Methods for treating nontuberculous mycobacterial disease

Abstract

The present invention relates to an inhalable pharmaceutical composition comprising clofazimine, or a pharmaceutically acceptable derivative thereof, a clofazimine salt, or a polymorph thereof, or a combination thereof, and a pharmaceutically acceptable carrier and / or excipient, for use in the treatment or prevention of pulmonary nontuberculous bacterial infections, wherein the clofazimine is present in an amount of 1 mg to 20 mg by weight of the composition, and the inhalable pharmaceutical composition provides an effective daily dose of up to 90 mg of clofazimine upon inhalation. Pharmaceutical combinations comprising clofazimine in the form of an aerosol for pulmonary inhalation, for administration alone or concomitantly with other drugs, are also provided as combination therapy in the treatment and / or prevention of pulmonary infections caused by mycobacteria and other Gram-positive bacteria, and pulmonary fungal infections.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 374,720, filed September 6, 2022, U.S. Provisional Patent Application No. 63 / 438,987, filed January 13, 2023, and U.S. Provisional Patent Application No. 63 / 449,908, filed March 3, 2023.

[0002] Technical Field Disclosed herein are methods and compositions for treating and preventing nontuberculous bacterial infections. In particular, the methods involve administering an inhalable composition for aerosolization comprising clofazimine in an inhalable solution, suspension, or dry powder, which is administered by nebulization or oral inhalation to a subject in need of treatment. [Background technology]

[0003] background Nontuberculous mycobacterial (NTM) lung disease is a serious infection caused by bacteria common in the environment that can result in decreased lung function, cough, fatigue, and reduced quality of life. Approximately 86,000 people in the United States are estimated to be living with NTM lung disease, with an 8% annual increase among women, older adults, and those at highest risk for underlying lung conditions.

[0004] Clofazimine is one of the three main drugs recommended by the World Health Organization for the treatment of leprosy, which is caused by Mycobacterium leprae, and in recent years has been increasingly used for the treatment of other mycobacterial infections, such as drug-resistant tuberculosis and infections caused by nontuberculous mycobacteria (NTM). Clofazimine has primarily been delivered in the form of oral capsules.

[0005] Clofazimine is a highly hydrophobic riminophenazine antibiotic (Log P=7.66) with antimycobacterial and anti-inflammatory activity that was first described in 1957. Its structural formula is: [ka]

[0006] The exact mechanism by which clofazimine exerts its antibacterial effects is unknown; however, it is known to preferentially bind to mycobacterial DNA, thereby inhibiting DNA replication and cell growth. Other suggested mechanisms of action include membrane damage / destabilization, generation of membrane-destabilizing lysophospholipids, interference with potassium transport, and / or intracellular redox cycling. Although clofazimine is impressively active in vitro against Mycobacterium tuberculosis (MTB), including multidrug-resistant strains, until recently it was generally considered ineffective for the treatment of pulmonary tuberculosis (see, e.g., Cholo M et al., J Antimicrob Chemother, 2012 Feb, 67(2):290-8).

[0007] Because clofazimine is practically insoluble in water and exhibits high membrane permeability, it is classified as a Biologics Classification System (BCS) Class II drug. To overcome the problems associated with poor oral absorption and poor bioavailability of the drug, various strategies have been applied, such as micronization, nanonization, supercritical fluid recrystallization, spray-freeze drying into liquid, solid dispersions, and solutions in optimizing oral dosage forms.

[0008] Because it is classified as a BCS Class II drug, clofazimine is generally considered an ideal candidate for formulation into a solid dispersion to improve oral bioavailability (see, e.g., Bhusnure et al. IJRPC 2014, 4(4), 906-918). Consistent with this, due to its lipophilicity, clofazimine is commonly administered as a microcrystalline suspension in an oil-wax base to improve oral absorption. Absorption in humans after oral administration is highly variable (45-62%). Adverse effects of clofazimine are dose-related and primarily affect the skin, eyes, gastrointestinal tract, and QT prolongation. Side effects include the development of reddish-brown discoloration of the skin and conjunctiva, which gradually resolves upon discontinuation. These are the result of chronic systemic accumulation.

[0009] Mycobacterium is a genus of Actinobacteria with its own genus, the family Mycobacteriaceae. Mycobacteria have a characteristic rod-like shape and waxy outer shell.

[0010] Therefore, Mycobacteria can be divided into three groups. Mycobacterium tuberculosis complex - the causative agent of tuberculosis Mycobacterium leprae - the causative agent of leprosy • Nontuberculous mycobacteria (NTM), which encompass all other mycobacteria that are not M. tuberculosis or M. leprae, including the Mycobacterium abscessus complex (MABSC) and the Mycobacterium avium complex (MAC).

[0011] Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex bacterium. As one of the oldest documented infectious agents in humans, TB remains a significant cause of mortality and morbidity worldwide, resulting in an estimated 10.4 million new cases of TB infection and 1.4 million deaths from active TB disease in 2015 (see, e.g., World Health Organization (WHO) Global Tuberculosis Report 2016). In addition to high morbidity and mortality, the incidence of multidrug-resistant tuberculosis (MDR-TB) is of increasing concern, with 580,000 patients presenting with drug-resistant TB infection in 2015. Comorbidities, such as human immunodeficiency virus (HIV), complicate treatment and were responsible for 1.2 million cases of TB in 2015.

[0012] To treat multidrug-resistant (MDR) infections, the WHO recommends implementing a 9- to 12-month treatment regimen of second-line anti-TB drugs. These regimens, such as the 9- to 12-month Bangladesh regimen, treat MDR-TB with a combination of gatifloxacin, ethambutol, pyrazinamide, and clofazimine, resulting in relapse-free cure in 87.9% of patients (see, for example, Sotgiu, G, et al., “Applicability of the shorter 'Bangladesh regimen' in high multidrug-resistant tuberculosis settings,” International Journal of Infectious Diseases (2017) 56 190-193).

[0013] Other studies investigating abbreviated TB treatments have demonstrated that clofazimine has no clinical benefit after two weeks of oral administration (see, e.g., Diacon, AH, et al., "Bactericidal Activity of Pyrazinamide and Clofazimine Alone and in Combinations with Pretomanid and Bedaquiline," American Journal of Respiratory and Critical Care Medicine (2015), 191(8), 943-953). The lack of activity was attributed to the drug's low bioavailability, as it was theorized to bind with high affinity to circulating serum proteins. Despite the fact that clofazimine has been empirically demonstrated to be effective in treating MDR-TB and extensively drug-resistant TB (XDR-TB), its poor bioavailability after systemic administration appears to limit its biological activity over short-term treatment (see, e.g., Swanson, RV, et al., "Pharmacokinetics and Pharmacodynamics of Clofazimine in a Mouse Model of Tuberculosis", Antimicrobial Agents and Chemotherapy (2015), 59(6), 3042-3051).

