, this inhalable composition has shown, in real-world clinical examples, an early conversion to sputum smear negativity — often within 7 to 15 days — accompanied by improved clinical outcomes and excellent patient tolerance. Even in patients with prior TB treatment failure, drug intolerance, hepatotoxicity, or complex pulmonary pathology, the administration of this inhalable formulation demonstrated accelerated resolution of symptoms and enhanced microbial clearance without the need to interrupt therapy. These results suggest that the invention provides a novel and effective adjunctive strategy for the management of both drug-sensitive and drug-resistant forms of pulmonary tuberculosis.

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023] Figure 1 : Ibu-Arginine diminished oxidative stress in murine macrophages treated with gamma-irradiated M. tuberculosis H37Rv. A) Determination of superoxide anion by DHE fluorescent probe. B) Expression of NOX-2 by Real time qPCR

[0024] Figure 2: Ibu-Arginine reduced M1 phenotype inflammatory markers expression induced by murine macrophage infection with gamma-irradiated M. tuberculosis H37Rv (MT). *p<0.05 ** p<0.01 *** p<0.001 vs Control, #p<0.05## p<0.01 ### p<0.001 vs MT

[0025] Figure 3: Chest X-ray image from Example 2

[0026] Figure 4: Chest X-ray image from Example 3

[0027] Figure 5: Chest X-ray image from Example 4

[0028] Figure 6: Chest X-ray image from Example 5 Figure 7: Chest X-ray image from Example 6

[0029] Figure 8: Chest X-ray image from Example 7

[0030] Figure 9: Chest X-ray image from Example 8

[0031] Figure 10: Chest X-ray image from Example 9

[0032] Figure 11 : Chest X-ray image from Example 10

[0033] Figure 12: Chest X-ray image from Example 11

[0034] Figure 13: Chest X-ray image from Example 12

[0035] BRIEF DESCRIPTION OF THE INVENTION

[0036] The present invention relates to a method for treating respiratory bacterial infections in a subject, comprising the administration, by inhalation, of an effective amount of a composition that includes a hypertonic saline solution and an ibuprofenate salt. In preferred embodiments, the composition further comprises arginine, resulting in a synergistic therapeutic effect.

[0037] The respiratory bacterial infection to be treated may include, but is not limited to, pneumonia, bronchiolitis, and tuberculosis. In particular embodiments, the infection is caused by bacteria such as Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, or Legionella pneumophila, Mycobacterium, Mycobacterium SPP. In another embodiment, the infection is caused by bacteria such as Mycobacterium avium or Mycobacterium abscessus. A preferred embodiment specifically targets tuberculosis, including cases caused by Mycobacterium tuberculosis. In another embodiment, the bacterial infection to be treated is a pulmonary infection with Mycobacterial species, including but not limited to: tuberculosis, multidrug resistant tuberculosis, M. avium complex (including M. intracelluare), M. abscessus, and M. kansasi.

[0038] The ibuprofenate salt used in the composition may be selected from sodium ibuprofenate, potassium ibuprofenate, or lithium ibuprofenate, with sodium ibuprofenate being particularly preferred. The concentration of the ibuprofenate salt in the hypertonic saline solution may range from 5 mM to 100 mM or, alternatively, from 5 mg / mL to 50 mg / mL. The saline solution preferably has a sodium chloride concentration from 0.3 M to 2.0 M and a pH between 7.0 and 9.0. In a preferred embodiment of the present invention the composition comprises ibuprofenate and arginine in a weight ratio ranging from 1 :1 to 1 :3, preferably in a ratio of 1 :2.

[0039] In a particularly advantageous embodiment, the invention provides a method of treating tuberculosis by inhalation or nebulization of a composition comprising ibuprofenate salt and arginine in a hypertonic saline solution. The composition may include from 5 to 20 mg / mL of ibuprofenate salt and from 10 to 40 mg / mL of arginine, and in one specific formulation comprises 10 mg / mL of ibuprofenate and 20 mg / mL of arginine. In a preferred regimen, the composition is administered by nebulization in a volume of 3 to 6 mL, preferably 4 mL, every from 4 to 10 hs, preferably every 8 hours.

[0040] Unexpectedly, the invention also provides immunomodulatory benefits by restoring the functional activity of alveolar macrophages infected with Mycobacterium tuberculosis. The composition reduces oxidative stress, downregulates M1 macrophage polarization markers, and re-establishes nitric oxide synthesis via endothelial nitric oxide synthase (eNOS), thus enhancing the host's innate immune response and reducing pathogen dissemination.

[0041] In another embodiment, the invention provides a method of treating tuberculosis by nebulization of an aqueous composition comprising 40 mg of ibuprofenate salt and 80 mg of arginine in a hypertonic saline solution with a salt concentration ranging from 0.3 M to 2.0 M, administered three times daily in conjunction with standard antibiotic therapy.

[0042] Furthermore, the present invention also comprises the use of a composition comprising ibuprofenate salt and arginine in a hypertonic saline solution for the manufacture of a medicament intended for the treatment of tuberculosis by inhalation or nebulization.

[0043] In a preferred embodiment of the present invention said composition is administered by nebulization, 4 ml of hypertonic solution with ibuprofenate salt in a concentration of 10 mg / ml, and arginine in a concentration of 20 mg / ml each from 4 to 10 hs.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] A method of treating a subject for a respiratory bacterial infection, preferably tuberculosis or bronchiolitis, main object of present invention, comprises administering to the subject by inhalation or nebulization a pharmaceutical composition to be applied on the pulmonary epithelium, comprises a non-steroidal anti-inflammatory drug (NSAID), solubilized in a aqueous solution at alkaline pH between 7.5 and 9.5; wherein also comprises a basic amino acid, such arginine.

[0046] “NSAID” refers to a non-steroidal anti-inflammatory drug which is selected from the group comprised by ibuprofen, naproxen, flurbiprofen, ketoprofen, diclofenac, diflunisal, etodolac, fenoprofen, indomethacin, meclofenamate, mefenaic acid, meloxicam, oxaprozim, piroxicam, sulindac, celecoxib, acetylated salicylated, and combinations thereof. Wherein said NSAID, is in a concentration from 5 mM to 500 mM, preferably in a concentration from 5 mM to 180 mM, more preferably in a concentration from 5 mM to 50 mM or, alternatively, from 40 mM to 60 mM. The NSAID is present in its salt form, wherein the carboxylic acid moiety is deprotonated and neutralized with a pharmaceutically acceptable cation. Said NSAID comprises as a counterion the monovalent cation selected from the group consisting of sodium, potassium, lithium and combination thereof. In one embodiment, the disclosed formulation comprises ibuprofen.