[0014] Treatment of pulmonary infections with inhaled antibiotics can result in higher drug concentrations in the lungs and reduced adverse effects compared to systemic delivery (e.g., Touw, DJ, et al., "Inhalation of antibiotics in cystic fibrosis", European Respiratory Journal (1995), 8, 1594-1604), which results in increased biological activity and efficacy (see, e.g., Hickey, AJ, "Inhaled drug treatment for tuberculosis: Past progress and future prospects", Journal of Controlled Release, (2016), 240, 127-134). In vivo mouse models have demonstrated that aerosolized clofazimine significantly improved bacillary clearance in TB infection models compared with oral clofazimine administration only 28 days after treatment initiation (see, e.g., Verma, RK, et al., "Inhaled microparticles containing clofazimine are efficacious in treatment of experimental tuberculosis in mice," Antimicrobial Agents and Chemotherapy (2013), 57(2), 1050-1052). This improved efficacy over a short period of time may be due to higher clofazimine concentrations in pulmonary macrophages within tuberculous granulomas, resulting from direct delivery of clofazimine to the site of lung infection.

[0015] Therefore, the use of aerosolized administration of clofazimine in patients with MDR TB or XDR-TB infection may further improve patient treatment outcomes and shorten the duration of current treatment regimens.

[0016] The group of nontuberculous mycobacteria (NTM), formerly known as atypical or ubiquitous mycobacteria, includes over 150 species. NTM can be found ubiquitously in nature and exhibit a wide diversity. NTM can be detected in soil, land, and drinking water, as well as in foods such as pasteurized milk or cheese. NTM are generally considered to have low pathogenicity. Nevertheless, they can cause severe illness in humans, especially in immunocompromised individuals or those with previous pulmonary disease. Currently, NTM are classified according to their growth rate, divided into slow-growing mycobacteria (SGM) and fast-growing mycobacteria (RGM).

[0017] The slow-growing Mycobacterium avium complex (MAC), which includes the species Mycobacterium avium, Mycobacterium chimaera, and Mycobacterium intracellulare, is one of the most important and most frequent pathogenic NTMs. It primarily causes pulmonary infections, as do Mycobacterium kansasii, Mycobaceterium malmoense, Mycobacterium xenopi, Mycobacterium simiae, Mycobacterium abscessus, Mycobacterium gordonae, Mycobacterium fortuitum, and Mycobacterium chelonae. Mycobacterium marinum is involved in skin and soft tissue infections, such as aquarium granulomatosis.

[0018] In particular, RGM causes severe, life-threatening chronic lung disease and is associated with disseminated and often fatal infections. Infections are typically caused by invasive procedures involving contaminated materials and catheters, non-sterile surgical procedures, or the injection and implantation of foreign bodies. Exposure to showerheads and whirlpools has also been reported as a risk of infection. NTM typically causes opportunistic infections in patients with chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and other immunocompromised patients.

[0019] In recent years, the rapidly growing (RGM) Mycobacterium abscessus complex (Mycobacterium abscessus complex, MABSC), which includes the subspecies Mycobacterium abscessus subsp. abscessus (Maabscessus), Mycobacterium abscessus bolletii, and Mycobacterium abscessus massiliense, has emerged as an important human pathogen and is associated with significantly higher mortality rates than any other RGM.

[0020] Mycobacterium abscessus infections in CF patients are particularly problematic because they lead to enhanced lung destruction and are often untreatable, with failure rates as high as 60-66% (see, for example, Obregon-Henao A et al., Antimicrobial Agents and Chemotherapy, November 2015, Vol. 59, No. 11, pp. 6904-6912; Qvist, T., Pressler, T., Hoiby, N. and Katzenstein, T. L., "Shifting paradigms of nontuberculous mycobacteria in cystic fibrosis," Respiratory Research (2014), 15(1): pp. 41-47).

[0021] Human infections with NTM have become increasingly relevant with the emergence of the human acquired immunodeficiency syndrome pandemic. Mycobacteria from the Mycobacterium avium complex (MAC) have been identified as a major cause of opportunistic infections in patients infected with the human immunodeficiency virus (HIV).

[0022] Some NTM species are known to form biofilms. Biofilms are bacterial microcolonies embedded in an extracellular matrix that provide stability and resistance to the human immune system. In recent years, some NTM species have been shown to form biofilms that enhance resistance to disinfectants and antibacterial agents. Biofilm assembly proceeds through several stages, including reversible attachment, irreversible adhesion, bacterial aggregation, organization, and signaling leading to biofilm formation, and eventual dispersal. During this process, bacteria develop a matrix containing extracellular polymeric substances (EPS), such as polysaccharides, lipids, and nucleic acids, to form a complex three-dimensional structure (see, for example, Sousa S. et al., International Journal of Mycobacteriology 4 (2015), 36-43). Specifically, mycobacterial EPS differs from other biofilms because mycobacteria do not produce exopolysaccharides (see, for example, Zambrano MM, Kolter R. Mycobacterial biofilms: a greasy way to hold it together. Cell. 2005). Mycobacterial biofilms vary between species but can contain mycolic acids, glycopeptidolipids, mycolyl-diacylglycerol, lipooligosaccharides, lipopeptides, and extracellular DNA (overview and original study: Rose SJ, Babrak LM, Bermudez LE (2015) Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics. PLoS ONE). Biofilm formation is known to increase resistance to antibacterial agents (see, for example, Faria S. et al., Journal of Pathogens, Vol. 2015, Article ID 809014).

[0023] As a novel approach for the treatment of NTM lung infections, delivery of aerosolized liposomal amikacin / inhaled amikacin solution sprayed by a jet nebulizer has been suggested (Rose S. et al, 2014, PLoS ONE, Volume 9, Issue 9, e108703, and Olivier K. et al, Ann Am Thorac Soc Vol 11, No 1, pp. 30-35), as well as inhalation of anti-TB drug dry powder microparticles for pulmonary delivery (Cholo M et al., J Antimicrob Chemother. 2012 Feb;67(2):290-8, and Fourie B. and Nettey O., 2015 Inhalation Magazine, Verma 2013 Antimicrob Agents Chemother).

[0024] Initial treatment with parenteral aminoglycosides, tigecycline, and other promising oral antibiotics such as linezolid, delamanid, and bedaquiline, as well as multiple combination regimens using inhaled amikacin after surgical intervention in selected cases, have shown promising results in the treatment of NTM lung disease (Lu Ryu et al., Tuberc Respir Dis 2016;79:74-84). However, the inventors particularly noted that due to the increasing incidence and prevalence of NTM infections, especially NTM lung disease, and limited treatment options, the development of novel dosage forms / pharmaceutical formulations that enhance the bioavailability of currently used oral antibiotics and result in better outcomes is necessary.