[0047] Given that ibuprofen has one chiral center, it has two possible enantiomers R and S. The invention contemplates the use of the R enantiomer, the use of the S enantiomer and mixtures thereof, including racemic mixtures (1 :1 mixtures of the R and S enantiomers). The designation of the R enantiomer or the S enantiomer indicates an optical purity of at least 90%, 95%, 98% or 99% by weight. “Optical purity” refers to the percent of the designated enantiomer relative to the combined weight of both enantiomers.

[0048] “Basic amino acids” refers to the amino acids that have basic side chains at neutral pH, such as; arginine, lysine, and histidine. Their side chains contain nitrogen and resemble ammonia, which is a base. In one embodiment, said basic amino acid, is in a concentration from 5 mM to 500 mM, preferably in a concentration from 25 mM to 300 mM, more preferably in a concentration from 50 mM to 250 mM, and even more preferable in a concentration from 80 mM to 150 mM or from 80 mM to 120 mM. Where said basic amino acid is preferably arginine.

[0049] “Aqueous solution” refers to the solution that uses a polar liquid as a solvent, preferably water, and has the NSAID and the basic amino acid solubilized in it. In another embodiment, said aqueous solution further comprises a salt suitable for human consumption, preferably Na2COs, KOI, or NaCI, more preferably NaCI. Wherein said aqueous solution is preferably hypertonic comprising a concentration of said salt suitable for human consumption from 0.3 M to 2 M, preferably from 0.4 M to 1.1 M, even more preferably from 0.9 M to 1 .05 M or 0.4 M to 0.6 M. Wherein said basic amino acid, in one embodiment of the present invention, preferably comprises concentrations between 25 and 500 mM, more preferably between 50 and 250 mM and more preferably between 80 and 150 mM. When a basic amino acid is present in the disclosed formulation and the formulation has a pH of 7 or greater, the basic amino acid is substantially or completely present as its free base, irrespective of whether the formulation is prepared with the free base or a salt thereof. As used herein, a reference to a basic amino acid such as “arginine” refers to the free base but includes any salt of the basic amino acid that may be present.

[0050] Furthermore, said composition comprises a pH in aqueous solution between pH 7.5 and pH 9.5, preferably between pH 8 and pH 9, more preferably the pH of said pharmaceutical composition is 8.5,

[0051] Its administration form is selected from the group comprised by inhalation, including but not limited to nebulization, aerosol spray, or vaporized mist, or nebulization.

[0052] The pharmaceutical composition disclosed is administered by inhalation, preferably by nebulization. Administering a formulation by “inhalation” refers to administering the formulation directly to the lungs through the mouth or / and nasal cavity, commonly by inhaling or nebulizing the formulation.

[0053] The administration of the pharmaceutical composition can be carried out by inhalation, preferbly by nebulization, in which a nebulizer changes liquid medicine into fine droplets (in aerosol or mist form) that are inhaled through a mouthpiece or mask. Nebulization can be accomplished by any suitable means, including by: 1 ) jet, which uses compressed gas to make an aerosol (tiny particles of medication in the air) or ultrasound, which makes an aerosol through high-frequency vibrations. In one embodiment, the nebulization is carried out with a piston nebulizer. In one embodiment, the nebulized droplets are of sufficient size to reach the alveoli, e.g., less than 0.1 microns or less than 0.8 microns or less than 0.5 microns.

[0054] Alternatively, the pharmaceutical composition may be delivered with an inhaler, e.g. through a metered dose inhaler (MDI), which “pushes out” a pre-measured spray of the pharmaceutical composition, with, for example, a hydrofluoroalkane aerosol spray. In another example, a soft mist inhaler (SMI) provides a pre-measured amount of the pharmaceutical formulation in a slow-moving mist.

[0055] Furthermore, the composition of the present invention may be prepared either in liquid state or as a powder or lyophilized by drying or lyophilization from the final aqueous solution of said pharmaceutical composition. Both the drying process and the lyophilization of pharmaceutical composition is well known in the prior art, therefore providing further details on the subject is not considered necessary.

[0056] The present invention is a method for the treatment of bacterial infections that produce lung disease such as; pneumonia, bronchiolitis, and tuberculosis.

[0057] The present invention has advantages, since with very low doses of NSAID and a basic amino acid, combined at the specific concentrations and at specific pH values, exhibits a synergic effect in the synthesis and release of nitric oxide (NO), which also improve the vasodilation and as consequence induce the improvement in the O2 saturation and finally an increase of the pulmonary function FEV-1 in patients. Unexpectedly, and as taught for the first time in the present invention, the composition of the invention not only contributes to the clearance of Mycobacterium tuberculosis infection but also enables the functional recovery of alveolar macrophages — cells which are typically hijacked, incapacitated, and used by the pathogen for dissemination. Remarkably, treatment with the inhaled ibuprofenate-arginine solution restores the redox balance of infected macrophages by Mycobacterium, reducing its oxidative damage, and downregulates pro-inflammatory M1 polarization markers. These effects result in a reprogramming of the immune microenvironment, allowing macrophages to resume their physiological roles in innate defense and coordination of adaptive immunity against this pathogenic bacterium.

[0058] This dual action — both antimicrobial and immunoreparative — has not been previously achieved by known therapies and represents a surprising and inventive advance in the treatment of tuberculosis with a different approach that the actual antibiotics therapy, allowing to avoid the increased capacity of this bacteria to develop antibiotic resistances. This treatment was evidenced by the results presented in Figures 1 and 2 and confirmed by examples from 2 to 12.

[0059] The alveolar macrophage plays a central role in the pathophysiology of pulmonary Tuberculosis (TB). This immune cell is a fundamental effector of the innate immune response and at the same time has the capacity to awaken and direct the adaptive immune response. The peculiarity of Mycobacterium tuberculosis (MT) is that it infects this cell line, blocks its defense capacity and uses it as a vehicle for dissemination.