[0025] The combination of oral clofazimine and amikacin has been shown to act synergistically in vitro against both Mycobacterium abscessus and Mycobacterium avium (see, e.g., van Ingen, J., et al., "In Vitro Synergy between Clofazimine and Amikacin in Treatment of Nontuberculous Mycobacterial Disease," Antimicrobial Agents and Chemotherapy 56(12), 6324-6327 (2012)). Furthermore, synergy has been demonstrated with the combination of oral clofazimine and bedaquiline, which is used against Mycobacterium tuberculosis (see, e.g., Cokol, M. et al., "Efficient Measurement and Factorization of High-Order Drug Interactions in Mycobacterium tuberculosis," Sciences Advances 2017:3:e170881, 11 October 2017). Synergistic effects have also been demonstrated with the combination of clofazimine and bedaquiline against the non-tuberculous bacterium Myocbacterium abscessus (Ruth, MM et al., “A Bedaquiline / Clofazimine Combination Regimen Might Add Activity to the Treatment of Clinically Relevant Non-Tuberculous Mycobacteria”, Journal of Antimicrobial Chemotherapy (2019), doi.org / 10.1093 / jac / dky526).

[0026] Fungal pathogens have emerged as a leading cause of human mortality. Current estimates suggest that deaths from invasive fungal infections are comparable to those of more well-known infectious diseases such as tuberculosis. Candida albicans, Cryptococcus neoformans, and Aspergillis fumigatus are the most common fungal pathogens in humans. Each of these species is responsible for hundreds of thousands of infections annually with unacceptably high mortality rates due to inadequate diagnosis and limited treatment options. Clofazimine has been shown to demonstrate efficacy as a combination agent against multiple fungi (see, e.g., Robbins, N., et al., “An Antifungal Combination Matrix Identifies a Rich Pool of Adjuvant Molecules that Enhance Drug Activity against Diverse Fungal Pathogens,” Cell Reports 13, 1481–1492, November 17, 2015). Fungi also play a role as commensals, colonizers and / or pathogens in cystic fibrosis (see, e.g., Chotirmall, SH and McElvaney, NG, “Fungi in the cystic fibrosis lung: Bystanders or pathogens?”, The International Journal of Biochemistry & Cell Biology 52 (2014), 161-173).

[0027] The low solubility of clofazimine in water results in low oral bioavailability and high microbial resistance. The specific techniques required to solubilize and stabilize the drug in pharmaceutical formulations in liquid aqueous carriers, such as for aerosolization for nebulizers, have become problematic. New treatment methods are needed to combat chronic NTM infection. Therefore, the present inventors have developed a new method for the treatment of NTM to achieve deep lung deposition of aerosol particles by increasing the efficacy of treatment and reducing adverse effects compared to oral and parenteral treatments.

Prior Technology Literature

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[0028] [Non-licensed document 1] Cholo M et al.,J Antimicrob Chemother,2012 Feb,67(2):290-8 [Non-licensed document 2] Bhusnure et al.IJRPC 2014,4(4),906-918 [Non-licensed document 3] Sotgiu,G,et al., "Applicability of the shorter 'Bangladesh regimen' in high multidrug-resistant tuberculosis settings", International Journal of Infectious Diseases (2017) 56 190-193

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[0029] overview Disclosed herein are methods and compositions for treating nontuberculous bacterial infections, comprising administering to a patient in need thereof a pharmaceutical composition for inhalation, the method comprising administering to the patient a therapeutically effective dose of a compound of formula N,5-bis(4-chlorophenyl)-3-propan-2-yliminophenazin-2-amine, clofazimine, or an isolated form of a clofazimine isomer, a polymorphic form of clofazimine selected from polymorphic forms I, II, III, and IV of clofazimine, and / or a combination thereof, or a pharmaceutically acceptable salt thereof, wherein the clofazimine compound is provided in the form of a suspension, solution, or dry powder, processes for their preparation, and methods of use and treatment involving the same. Additionally, the present disclosure provides a pharmaceutical composition in therapeutic combination with one or more active agents, including clofazimine, in the form of an aerosol for pulmonary inhalation. For example, the present disclosure provides methods for the treatment of NTM infections with compositions for inhalation by nebulization or by inhalation of dry powder without serious adverse events to the patient being treated, including the absence of laboratory abnormalities, cardiac complications such as QT wave prolongation, etc.

[0030] In one embodiment, the method of treatment comprises administering to a patient in need of treatment an inhalable pharmaceutical composition containing up to 10 mg, up to 30 mg, up to 60 mg, up to 90 mg, or up to about 100 mg of clofazimine compound, its isomer, pharmaceutically acceptable salt, or polymorphic form, which is administered once daily to the patient in one or more inhalations for a period of one week, about six months, or longer, which may be continuous throughout the period or discontinuously, e.g., allowing the patient one or more rest periods for intervals of up to about one month between dosing regimens. In one embodiment, the patient is administered a pharmaceutical composition containing clofazimine or a polymorphic form thereof in an amount of about 20 mg to about 100 mg, or about 20 mg to about 90 mg, about 25 mg to about 100 mg, or about 30 mg to about 90 mg, depending on the patient's needs, for a period of six months or longer. In one embodiment, a patient is administered a pharmaceutical composition comprising clofazimine for a period of 6 months or longer, and the interval between treatments is an interval during which the patient does not receive clofazimine treatment, e.g., the resting period can be about 1 week, 2 weeks, 3 weeks, 1 month, or longer. The resting period can be determined for each individual patient with NTM depending on the severity of the disease or infection and can include periods from several days up to 1 month or longer.

[0031] In one embodiment, the method can be a combination treatment in which a patient is administered an inhalable aerosolized clofazimine composition in combination with one or more other drugs, including steroids, antibiotics, etc. In this embodiment, the combinations and compositions provided herein can be used to treat and / or prevent pulmonary infections caused by mycobacteria and other gram-positive bacteria, as well as pulmonary fungal infections. The combination treatment involves administration of an inhalable clofazimine composition with the concurrent or sequential administration of one or more drugs, which can be administered by different routes, including inhalation or intravenous, subcutaneous, or oral administration. In this embodiment, the one or more drugs include anti-inflammatory drugs, including antibiotics, including amikacin, streptomycin, kanamycin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and other aminoglycosides active against NTM pulmonary infections, including para-aminosalicylates, and mixtures thereof, including ibuprofen, prednisone, etc.

[0032] In one embodiment of the present invention, (a) a therapeutically effective dose of a clofazimine compound, a clofazimine isomer, a clofazimine polymorphic form, or a pharmaceutically acceptable derivative or salt thereof; (b) a nonionic surfactant (e.g., polysorbate 80) having a hydrophilic-lipophilic balance value greater than 10; (c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline, and aqueous electrolyte solutions; The clofazimine compound, isomer, polymorphic form, or pharmaceutically acceptable derivative or salt thereof is provided in the form of particles in a suspension; The particles of clofazimine or a pharmaceutically acceptable derivative or salt thereof have a median size of less than 5 μm and a D90 of less than 6 μm.

[0033] In another embodiment of the invention, the particles of clofazimine or a pharmaceutically acceptable derivative or salt thereof have an average size of less than 2 μm and a D90 of less than 3 μm. In another embodiment of the present invention, (a) a therapeutically effective dose of clofazimine; (b) a nonionic surfactant having a hydrophilic-lipophilic balance value greater than 10; (c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline, and aqueous electrolyte solutions; The clofazimine is provided in the form of particles in a suspension, The particles of clofazimine have a median size of less than 5 μm and a D90 of less than 6 μm.

[0034] In another embodiment, the particles of clofazimine have a median size of less than 2 μm and a D90 of less than 3 μm.The clofazimine compositions of the invention are stable at room temperature for up to about 1 year or longer.

[0035] Aerosolization of the compositions of the present invention with a suitable nebulizer significantly increases delivery of aerosolized clofazimine to the lower lung (i.e., the central and lower peripheral bronchi, bronchioles, and alveoli), thereby substantially enhancing therapeutic efficacy.

[0036] Furthermore, the inhalation device should preferably also be adapted for localized pulmonary delivery of an aerosol with an optimal particle size distribution for uniform deposition in the lower lung.

[0037] Thus, the present invention provides aerosols having aerosol particles sized to facilitate delivery to the alveoli and bronchioles. A suitable aerodynamic particle size for targeting the alveoli and bronchioles is 1 to 5 μm. Larger particles are preferentially deposited in the upper lung, i.e., the bronchi and trachea, and the mouth and throat, i.e., the oropharyngeal region. Thus, the inhalation device is configured to generate aerosols having a mass median aerodynamic diameter (MMAD) in the range of about 1 to about 5 μm, preferably about 1 to about 3 μm. In further embodiments, the particle size distribution is narrow, with a geometric standard deviation (GSD) of less than about 3.

[0038] Local pulmonary delivery of the clofazimine compositions of the present invention reduces the amount of compound that needs to be administered to a patient to achieve a therapeutically effective dose, thus reducing the serious side effects or toxicity caused by orally administered suspensions, capsules, or tablets. The reduced pulmonary administration of clofazimine treatment to patients in need thereof herein is to the local lung tissue and therefore less toxic by reducing the amount of drug absorbed into the patient's systemic circulation, which can cause a variety of adverse effects, ranging from inconvenient to life-threatening, including skin / conjunctival discoloration, ichthyosis, loss of appetite, diarrhea, corneal xerosis, and reduced lymph node enlargement, most of which are reversible upon cessation of treatment.

[0039] Also disclosed is an inhalable pharmaceutical composition comprising clofazimine, or a pharmaceutically acceptable derivative of clofazimine, a clofazimine salt, or a polymorph of clofazimine, or a combination thereof, and a pharmaceutically acceptable carrier and / or excipient, for use in treating or preventing a pulmonary nontuberculous bacterial infection, wherein the clofazimine is present in an amount of 1 mg to 20 mg by weight of the composition, and the inhalable pharmaceutical composition provides an effective daily dose of up to 90 mg of clofazimine upon inhalation.The treated or prophylactically dissuaded pulmonary nontuberculous bacterial infection may be caused by a mycobacterium selected from the group consisting of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium leprae, and combinations thereof. In one example, the nontuberculous bacterial infection is an opportunistic infection selected from the group consisting of Mycobacterium avium complex pulmonary disease and opportunistic nontuberculous infections associated with one or more of the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, or acquired immunodeficiency syndrome, or a combination thereof. Concomitant conditions may be treated, such as opportunistic nontuberculous mycobacterial infections in patients with cystic fibrosis. The inhalable pharmaceutical composition may be used to treat or prophylactically suppress infections caused by mycobacteria or other gram-positive bacteria, administered by inhalation before, simultaneously with, or after administration of a drug selected from the group consisting of bedaquiline or a pharmaceutically acceptable salt of a derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof.

[0040] In one example, clofazimine is at least about 90% orthorhombic polymorph III.

[0041] The inhalable pharmaceutical composition can also be used to treat or as prophylaxis against nontuberculous bacterial infections of the lungs, and can be delivered by inhalation for treatment or prophylaxis with a high lung deposition rate of at least about 30%.

[0042] In yet another embodiment, an inhalable pharmaceutical composition is disclosed comprising clofazimine, or a pharmaceutically acceptable derivative of clofazimine, a clofazimine salt, or a polymorph of clofazimine, or combinations thereof, and a pharmaceutically acceptable carrier and / or excipient for use in the treatment or prevention of pulmonary nontuberculous bacterial infections, wherein the clofazimine is present in an amount of 1 mg to 20 mg weight % of the composition and delivered by an inhaler configured to cause a high lung deposition rate of at least 30%. [Brief explanation of the drawings]