[0060] Meanwhile MT efficiently escapes phagosomal degradation and inactivation through NADPH-Oxidase 2 (NOX2)-derived Reactive Oxygen Species (ROS) production, macrophages trigger excessive mitochondrial ROS production while leading to cell death to minimise the spread of MT infection. Loss of macrophage functionality can occur due to numerous noxae, such as viruses / bacteria / environmental pollution. Reducing oxidative stress and restoring NO synthesis by eNOS and not via iNOS is a fundamental tool in the treatment of chronic or acute lung diseases.

[0061] The effect of the composition used in the method of present invention: (hypertonic saline solution comprising an ibuprofenate salt with arginine) ibuprofen-arginine solution on oxidative stress and inflammation markers in mouse macrophages (RAW) infected with Gamma-Irradiated MT Strain H37Rv was evaluated. Since macrophages undergo different polarization phenotypes the effect of said composition on the phenotypic changes associated with M1 macrophage polarization was evaluated.

[0062] For said evaluation Cell culture: mouse macrophages (RAW) were cultured in RPMI medium supplemented with 10% SBF, penicillin / streptomycin, at 37° C in a humidified atmosphere at 5% CO2. Cells were infected with Mtb- H37Rv (10 pg / mL) for 24 hr.

[0063] Determination of reactive oxygen species: ROS production was measured by fluorometric assay, using the DHE (dehydroethidium) probe. Cells were cultured in a 24- well plate until confluence was reached, and different treatments were applied for 1 hr. Then, cells were incubated with the probe at 37°C for 30 min. Fluorescence values were obtained in a plate reader (Thermo Fluorometer) and observed under a fluorescence microscope. Superoxide anion generation was also measured by flow cytometry, using a FACsARIA flow cytometer.

[0064] An analysis of gene expression of the NADPH Oxidase subunit (NOX 2) responsible for the generation of ROS and inflammation-associated genes: iNOS, TNF-Alpha, IL-6 and MCP-1 were determined by real-time qPCR. mRNA was isolated using Trizol, and cDNA synthesis was performed using M-MLV reverse transcriptase. Expression levels were normalized using a constitutive gene (Eef2). Relative quantification was performed using the comparative deltaCT method.

[0065] As a result, infection of Raw cells by Mtb induces an increase in oxidative stress observed by increased fluorescence of the DHE probe. This effect is highly inhibited by pretreatment with 20 and 50 pM Ibu-arginine solution (Fig. 1A). We then measured the expression of the NOX-2 subunit responsible for ROS generation. Infected macrophages showed a significant increase of NOX-2 expression which was significantly decreased by Ibuprofen-arginine (10-50-100 pM) treatment (Fig. 1 B; ***p < 0.001 vs Control; ## p < 0.01 and ### p < 0.001 vs Mycobacterium tuberculosis (MT)) Macrophages are primary host cells of MT infection and thus have a central role in immune control of tuberculosis. Murine macrophages infected with MT showed increased levels of M1 phenotypic polarization further enhanced secretion of M1 -related proteins. Regulating macrophage polarization, cytokines induced during infection result in diverse physiological changes that further define the outcome of the disease condition.

[0066] Based on the above, the gene expression of M1 polarization markers was determined. We found that MT infection significantly increased the expression of iNOS, II- 6, TNF-a and MCP-1 .

[0067] Ibuprofen-arginine 100 pM significantly reduced the expression of all these markers. Lower concentrations (10 pM and 50 pM) were also effective in significantly decreasing IL- 6 expression (Fig. 2).

[0068] A “subject” is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

[0069] “T reat,” “treating,” or “treatment,” when used in connection with a subject with a lung disease, includes improving the effects or symptoms of infection or shortening the duration of the infection. In instances where the subject has become hypoxic, “treat,” “treating,” or “treatment,” refers to returning blood oxygenation to normal or near normal more rapidly than in the absence of treatment. In instances where the subject has progressed to severe disease, “treat,” “treating,” or “treatment,” refers to lessening the likelihood of requiring intubation, decreasing the time requiring intubation, decreasing recovery time and / or reducing mortality rate.

[0070] “Effective amount” means an amount when administered to the subject with a lung disease which results in beneficial or desired results, including improvement of the effects or symptoms of said lung diseases, including normalizing blood oxygenation levels, shortening recovery time, decreasing the likelihood of requiring intubation in severe disease and / or decreasing mortality.

[0071] The precise amount of the pharmaceutical solution administered to provide an “effective amount” to the subject will depend on the type, and severity of the respiratory bacterial infection, and on the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Suitable dosages are known for approved therapeutic agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of lung disease being treated and the amount of pharmaceutical composition being used by following, for example, dosages reported in the literature and recommended in the Physician’s Desk Reference (57th ed., 2003). For example, an “effective amount” can be between 1 mL to 50 mL of the pharmaceutical composition used in the disclosed methods. Alternatively, an “effective amount” is between 1 mL to 25 mL of the pharmaceutical composition used in the disclosed methods. In another alternative, “effective amount” is between 1 mL and 10 mL of the pharmaceutical composition used in the disclosed methods. In another alternative, “effective amount” is between 3 mL and 7 mL of the pharmaceutical composition used in the disclosed methods. In yet another alternative, an “effective amount” is 4 mL of the pharmaceutical composition used in the disclosed methods. An effective amount is administered between 1 and 5 times daily, alternatively from 1 to 3 times daily, alternatively 2 times daily, alternatively 3 times daily. The time of administration varies between 5 minutes and 1 hour and alternatively between 5 minutes and 30 minutes. In yet another alternative, the time of administration varies between 10 minutes and 20 minutes.

[0072] A small but significant percentage of subjects with a respiratory bcterial infection progress to severe and even life-threatening disease such as pneumonia or acute respiratory distress syndrome (ARDS) (referred to herein as “severe disease”). Pneumonia is an infection that inflames the air sacs (alveoli) in one or both lungs. The air sacs may fill with fluid or pus (purulent material), causing cough with phlegm or sputum, fever, chills, and difficulty breathing. ARDS is also characterized by fluid build-up in the air sacs in the lungs, but it is also accompanied by hyperinflammation, which may induce a condition sometimes referred to as “cytokine storm” or systemic inflammation which can lead to respiratory failure and death. Symptoms of ARDS include severe shortness of breath, labored and unusually rapid breathing, low blood pressure and / or confusion and extreme tiredness.

[0073] The present invention relates to a method for treating respiratory bacterial infections in a subject, comprising the administration, by inhalation, of an effective amount of a composition that includes a hypertonic saline solution and an ibuprofenate salt. In preferred embodiments, the composition further comprises arginine, resulting in a synergistic therapeutic effect.

[0074] The respiratory bacterial infection to be treated may include, but is not limited to, pneumonia, bronchiolitis, and tuberculosis. In particular embodiments, the infection is caused by bacteria such as Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, or Legionella pneumophila, Mycobacterium, Mycobacterium SPP. A preferred embodiment specifically targets tuberculosis, including cases caused by Mycobacterium tuberculosis. In another embodiment, the infection is caused by bacteria such as Mycobacterium avium or Mycobacterium abscessus. In another embodiment, the bacterial infection to be treated is a pulmonary infection with Mycobacterial species, including but not limited to: tuberculosis, multidrug resistant tuberculosis, M. avium complex (including M. intracelluare), M. abscessus, and M. kansasi.

[0075] The ibuprofenate salt used in the composition may be selected from sodium ibuprofenate, potassium ibuprofenate, or lithium ibuprofenate, with sodium ibuprofenate being particularly preferred. The concentration of the ibuprofenate salt in the hypertonic saline solution may range from 5 mM to 100 mM or, alternatively, from 5 mg / mL to 50 mg / mL. The saline solution preferably has a sodium chloride concentration from 0.3 M to 2.0 M and a pH between 7.0 and 9.0.

[0076] In a preferred embodiment of the present invention the composition comprises ibuprofenate and arginine in a weight ratio ranging from 1 :1 to 1 :3, preferably in a ratio of 1 :2.

[0077] In a particularly advantageous embodiment, the invention provides a method of treating tuberculosis by inhalation or nebulization of a composition comprising ibuprofenate salt and arginine in a hypertonic saline solution. The composition may include from 5 to 20 mg / mL of ibuprofenate salt and from 10 to 40 mg / mL of arginine, and in one specific formulation comprises 10 mg / mL of ibuprofenate and 20 mg / mL of arginine. In a preferred regimen, the composition is administered by nebulization in a volume of 3 to 6 mL, preferably 4 mL, every from 4 to 10 hs, preferably every 8 hours.

[0078] In another embodiment, the formulation used in the disclosed methods is administered by inhalation or nebulization and comprises from 30 mM to 70 mM of ibuprofenate salt, from 75 mM to 125 mM of arginine and from 0.2 M to 0.8 M of NaCI. Alternatively, the formulation used in the disclosed methods is administered by inhalation or nebulization and comprises from 40 mM to 60 mM of ibuprofenate salt, from 90 mM to 1 10 mM of arginine and from 0.4 M to 0.6 M of NaCI. Alternatively, the formulation used in the disclosed methods is administered by inhalation or nebulization and comprises 50 mM of ibuprofenate salt, 100 mM of arginine and from 0.5 M of NaCI. In the embodiment described the pH of the formulation is from 7.0 to 9.0.

[0079] In the embodiments described in the prior two paragraphs, the composition is preferably administered by nebulization in a volume of 3 to 6 mL, more preferably 4 mL, every from 4 to 10 hs, preferably every 8 hours, for example, two or three times daily.

[0080] Unexpectedly, the invention also provides immunomodulatory benefits by restoring the functional activity of alveolar macrophages infected with Mycobacterium tuberculosis. The composition reduces oxidative stress, downregulates M1 macrophage polarization markers, and re-establishes nitric oxide synthesis via endothelial nitric oxide synthase (eNOS), thus enhancing the host's innate immune response and reducing pathogen dissemination.

[0081] In another embodiment, the invention provides a method of treating tuberculosis by nebulization of an aqueous composition comprising 40 mg of ibuprofenate salt and 80 mg of arginine in a hypertonic saline solution with a salt concentration ranging from 0.3 M to 2.0 M, administered three times daily in conjunction with standard antibiotic therapy.

[0082] Furthermore, the present invention also comprises the use of a composition comprising ibuprofenate salt and arginine in a hypertonic saline solution for the manufacture of a medicament intended for the treatment of tuberculosis by inhalation or nebulization.

[0083] In a preferred embodiment of the present invention said composition is administered by nebulization, 4 ml of hypertonic solution with ibuprofenate salt in a concentration of 10 mg / ml, and arginine in a concentration of 20 mg / ml each from 4 to 10 hs.

[0084] The disclosed formulations are safe for long term administration. As such, they can be administered acutely and for relatively short durations of time (treatment of typical bacterial pneumonias, with 7 to 21 days treatment in combination with standard of care antibiotics) or for long periods of time, for example, for 6 to 12 months or longer, as needed, for treatment of chronic mycobacterial infections.

[0085] In one aspect, the disclosed methods treat patients with high active bacterial load such as patients with high active Mycobacterium load load (e.g., patients with active pulmonary tuberculosis who have a high bacterial load), In one example, the bacterial load is high enough to be detectable through staining and microscopy (BAAR). For Mycobacterium such as those causing tuberlosis (e.g., Mycobacterium tuberculosis), treatment can be continued until the bacteria causing the infection are no longer detectable by means of an Acid-Fast Bacillus (AFB) test, for example, in the sputum. Low blood oxygenation levels also accompany pneumonia, tuberculosis and ARDS and are responsible, at least in part, for the severe symptoms associated with these conditions. Oxygen saturation levels offer an integrated assessment of pulmonary and cardiac function, and its non-invasive measurement with transdermal pulse oximetry has become a routine component of the assessment of disease severity. “Low blood oxygenation levels” in a subject refers to a pulse-oximetry oxygen saturation level of less than 95%, the lower limit of normal for healthy subjects (patients with hypercapneic respiratory disease live with lower chronic oxygen saturation levels). Oxygen saturation of < 92% is considered an urgent matter, requiring immediate intervention, particularly when this reflects an acute change from baseline normal values as is often observed in patients with pneumonitis. A subject with a low blood oxygenation level is also referred to herein as being “hypoxic”. Subjects with respiratory viral or bacterial infection diseases who have progressed to pneumonia, tuberculosis or ARDS, when treated according to the disclosed methods, have shown improved blood oxygenation, including restoration of blood oxygenation levels to normal and with relief of the severe symptoms associated with ARDS.