[0043] [Figure 1] Figures 1A and 1B show semi-log plots of the mean ± standard deviation clofazimine concentrations in dog plasma after a single dose (Day 1) or 28 consecutive daily doses (Day 28) of low-dose (Group 3), medium-dose (Group 4), or high-dose (Group 5) clofazimine inhalation solution (CIS). [Figure 2] Figure 2 shows a log-log plot of the dose-dependence of plasma clofazimine pK in dog plasma. (Left panel - 2A) Plasma Cmax was proportional to dose (1.44) at SDs 1 and 28. Cmax at SD 28 was approximately 2.56-fold higher than Cmax at SD 1. Figure 2B (right panel) AUC0-24 was proportional to dose (1.47) at SDs 1 and 28. AUC0-24 at SD 28 was approximately 3.76-fold higher than that at SD 1. [Figure 3] Figure 3 shows a semi-logarithmic plot of the mean 6 standard deviation clofazimine concentrations in dog plasma for the recovery period after 28 consecutive daily doses (day 28) of low-dose (group 3), medium-dose (group 4), or high-dose (group 5) CIS. [Figure 4]FIG. 4 shows a graph illustrating the lung and plasma levels of clofazimine in study dogs after administration on days 29, 56, and 84 of the study. [Figure 5] FIG. 5 shows a graph illustrating clofazimine concentrations in lung and plasma of dogs in SD29 for low, medium and high mg / kg treatments. [Figure 6] FIG. 6 shows a graph illustrating the mean clofazimine plasma concentrations (semi-log scale) of human subjects treated with single ascending doses of clofazimine at various time points after administration of clofazimine for clofazimine doses of 30 mg, 60 mg, and 90 mg. [Figure 7] FIG. 7 shows a graph illustrating the mean clofazimine plasma concentrations (semi-log scale) of human subjects treated with single ascending doses of clofazimine at various time points after administration of clofazimine for 30 mg and 90 mg clofazimine doses. [Figure 8] Figure 8 shows a graph illustrating the effect between detergents used in experiments measuring cell viability. Three cell lines, A549 (dark bars), Calu-3 (light gray bars), and hAELVi (lighter gray bars), were tested for viability in medium containing an ultra-purified detergent (PS80) containing approximately 70% oleic acid. HBSS represents the negative control, and 1% Triton® X-100 was used as a positive control. The data are shown in the graph. [Figure 9] Figure 9 shows a graph illustrating the effect between detergents used in experiments measuring cell viability. Three cell lines, A549 (dark bars), Calu-3 (light gray bars), and hAELVi (lighter gray bars), were tested for viability in medium containing an ultra-purified detergent (PS80) containing approximately 99% oleic acid. HBSS represents the negative control, and 1% Triton X-100 was used as a positive control. The data are shown in the graph. DETAILED DESCRIPTION OF THE INVENTION

[0044] Detailed Description This disclosure describes the unexpected discovery of a therapeutically effective pharmaceutical formulation for pulmonary delivery of clofazimine or an isomer that can be easily aerosolized. The formulation facilitates pulmonary aerosolization of clofazimine in the form of a suspension, achieving lower (i.e., deeper) pulmonary deposition of the active agent in the alveolar capillaries, thereby significantly increasing the bioavailability of the highly hydrophobic BCS Class II drug, resulting in significantly increased therapeutic efficacy coupled with reduced systemic side effects. In an embodiment particularly effective with respect to antimycobacterial, nontuberculous bacterial, and anti-inflammatory activity, the orthorhombic polymorph III of clofazimine is used in substantially pure form, at least about 85%, more preferably at least about 90%, more preferably at least about 95%, more preferably at least about 97%, and even more preferably at least about 99%.

[0045] In another aspect, this finding provides improved antibiotic therapy for infections caused by mycobacteria and gram-positive bacteria, particularly pulmonary infections by NTMs, such as opportunistic infections in immunocompromised patients, including those with cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and HIV.

[0046] The present disclosure provides more effective therapeutic regimens that aim to prevent / overcome and / or reduce the systemic side effects caused by established oral treatment regimens for pulmonary infections by Gram-positive bacteria, particularly pulmonary TB and NTM infections, and also to reduce the dose and duration of treatment with clofazimine required to treat the infection.

[0047] It will be understood by those skilled in the art that the present application also discloses each and every individual feature disclosed herein and any combination thereof.

[0048] Definition: As used herein, unless otherwise defined, the term "clofazimine" can include clofazimine compounds, clofazimine isomers, clofazimine polymorphs, including polymorphic Forms I, II, III, or IV, clofazimine derivatives, clofazimine analogs, or pharmaceutically acceptable salts thereof, and / or combinations thereof.

[0049] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compounds of the present invention and is not biologically or otherwise undesirable. In many cases, the compounds herein are capable of forming acid and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids that can form salts include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids capable of forming salts include, for example, acetic acid, propionic acid, naphthoic acid, oleic acid, palmitic acid, pamoic (emboic) acid, stearic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, ascorbic acid, glucoheptonic acid, glucuronic acid, lactic acid, lactobionic acid, tartaric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0050] Pharmaceutically acceptable base addition salts can be formed using inorganic and organic bases. Inorganic bases that can form salts include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum, with ammonium, potassium, sodium, calcium, and magnesium salts being particularly preferred. Organic bases that can form salts include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, specifically, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, histidine, arginine, lysine, benethamine, N-methyl-glucamine, and ethanolamine. Other acids include dodecylsulfuric acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, and saccharin.

[0051] According to this, apart from the free base, preference is given to using the methanesulfonates, maleates, isonicotinates, nicotinates, malonates and salicylates, especially clofazimine mesylate.

[0052] As used herein, the term "pharmaceutically acceptable derivative" refers to, for example, compounds disclosed in U.S. Pat. No. 9,540,336, the disclosure of which is incorporated herein in its entirety. Furthermore, derivatives refer to compounds as described in Lu, Y., Zhen, M., Wang, B., Fu, L., Zhao, W., Li, P., Xu, J., Zhu, H., Jin, H., Yin, D., Huang, H., Upton, A. M., and Ma, Z., "Clofazimine Analogs with Efficacy Against Experimental Tuberculosis and Reduced Potential for Accumulation," Antimicrobial Agents and Chemotherapy (2011), 55(11): pp. 5185-5193. Furthermore, the term "pharmaceutically acceptable derivative" of a compound refers to, for example, a prodrug of the compound. In general, a prodrug is a derivative of a compound that can provide the active form of the compound upon administration. Such derivatives can be, for example, esters or amides of carboxyl groups, carboxyl esters of hydroxyl groups, or phosphate esters of hydroxyl groups.

[0053] "Therapeutically effective amount," "therapeutically effective dose," or "pharmaceutically effective amount" refers to an amount of clofazimine, or a pharmaceutically acceptable salt or derivative thereof, disclosed herein that has a therapeutic effect. A dose of clofazimine useful for treatment is a therapeutically effective amount. Thus, as used herein, a therapeutically effective amount refers to an amount of clofazimine that produces the desired therapeutic effect as determined by clinical trial results and / or model animal infection studies.

[0054] The amount and daily dose of the clofazimine composition administered to a patient can be determined and will vary depending on several factors, such as the particular microbial strain involved. The dosage may further depend on the patient's height, weight, sex, age, and medical history. For prophylactic treatment, a therapeutically effective amount is an amount effective to prevent microbial infection.

[0055] A "therapeutic effect" alleviates one or more symptoms of an infection to some extent, including curing the infection. "Cure" means that symptoms of an active infection are eliminated, including the complete or substantial elimination of excess viable members of the microorganisms involved in the infection to a point at or below the threshold of detection by conventional measurements. However, even after a cure is achieved, certain long-term or permanent effects of the infection may exist (such as extensive tissue damage). As used herein, "therapeutic effect" is defined as a statistically significant reduction in bacterial burden in a host, the emergence of resistance, or an improvement in infection symptoms, as measured by human clinical results or animal studies.