[0086] A second common measure of cardiopulmonary status is the respiratory rate, which in healthy adults is typically less than or equal to 20 breaths / minute. Subjects with respiratory bacterial infection that have progressed to pneumonia or ARDS frequently present with respiratory rates far in excess of the normal range (up to 21 -25 breathes / minute, 25-30 breath es / minute or, in more severe cases, 30 to 40 breaths / minute), and such subjects, when treated according to the disclosed methods, have shown improvement in respiratory rate to the normal range. This is one component of the alleviation of severe symptoms associated with ARDS described above.

[0087] A third measure of cardiopulmonary status is the heart rate, which in healthy adults at rest is typically less than 90 beats per minute, but may be markedly elevated in subjects with respiratory bacterial infections that have progressed to pneumonia or ARDS. Subjects with respiratory infections that have progressed to tuberculosis, pneumonia or ARDS frequently present with heart rates far in excess of normal range (from 91 -110 beats / minute, in more severe cases from 11 1 -130 beats / minute, to over 131 beats / minute in the most severe cases). Subjects with pneumonia or ARDS, when treated according to the disclosed methods, have shown improvement in heart rate to the normal range.

[0088] The National Early Warning Score (NEWS2) is an accepted assessment tool for identifying subjects who have or are likely to develop acute illness. See, for example, Royal College of Physicians, National Early Warning Score (NEWS) 2: Standardizing the Assessment of Acute-Illness Severity in the NHS. Updated Report of the Working Party, London :RCP, 2017. Specifically, a NEWS2 Score of 0-4 indicates a low level of clinical risk for the subject; a NEWS2 Score of 5-6 indicates a moderate level of clinical risk for the subject; and a NEWS2 Score of 7 or more indicates a high level of clinical risk for the subject. The disclosed methods can be used to treat a subject with a NEWS2 Score of 0-4, 5-6 or 7 or more to reduce the likelihood of increasing the scope or to reduce the score to bring the subject to an improved condition with a lower score.

[0089] In particularly severe cases, subjects with ARDS require breathing assistance and are put on a mechanical ventilator, i.e., the subject is “intubated”. The disclosed methods can increase blood oxygenation and are useful in reducing the likelihood that a hypoxic subject with severe disease who is not yet intubated will subsequently require intubation. The disclosed methods are also useful in increasing blood oxygenation in intubated patients, thereby increasing the likelihood of recovery and decreasing the amount of the time the subject spends on a ventilator.

[0090] Considering the previous information mentioned and the results shown in the examples (see below), It has been demonstrated the composition of the present invention shows important improvement for the treatments of various diseases that affect specifically lungs: pneumonia, bronchiolitis, tuberculosis. In all these pathologies the nebulization with this composition containing a mix of Ibuprofen-salt at a pH 8.5, preferably with arginine produces a substantial anti-inflammatory effect and an improvement of oxygen pressure (PO2); respiratory frequency and cardiac frequency.

[0091] EXAMPLES

[0092] Example 1 : Preparation of the favorite composition of the present invention (Ibu-Arg solution).

[0093] The preferred method of preparation of the formulation used for the embodiment of the present invention, which proves to be the best known to the inventors, is described, but is not the only possible one. a) dissolving Na2COs in 70% of the final volume of purified water, heated at 45eC; b) adding 40 mg ibuprofen (sifted to a fine powder) to the solution of step a) and stirring until dissolution; c) adding 80 mg arginine hydrochloride to the solution of step b); d) adding NaCI to the solution of step c) and stirring until dissolution. e) adding Na2COs solution to the solution of step d) to reach a pH value of 8.5; f) cooling the solution obtained in step e) to room temperature and adding purified water to complete the final volume of 4 ml of the solution; g) filtering the preparation obtained in step f), through a 0.22-micron pore filter and obtain the Ibu-Arg solution of the invention.

[0094] Clinic tests

[0095] In the following examples, several persons with tuberculosis have been treated with administration, by inhalation, an effective amount of a hypertonic saline solution comprising an ibuprofenate salt. The treatment of a cohort of 11 patients diagnosed with pulmonary tuberculosis, was managed at the Pulmonology Department of a high-complexity general referral hospital. This institution participates in the Provincial Tuberculosis Program, which adheres to the Argentine National Tuberculosis Program and follows the guidelines issued by the World Health Organization (WHO).

[0096] The National Program recommends control with sputum smear microscopy (AFB testing) at various stages of disease progression, particularly in cases with poor clinical evolution or upon completion of the intensive treatment phase (i.e., after 60 days). At this stage, sputum positivity rates are reported to be approximately 75-80% in drug-sensitive tuberculosis (DS-TB) and 40-60% in multidrug-resistant tuberculosis (MDR-TB).

[0097] In the province of Salta, a total of 905 new cases of tuberculosis were diagnosed and patients were treated with the (HRZE) / (HR) antibiotic regimen. From this population, 1 1 cases were selected to receive, complementarily, the composition used in the present invention of example 1 : ibuprofen / arginine 40 / 80 mg in 4 ml vials (Ibu-Arg solution), administered via inhalation every 8 hours (only one case received it every 6 hours) as adjuvant therapy. In this group, a very early conversion to sputum smear negativity was observed (on average: 9 days), without the need for adverse event treatment discontinuation, showing that inhaled Ibu-Arg solution is both safe and potentially highly effective in the epidemiological and clinic management of pulmonary tuberculosis. EXAMPLE 2: Clinical Application of Inhaled Ibu-Arg solution in a Patient with Pulmonary Tuberculosis

[0098] A 33-year-old male patient (body weight: 71 kg) was managed on an outpatient basis following a diagnosis of pulmonary tuberculosis. The patient tested negative for HIV and reported a history of pulmonary tuberculosis treated a decade earlier.

[0099] Radiographic imaging revealed bilateral cavitary lesions consistent with active pulmonary tuberculosis. On April 17, 2023, sputum smear microscopy was positive for acidfast bacilli (AFB++) and culture confirmed the presence of Mycobacterium tuberculosis complex.

[0100] Standard first-line antituberculous therapy was initiated on April 24, 2023. Concurrently, the patient began inhalation therapy with a hypertonic saline solution containing ibuprofen / arginine (Ibu-Arg solution), administered at a dose of 40 mg Ibu every 8 hours via nebulization. The patient demonstrated marked symptomatic improvement within the first 48 hours of therapy. Tolerance to both the standard anti-tuberculosis regimen and Ibu-Arg solution was excellent, with no adverse effects reported.

[0101] Follow-up sputum smear microscopy conducted on May 3, 2023 showed a complete conversion to AFB-negative status. Thus, the total time to sputum smear conversion was 9 days. This rapid conversion suggests that Ibu-Arg solution, administered by inhalation as an adjuvant to standard therapy, may contribute to accelerated bacteriological clearance in pulmonary tuberculosis.

[0102] (See Figure 3 for chest radiograph corresponding to this patient.)

[0103] EXAMPLE 3: Clinical Application of Inhaled Ibu-Arg solution in a Patient with Unilateral Pulmonary Tuberculosis and Comorbid Diabetes

[0104] A 59-year-old female patient (body weight: 46 kg) was managed on an outpatient basis following a diagnosis of pulmonary tuberculosis. The patient was HIV negative and had a history of poorly controlled Type 2 diabetes mellitus, managed with oral hypoglycemic agents.

[0105] Radiological evaluation indicated unilateral right-sided pulmonary tuberculosis without cavitary lesions. Sputum smear microscopy performed on April 22, 2023, was positive for acid-fast bacilli (AFB+). Culture data were not available. On May 9, 2023, standard first-line antituberculous therapy was initiated. Simultaneously, the patient began nebulized administration of Ibu-Arg solution (ibuprofen / arginine) every 8 hours. At treatment initiation, the patient presented marked respiratory symptoms, including hemoptysis. Within 48 hours of starting therapy, a notable symptomatic improvement was observed.

[0106] The patient experienced mild gastritis related to the standard anti-tuberculosis drugs; however, tolerance to inhaled Ibu-Arg solution was excellent, and no adverse events were attributed to its administration.

[0107] Follow-up sputum smear microscopy on May 18, 2023, confirmed conversion to AFB-negative status. The time to sputum smear conversion was 9 days, suggesting that inhaled Ibu-Arg solution may facilitate early bacteriological clearance even in patients with significant comorbid conditions such as diabetes.

[0108] (See Figure 4 for chest radiograph corresponding to this patient.)

[0109] EXAMPLE 4: Clinical Application of Inhaled Ibu-Arg solution in a Hospitalized Patient with Cavitary Pulmonary Tuberculosis and Comorbid Diabetes

[0110] A 49-year-old male patient (body weight: 51 kg) was hospitalized following a diagnosis of pulmonary tuberculosis. The patient was HIV negative and had a medical history of poorly controlled Type 2 diabetes mellitus treated with oral hypoglycemic agents. On admission, the patient presented with dyspnea and notable weight loss.

[0111] Sputum smear microscopy performed on May 8, 2023, was strongly positive for acid-fast bacilli (AFB+++). Radiological imaging confirmed bilateral cavitary pulmonary tuberculosis. Culture results were not available.

[0112] The patient was started on standard first-line antituberculous therapy on May 9, 2023. Nebulized administration of Ibu-Arg solution (ibuprofen / arginine) at a dose of 40 mg Ibu every 8 hours was initiated on May 19, 2023, as adjuvant therapy.

[0113] Clinical evolution was characterized by slow but progressive improvement. The patient experienced gastritis and vomiting associated with the standard anti-TB regimen; however, Ibu-Arg solution was well tolerated, with no reported adverse effects related to its use. Sputum smear microscopy conducted on June 6, 2023, confirmed conversion to AFB-negative status. The time to sputum smear conversion was 18 days from initiation of standard treatment and 18 days from Ibu-Arg solution introduction, indicating potential contribution of the adjuvant therapy in bacteriological clearance in a clinically complex patient.

[0114] (See Figure 5 for chest radiograph corresponding to this patient.)

[0115] EXAMPLE 5: Clinical Application of Inhaled Ibu-Arg solution in a Young Outpatient with Cavitary Pulmonary Tuberculosis and Familial Exposure

[0116] A 20-year-old female patient (body weight: 41 kg) was managed on an outpatient basis after presenting with pulmonary tuberculosis. The patient was HIV negative and had a positive epidemiological history due to familial exposure to tuberculosis.

[0117] The Sputum smear microscopy conducted on May 22, 2023, was strongly positive for acid-fast bacilli (AFB+++). Radiological imaging revealed bilateral cavitary pulmonary tuberculosis. Culture results were not available.

[0118] Standard first-line antituberculous therapy was initiated on May 30, 2023. Simultaneously, the patient began inhalation therapy with Ibu-Arg solution (ibuprofen / arginine), administered at a dose of 40 mg ibuprofen and 80 mg arginine every 8 hours via nebulization.

[0119] During the clinical course, the patient exhibited intolerance to the initial anti-TB drug regimen, requiring modification of the treatment plan. In contrast, tolerance to inhaled Ibu- Arg solution was excellent, and progressive clinical improvement was observed.

[0120] Follow-up sputum smear microscopy performed on June 7, 2023, showed conversion to AFB-negative status. The total time to sputum smear conversion was 8 days, suggesting that Ibu-Arg solution may accelerate bacterial clearance even in cases with initial intolerance to standard therapy.

[0121] (See Figure 6 for chest radiograph corresponding to this patient.) EXAMPLE 6: Clinical Application of Inhaled Ibu-Arg solution in an Outpatient with Cavitary Pulmonary Tuberculosis Confirmed by Culture

[0122] A 27-year-old female patient (body weight: 39.5 kg) was managed on an outpatient basis following a diagnosis of pulmonary tuberculosis. The patient was HIV negative and presented with general symptoms consistent with active disease.

[0123] Sputum smear microscopy performed on May 30, 2023, was strongly positive for acid-fast bacilli (AFB+++). Radiological examination revealed bilateral pulmonary tuberculosis with cavitary involvement in the right lung. Microbiological culture was positive for Mycobacterium tuberculosis.

[0124] The patient began standard first-line antituberculous therapy on May 30, 2023. Simultaneously, the patient commenced nebulized administration of an Ibu-Arg solution containing 40 mg of ibuprofen and 80 mg of arginine every 8 hours.

[0125] Clinical progression showed good overall tolerance to the anti-TB regimen. The patient demonstrated excellent tolerance to the Ibu-Arg solution with steady clinical improvement and rapid weight gain over the course of treatment.

[0126] Follow-up sputum smear microscopy performed on June 14, 2023, confirmed conversion to AFB-negative status. The total time to sputum smear conversion was 15 days.

[0127] (See Figure 7 for chest radiograph corresponding to this patient.)