[0056] As used herein, "treat," "treatment," or "treating" refers to administering a pharmaceutical composition / combination for prophylactic and / or therapeutic purposes.

[0057] The term "prophylactic treatment" refers to treating a patient who is not yet infected but who is susceptible to or otherwise at risk for a particular infection. The term "therapeutic treatment" refers to administering treatment to a patient who already has an infection. Thus, in a preferred embodiment, treating is the administration of a therapeutically effective amount of clofazimine to a mammal (either for therapeutic or prophylactic purposes).

[0058] Unless otherwise stated herein, the term "inhalation" is meant to refer to pulmonary inhalation.

[0059] Unless otherwise stated herein, the term "infection" as used herein is meant to refer to a pulmonary infection.

[0060] Unless otherwise stated, the term "substantially," when used to refer to the purity of a compound, indicates a purity of the compound of 95% or greater purity.

[0061] Unless otherwise specified, the term "appropriate particle size" refers to a particle size of clofazimine in a composition or a particle size of a composition that provides a desired therapeutic effect when administered to a patient.

[0062] Unless otherwise specified, the term "appropriate concentration" refers to the concentration of ingredients in a composition or combination that provides a pharmaceutically acceptable composition or combination.

[0063] Pharmaceutical Compositions and Combinations The following water grades are particularly applicable to the present invention: Sterile Purified Water, Sterile Water for Injection, Sterile Water for Irrigation, Sterile Water for Inhalation (USP) and corresponding water grades according to e.g. the European Pharmacopoeia or National Formulary.

[0064] The aqueous electrolyte solution used in accordance with the present invention as the aqueous liquid carrier may further comprise sodium chloride, potassium chloride, lithium chloride, magnesium chloride, calcium chloride or mixtures thereof.

[0065] The aqueous liquid carrier is preferably isotonic saline (about / approximately 150 mM NaCl, preferably 0.9% NaCl corresponding to 154 mM NaCl).

[0066] Clofazimine has been shown to exist in at least four polymorphic forms (see, e.g., Bannigan, et al., "Investigation into the Solid and Solution Properties of Known and Novel Polymorphs of the Antimicrobial Molecule Clofazimine," Cryst. Growth Des. 2016, 16(12), pp. 7240-7250). Clofazimine can exist in triclinic form FI, monoclinic form FII, and orthorhombic form FIII. An additional form, FIV, has also been observed only at elevated temperatures.

[0067] Therefore, in a further embodiment of the present invention, (a) a therapeutically effective dose of clofazimine; (b) a nonionic surfactant having a hydrophilic-lipophilic balance value greater than 10; (c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline, and aqueous electrolyte solutions; The clofazimine is provided in the form of particles in a suspension, Pharmaceutical compositions are provided in which the particles of clofazimine have a median size of less than 5 μm and a D90 of less than 6 μm, preferably a median size of less than 2 μm and a D90 of less than 3 μm, and the clofazimine is provided in a polymorphic form or forms selected from triclinic form FI, monoclinic form FII and orthorhombic form FIII and mixtures of such forms.

[0068] In another embodiment, clofazimine is provided substantially in orthorhombic form FIII.

[0069] In further embodiments, there is provided a pharmaceutical composition according to any of the composition embodiments described herein, wherein the non-ionic surfactant is selected from the group consisting of polysorbate 20 (e.g., Tween® 20), polysorbate 60 (e.g., Tween® 60), polysorbate 80 (e.g., Tween® 80), stearyl alcohol, polyethylene glycol derivatives of hydrogenated castor oil having a hydrophilic-lipophilic balance value of 14 to 16 (e.g., Cremophor® RH40), hydrogenated castor oil having a hydrophilic-lipophilic balance value of 15 to 17 (e.g., Cremophor® RH40), and the like. polyethylene glycol derivatives (e.g., Cremophor® RH60), sorbitan monolaurate (e.g., Span® 20), sorbitan monopalmitate (e.g., Span® 40), sorbitan monostearate (e.g., Span® 60), polyoxyethylene (20) oleyl ether (e.g., Brij® 020), polyoxyethylene (20) cetyl ether (e.g., Brij® 58), polyoxyethylene (10) cetyl ether (e.g., Brij® 59), Brij® C10), polyoxyethylene (10) oleyl ether (e.g., Brij® O10), polyoxyethylene (100) stearyl ether (e.g., Brij® S100), polyoxyethylene (10) stearyl ether (e.g., Brij® S10), polyoxyethylene (20) stearyl ether (e.g., Brij® S20), polyoxyethylene (4) lauryl ether (e.g., Brij® L4), polyoxyethylene (20) cetyl ether (e.g., Bri j (registered trademark) 93), polyoxyethylene (2) cetyl ether (e.g., Brij (registered trademark) S2), caprylocaproyl polyoxyl-8 glyceride (e.g., Labrasol (registered trademark)), polyethylene glycol (20) stearate (e.g., Myrj (registered trademark) 49), polyethylene glycol (40) stearate (e.g., Myrj (registered trademark) S40), polyethylene glycol (100) stearate (e.g., Myrj (registered trademark) S100), polyethylene glycol (8) stearate (e.g., Myrj (registered trademark) S8),and polyoxyl 40 stearate (e.g., Myrj™ 52), and mixtures thereof.

[0070] In embodiments thereof, compositions for treating pulmonary infections include pharmaceutical compositions comprising an active agent, including an antimicrobial agent such as clofazimine, and a surfactant, including polysorbate 80, wherein the surfactant comprises one or more fatty acids, including myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid, and / or combinations thereof. In some embodiments, the surfactant comprises palmitic acid in an amount up to about 18% by weight of the composition, palmitoleic acid in an amount up to about 10% by weight, or stearic acid in an amount up to about 6% by weight. In certain embodiments, the surfactant comprises oleic acid in an amount up to 100% by weight of the composition. In one embodiment, the surfactant in the composition comprises an oleic acid content ranging from about 70% by weight to about 99% by weight. In some embodiments, the surfactant comprises from about 80% to about 99% by weight of the composition, or from about 83% to about 95% by weight, or from about 85% to about 93% by weight.

[0071] In one embodiment, a composition for treating a pulmonary infection comprises an active agent comprising clofazimine, a saline solution, and a surfactant comprising a fat content other than oleic acid that is less than 10% by weight of the composition.

[0072] In an exemplary embodiment, a dose of a composition for treating pulmonary diseases, including pulmonary infections, is provided, the composition comprising clofazimine in an amount of up to 150 mg, about 1% to about 7% saline solution, and a surfactant comprising about 80% to about 99.5% oleic acid.

[0073] In another embodiment, there is provided a pharmaceutical composition according to any of the composition embodiments described herein, wherein the non-ionic surfactant is polysorbate 80 and the aqueous liquid carrier is distilled water, hypertonic saline, or isotonic saline. In another embodiment of the invention, there is provided a pharmaceutical composition wherein the hypertonic saline is 1% to 7% (w / v) sodium chloride. In a further embodiment of the invention, there is provided a pharmaceutical composition wherein the non-ionic surfactant is polysorbate 80 and the aqueous liquid carrier is isotonic saline.

[0074] In another embodiment, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the osmolality of the composition is in the range of 200-700 mOsm / kg. In a further embodiment, the osmolality of the composition is in the range of 300-400 mOsm / kg.

[0075] In further embodiments, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the nonionic surfactant is in the range of 0.001% to 5% (v / v), 0.05% to about 2.5%, or about 0.01% to about 1% of the total composition, and the amount of clofazimine is in the range of 0.1% to 30% (w / v) of the total composition.

[0076] In another embodiment of the present invention, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the pharmaceutical composition comprises: (1) homogenizing a suspension of clofazimine, a non-ionic surfactant, and water to obtain a suspension containing clofazimine of appropriate particle size; (2) adjusting the pH of the suspension obtained from (1) to a pH of 5.5 to 7.5; (3) adjusting the sodium chloride concentration to an appropriate concentration; (4) adjusting the osmolality to an appropriate level.

[0077] In a further embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154 mM sodium chloride. In another embodiment, the homogenization in step (1) is carried out by high-pressure homogenization, high-shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes. In another aspect, the homogenization of clofazimine is carried out in multiple homogenization steps. In another embodiment, suitable particle sizes for clofazimine are particles having an average size of less than 5 μm and a D90 of less than 6 μm. In a further embodiment, suitable particle sizes for clofazimine are particles having an average size of less than 2 μm and a D90 of less than 3 μm.

[0078] In a further embodiment there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the pharmaceutical composition comprises: (1) homogenizing a suspension of clofazimine in a non-aqueous liquid to obtain a suspension containing clofazimine of appropriate particle size; (2) isolating clofazimine; (3) adding clofazimine to a nonionic surfactant and water; (4) adjusting the pH of the suspension obtained from (3) to a pH between pH 5.5 and pH 7.5; (5) adjusting the sodium chloride concentration to an appropriate concentration.

[0079] In a further embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154 mM sodium chloride. In a further embodiment, the homogenization in step (1) is carried out by high-pressure homogenization, high-shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes. In another embodiment, the homogenization of clofazimine is carried out in multiple homogenization steps. In another embodiment, suitable particle sizes for clofazimine are particles having an average size of less than 5 μm and a D90 of less than 6 μm. In a further embodiment, suitable particle sizes for clofazimine are particles having an average size of less than 2 μm and a D90 of less than 3 μm.

[0080] In a further embodiment there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the composition comprises: (1) micronizing clofazimine to obtain clofazimine of a suitable particle size; (2) adding clofazimine to a nonionic surfactant and water; (3) adjusting the pH of the suspension obtained from (2) to a pH of 5.5 to 7.5; (4) adjusting the sodium chloride concentration to an appropriate concentration.

[0081] In a further embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154 mM sodium chloride.

[0082] In another embodiment, the micronization of clofazimine is carried out by jet milling, spray drying, ball milling, or supercritical fluid processing. In another embodiment, the micronization of clofazimine is carried out in multiple micronization steps. In another embodiment, suitable particle sizes for clofazimine are particles having an average size of less than 5 μm and a D90 of less than 6 μm. In a further embodiment, suitable particle sizes for clofazimine are particles having an average size of less than 2 μm and a D90 of less than 3 μm.

[0083] In a further embodiment, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the composition is prepared by a process comprising homogenizing a suspension of clofazimine in water containing a non-ionic surfactant, an appropriate concentration of sodium chloride, and adjusted to a pH of between pH 5.5 and pH 7.5 to obtain clofazimine of a suitable particle size. In a further embodiment, the pH is adjusted to 7.4, and the sodium chloride concentration is adjusted to 154 mM sodium chloride. In a further embodiment, the homogenization is performed by high-pressure homogenization, high-shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes. In another embodiment, the homogenization of clofazimine is performed in multiple homogenization steps. In another embodiment, the suitable particle size of clofazimine is particles having an average size of less than 5 μm and a D90 of less than 6 μm. In a further embodiment, the suitable particle size of clofazimine is particles having an average size of less than 2 μm and a D90 of less than 3 μm.

[0084] In another embodiment, there is provided a process for the preparation of a pharmaceutical composition according to any of the composition embodiments described herein, the process comprising: (1) homogenizing a suspension of clofazimine, a non-ionic surfactant, and water to obtain a suspension containing clofazimine of appropriate particle size; (2) adjusting the pH of the suspension obtained from (1) to a pH of 5.5 to 7.5; (3) adjusting the sodium chloride concentration to an appropriate concentration; (4) adjusting the osmolality to an appropriate level.

[0085] In another embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154 mM sodium chloride. In a further embodiment, homogenization is performed by high-pressure homogenization, high-shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes. In a further embodiment, homogenization of the clofazimine is performed in multiple homogenization steps. In a further embodiment, suitable particle sizes for clofazimine are particles having an average size of less than 5 μm and a D90 of less than 6 μm. In another embodiment, suitable particle sizes for clofazimine are particles having an average size of 2 μm and a D90 of less than 3 μm.

[0086] In another embodiment, a process for preparing any of the pharmaceutical composition embodiments as described herein is provided, comprising the steps of: (1) homogenizing a suspension of clofazimine in a non-aqueous liquid to obtain a suspension containing clofazimine of an appropriate particle size; (2) isolating clofazimine; (3) adding clofazimine to a nonionic surfactant and water; (4) adjusting the pH of the suspension obtained from (3) to a pH between pH 5.5 and pH 7.5; (5) A step of adjusting the sodium chloride concentration to an appropriate concentration.

[0087] In another embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154 mM sodium chloride. In a further embodiment, homogenization is performed by high-pressure homogenization, high-shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes. In a further embodiment, homogenization of the clofazimine is performed in multiple homogenization steps. In a further embodiment, suitable particle sizes for clofazimine are particles having an average size of less than 5 μm and a D90 of less than 6 μm. In another embodiment, suitable particle sizes for clofazimine are particles having an average size of 2 μm and a D90 of less than 3 μm.

[0088] In a further embodiment, there is provided a process for the preparation of a pharmaceutical composition according to any one of the pharmaceutical composition embodiments described herein, comprising: (1) micronizing clofazimine to obtain clofazimine of a suitable particle size; (2) adding clofazimine to a nonionic surfactant and water; (3) adjusting the pH of the suspension obtained from (2) to a pH of 5.5 to 7.5; (4) adjusting the sodium chloride concentration to an appropriate concentration.