[0128] EXAMPLE 7: Clinical Application of Inhaled Ibu-Arg solution in a Patient with Hepatotoxicity During Treatment of Cavitary Pulmonary Tuberculosis

[0129] A 33-year-old female patient (body weight: 43.5 kg) was managed on an outpatient basis following a diagnosis of pulmonary tuberculosis. The patient was HIV negative and presented with symptoms consistent with active infection.

[0130] Sputum smear microscopy performed on May 23, 2023, was strongly positive for acid-fast bacilli (AFB+++). Radiological studies revealed right-sided pulmonary tuberculosis with cavitation. Microbiological culture confirmed infection with Mycobacterium tuberculosis. Standard first-line antituberculous therapy was initiated on May 23, 2023. Concurrently, the patient began inhalation therapy with Ibu-Arg solution (ibuprofen / arginine) at a dose of 40 mg ibu ., 80 mg arg. every 8 hours via nebulization.

[0131] The patient developed anorexia, gastritis, and signs of toxic hepatitis during the course of antituberculous treatment. The standard regimen was adjusted, resulting in a gradual decrease in liver transaminase levels. Despite these complications, the patient exhibited excellent tolerance to Ibu-Arg solution, which was not discontinued at any point.

[0132] Follow-up sputum smear microscopy performed on June 15, 2023, confirmed conversion to AFB-negative status. The time to sputum smear conversion was 23 days.

[0133] (See Figure 8 for chest radiograph corresponding to this patient.)

[0134] EXAMPLE 8: Clinical Application of Inhaled Ibu-Arg Solution in a Patient with Cavitary Pulmonary Tuberculosis and Transient Hepatic Toxicity

[0135] A 40-year-old female patient (body weight: 59.3 kg) was managed on an outpatient basis after clinical suspicion of tuberculosis. A chest CT scan performed on May 5, 2023 revealed bilateral pulmonary lesions suggestive of cavitary tuberculosis.

[0136] Sputum smear microscopy on May 30, 2023 was positive for acid-fast bacilli (AFB++), and culture later confirmed the presence of Mycobacterium tuberculosis. Radiological classification confirmed bilateral cavitary pulmonary tuberculosis, consistent with CT findings.

[0137] Standard first-line antituberculous therapy was initiated on June 2, 2023. Concurrently, the patient began inhaled administration of Ibu-Arg solution (comprising 40 mg of ibuprofen and 80 mg of arginine) every 8 hours.

[0138] During the course of treatment, the patient developed anorexia, gastritis, and hemoptysis. Elevations in liver transaminase levels led to a temporary discontinuation of systemic antibiotics for 7 days. Despite these complications, the patient exhibited excellent tolerance to the Ibu-Arg solution, which was continued without interruption.

[0139] Sputum smear microscopy on June 9, 2023 confirmed conversion to AFB-negative status. A confirmatory smear on June 12, 2023 remained negative. The time to sputum smear conversion was recorded as 10 days. (See Figure 9 for chest radiograph corresponding to this patient.)

[0140] EXAMPLE 9: Clinical Application of Inhaled Ibu-Arg Solution in a Patient with Suspected Tuberculous Pleural Effusion and Subacute Pulmonary Presentation

[0141] A 30-year-old female patient (body weight: 48 kg) was admitted to hospital on May 31 , 2023. The patient was HIV negative and had a positive epidemiological background, as her mother had been diagnosed with pulmonary tuberculosis.

[0142] Two weeks prior to admission, the patient developed right-sided pleuritic chest pain. A chest X-ray revealed a parapneumonic pleural effusion. An initial pleural fluid aspiration performed on May 15, 2023, was non-diagnostic. Treatment with amoxicillin-clavulanic acid was initiated. Due to lack of clinical improvement, partial drainage of the effusion was performed on May 24, 2023, and the antibiotic regimen was modified. However, the effusion recurred by May 31 , 2023, prompting readmission and a second drainage procedure.

[0143] Pleural fluid samples were tested for acid-fast bacilli (AFB) and cultured for Mycobacterium tuberculosis, both of which were negative. Empirical antituberculous therapy was started on clinical grounds. Simultaneously, the patient began inhaled administration of Ibu-Arg solution (comprising 40 mg of ibuprofen and 80 mg of arginine) every 8 hours via nebulization.

[0144] The patient demonstrated progressive clinical improvement. After 7 days of treatment, the pleural effusion had not recurred. Tolerance to both the standard antibiotic regimen and Ibu-Arg solution was excellent, with no adverse events reported.

[0145] At the time of reporting, pleural fluid culture results were still pending. The pulmonary condition was classified as non-cavitary pneumonia with a subacute course, most likely of tuberculous origin.

[0146] Time to Sputum Smear Conversion: Indeterminate (no positive AFB smear reported).

[0147] (See Figure 10 for chest radiograph corresponding to this patient.) EXAMPLE 10: Clinical Application of Inhaled Ibu-Arg Solution in a Patient with Bilateral Cavitary Pulmonary Tuberculosis and Interstitial Infiltrate

[0148] A 46-year-old female patient (body weight: 58.4 kg) was managed on an outpatient basis after referral due to a sputum smear that tested strongly positive for acid-fast bacilli (AFB+++) on June 6, 2023. The patient was HIV negative.

[0149] Radiological evaluation revealed bilateral cavitary pulmonary tuberculosis in addition to a left pulmonary interstitial infiltrate. Microbiological culture results were not available.

[0150] Standard first-line antituberculous therapy was initiated on June 9, 2023. Simultaneously, the patient began nebulized administration of Ibu-Arg solution (containing 40 mg ibuprofen and 80 mg arginine) every 8 hours.

[0151] The patient demonstrated progressive clinical improvement. Tolerance to the antituberculous regimen was excellent. Likewise, inhaled Ibu-Arg solution was well tolerated, with no adverse effects reported during the treatment period.

[0152] Sputum smear microscopy performed on June 29, 2023, confirmed conversion to AFB-negative status. The time to sputum smear conversion was 20 days.

[0153] (See Figure 1 1 for chest radiograph corresponding to this patient.)

[0154] EXAMPLE 11 : Clinical Application of Inhaled Ibu-Arg Solution in a Patient with Multidrug-Resistant Pulmonary Tuberculosis and Prior Treatment History

[0155] A 34-year-old female patient (body weight: 46 kg) was managed on an outpatient basis following referral for a sputum smear that tested strongly positive for acid-fast bacilli (AFB+++) on June 6, 2023. The patient was HIV negative and had a prior medical history of dengue infection.

[0156] The patient had a documented diagnosis of multidrug-resistant pulmonary tuberculosis and had completed two prior antituberculous regimens. The first standard 4- drug treatment was completed under supervision in September 2021 . A second 4-drug regimen, also supervised, was completed on May 29, 2023.

[0157] On June 17, 2023, a new sputum smear was again positive for AFB+++, with viable bacilli. Radiological findings included bilateral non-cavitary pulmonary interstitial infiltrates, decreased left lung volume, and possible moderate pleural effusion as observed on June 21 . Subsequent imaging showed a more aerated left lung without evident pleural effusion, indicating radiological improvement.

[0158] A third course of standard first-line antituberculous therapy was initiated on June 21 , 2023. At the same time, the patient began inhaled administration of Ibu-Arg solution (comprising 40 mg ibuprofen and 80 mg arginine) every 8 hours via nebulization.

[0159] Clinical progress was favorable, with evident radiological and symptomatic improvement. The patient tolerated the new antibiotics well. Tolerance to the Ibu-Arg solution was also excellent, with no adverse events reported.

[0160] Follow-up sputum smear microscopy performed on July 3, 2023, was negative for AFB. The time to sputum smear conversion was 7 days.

[0161] (See Figure 12 for chest radiograph corresponding to this patient.)

[0162] EXAMPLE 12: Clinical Application of Inhaled Ibu-Arg Solution in an Adolescent Patient with History of Miliary and Meningeal Tuberculosis

[0163] A 15-year-old male patient (body weight: 66 kg) was managed on an outpatient basis after presenting with unilateral cavitary pulmonary tuberculosis. The patient had a medical history of intellectual developmental delay and had completed a supervised 4-drug treatment regimen for miliary pulmonary and meningeal tuberculosis in November 2022. The prior diagnosis had been confirmed by positive cerebrospinal fluid smear for Mycobacterium tuberculosis. The patient also had a positive epidemiological background, as his brother had previously been diagnosed with pulmonary tuberculosis.

[0164] Radiological classification identified a cavitary lesion in the left lung. Sputum smear and culture were both positive for Mycobacterium tuberculosis.

[0165] Standard first-line antituberculous therapy was initiated on June 29, 2023. In parallel, the patient began nebulized administration of Ibu-Arg solution (comprising 40 mg ibuprofen and 80 mg arginine) every 6 hours.

[0166] The patient demonstrated progressive clinical improvement with excellent tolerance to both the antibiotic regimen and the Ibu-Arg solution. No adverse effects were reported. Follow-up sputum smear microscopy on July 10, 2023 confirmed conversion to AFB- negative status. The time to sputum smear conversion was 11 days.

[0167] (See Figure 13 for chest radiograph corresponding to this patient.)

Claims

ClaimsWhat is claimed is:1 . A method of treating a subject suffering from a respiratory bacterial infection, comprising administering to the subject by inhalation an effective amount of a composition comprising a hypertonic saline solution and an ibuprofenate salt.

2. The method of claim 1 , wherein said effective amount of said composition further comprises arginine.

3. The method of claim 1 or 2, wherein the respiratory bacterial infection is selected from the group consisting of pneumonia, bronchiolitis, and tuberculosis.

4. The method of claim 1 or 2, wherein the respiratory bacterial infection is caused by a Mycobacterium bacterium.

5. The method of claim 1 or 2, wherein the respiratory bacterial infection is caused by a bacterium selected from the group consisting of, Mycobacterium tuberculosis, Mycobacterium SPP, Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila.

6. The method of claim 1 or 2, wherein the respiratory bacterial infection is caused by a bacterium selected from the group consisting of Mycobacterium avium and Mycobacterium abscessus.

7. The method of claim 1 or 2, wherein the respiratory bacterial infection is tuberculosis.

8. The method of claim 1 or 2, wherein the respiratory bacterial infection is tuberculosis caused by Mycobacterium tuberculosis.

9. The method of claim 1 or 2, wherein the ibuprofenate salt is selected from the group consisting of sodium ibuprofenate, potassium ibuprofenate, and lithium ibuprofenate.

10. The method of claim 9, wherein the ibuprofenate salt is sodium ibuprofenate.11 . The method of claim 9, wherein the concentration of the ibuprofenate salt in the solution is from 5 mM to 100 mM.

12. The method of claim 9, wherein the concentration of the ibuprofenate salt in the solution is from 5 mg / ml to 50 mg / ml.

13. The method of claim 1 or 2, wherein the concentration of NaCI in the hypertonic saline solution is from 0.3 M to 2.0 M.

14. The method of claim 1 or 2, wherein the pH of said composition is from 7.0 to 9.0.

15. The method of claim 2, wherein the weight ratio of ibuprofenate salt to arginine is from 1 :1 to 1 :3.

16. The method of claim 15, wherein the weight ratio of ibuprofenate salt to arginine is 1 :2.

17. A method of treating a subject suffering from tuberculosis, comprising administering to the subject, by inhalation or nebulization, a composition comprising an ibuprofenate salt and arginine in a hypertonic saline solution.

18. The method of claim 17, wherein said composition comprises ibuprofenate salt from 5 to 20 mg / ml, and arginine frornl O to 40 mg / ml.

19. The method of claim 17, wherein said composition comprises ibuprofenate salt in a concentration of 10 mg / ml, and arginine in a concentration of 20 mg / ml.

20. The method of claim 17 or 18, wherein the composition is administered by nebulization in a volume of 3 to 6 mL every 8 hours..

21. The method of claim 2 or 17, wherein said composition restores functional activity of alveolar macrophages infected with Mycobacterium tuberculosis by reducing oxidative stress, downregulating M1 macrophage polarization markers, and re-establishing nitric oxide synthesis via eNOS, thereby enhancing the host’s innate immune response and limiting pathogen dissemination.

22. A method of treating a subject suffering from tuberculosis, comprising administering to the subject, by nebulization, an aqueous composition comprising 40 mg of ibuprofenate salt and 80 mg of arginine in a hypertonic saline solution having a salt concentration between 0.3 M and 2.0 M, three times daily, in combination with standard antibiotic therapy..

23. Use of a composition comprising ibuprofenate salt and arginine in a hypertonic saline solution for the manufacture of a medicament for the treatment of tuberculosis by inhalation.

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