[0089] In another embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154 mM sodium chloride. In a further embodiment, the micronization of clofazimine is carried out by jet milling, spray drying, ball milling, or supercritical fluid processing. In a further embodiment, the micronization of clofazimine is carried out in multiple micronization steps. In a further embodiment, suitable particle sizes for clofazimine are particles having an average size of less than 5 μm and a D90 of less than 6 μm. In another embodiment, suitable particle sizes for clofazimine are particles having an average size of 2 μm and a D90 of less than 3 μm.

[0090] In another embodiment, provided herein is a process for preparing a pharmaceutical composition according to any one of the pharmaceutical composition embodiments described herein, comprising homogenizing a suspension of clofazimine in water containing a non-ionic surfactant, a suitable concentration of sodium chloride, and adjusted to a pH of between pH 5.5 and pH 7.5 to obtain clofazimine of a suitable particle size. In another embodiment, the pH is 7.4, and the suitable concentration of sodium chloride is 154 mM sodium chloride. In a further embodiment, the homogenization is performed by high-pressure homogenization, high-shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes. In a further embodiment, the homogenization of clofazimine is performed in multiple homogenization steps. In a further embodiment, the suitable particle size of clofazimine is particles having an average size of less than 5 μm and a D90 of less than 6 μm. In another embodiment, the suitable particle size of clofazimine is particles having an average size of 2 μm and a D90 of less than 3 μm.

[0091] In a further embodiment, there is provided a process for preparing a pharmaceutical composition according to any one of the composition embodiments described herein, comprising: (a) homogenizing a suspension of clofazimine, a non-ionic surfactant, and water to obtain a suspension containing clofazimine of an appropriate particle size; (b) adjusting the pH of the resulting suspension to a pH of between pH 5.5 and pH 7.5; (c) adjusting the sodium chloride concentration to an appropriate concentration; and (d) adjusting the osmolality to an appropriate level, wherein steps (b), (c), and (d) may be performed in the following order: (b), (c), (d); (b), (d), (c); (c), (b), (d); (c), (d), (b); (d), (b), (c); or (d), (c), (b).

[0092] In another embodiment, there is provided a process for preparing a pharmaceutical composition according to any one of the composition embodiments described herein, comprising: (a) homogenizing a suspension of clofazimine in a non-aqueous liquid to obtain a suspension containing clofazimine of an appropriate particle size; (b) isolating clofazimine; (c) adding clofazimine to a non-ionic surfactant and water; (d) adjusting the pH of the resulting suspension to a pH of between pH 5.5 and pH 7.5; and (e) adjusting the sodium chloride concentration to an appropriate concentration, wherein steps (d) and (e) may be performed in the order of (d), (e); or (e), (d).

[0093] In another embodiment, there is provided a process for preparing a pharmaceutical composition according to any of the composition embodiments described herein, comprising: (a) micronizing clofazimine to obtain clofazimine of a suitable particle size; and (b) adding clofazimine to water containing a non-ionic surfactant, a suitable concentration of sodium chloride, and adjusted to a pH of between 5.5 and 7.5.

[0094] In another embodiment, a pharmaceutical combination in the form of an aerosol for inhalation prepared by aerosolizing a composition according to any one of the composition embodiments described herein with a nebulizing device selected from an ultrasonic nebulizer, an electrospray nebulizer, a vibrating membrane nebulizer, a jet nebulizer, and a mechanical soft mist inhaler, A pharmaceutical combination is provided in which the aerosol particles generated by the nebulizer device have a mass median aerodynamic diameter of 1 to 5 μm. In a further embodiment, the inhaled aerosol is for lower lung deposition. In another embodiment, the nebulizer device has an output rate of 0.1 to 1.0 ml / min. In another embodiment, the total inhaled volume is 1 ml to 5 ml.

[0095] In another embodiment, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the pharmaceutical composition is for use in combination with an agent for dispersing and / or disrupting biofilms and a mucolytic and / or mucoactive agent, and / or an agent that reduces biofilm formation selected from nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth thiol, ethylenediaminetetraacetic acid, gentamicin-loaded phosphatidylcholine-decorated gold nanoparticles, a chelating agent, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides. In another embodiment, the composition for use is administered prior to, concurrently with, or subsequent to the administration of a drug selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof.

[0096] In another embodiment, there is provided a pharmaceutical combination according to any of the combination embodiments described herein, wherein the pharmaceutical combination is for use in combination with an agent for dispersing and / or disrupting biofilms and a mucolytic and / or mucoactive agent, and / or an agent that reduces biofilm formation selected from nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth thiol, ethylenediaminetetraacetic acid, gentamicin-loaded phosphatidylcholine-decorated gold nanoparticles, a chelating agent, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides. In another embodiment, the combination for use is used to administer a composition of the present invention before, simultaneously with, or following administration of a drug selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof. In another embodiment, the composition is administered before, simultaneously, or following administration of a drug selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof. In a further embodiment, the composition is administered before, simultaneously, or following administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.

[0097] In another embodiment, a pharmaceutical composition according to any one of the composition embodiments described herein is provided for use in treating and / or preventing pulmonary infections caused by mycobacteria or other Gram-positive bacteria. In a further embodiment, the infection is caused by a Mycobacterium species selected from nontuberculous mycobacteria and the Mycobacterium tuberculosis complex, and combinations thereof. In a further embodiment, the nontuberculous mycobacteria is selected from Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium leprae, and combinations thereof. In another embodiment, the infection is an opportunistic infection selected from MAC pulmonary disease and nontuberculous infections in patients with cystic fibrosis, chronic obstructive pulmonary disease, or acquired immunodeficiency syndrome. In another embodiment, the infection is an opportunistic nontuberculous mycobacterial infection in a patient with cystic fibrosis. In another embodiment, the composition for use is administered before, concurrently with, or following administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof. In another embodiment, the composition is administered before, concurrently, or following administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof. In a further embodiment, the composition is administered before, concurrently, or following administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.

[0098] In another embodiment, a pharmaceutical combination according to any of the combination embodiments described herein is provided for use in the treatment and / or prevention of pulmonary infections caused by mycobacteria or other Gram-positive bacteria. In a further embodiment, the infection is caused by a Mycobacterium species selected from nontuberculous mycobacteria and the Mycobacterium tuberculosis complex, and combinations thereof. In a further embodiment, the nontuberculous mycobacteria is selected from Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium leprae, and combinations thereof. In another embodiment, the infection is an opportunistic infection selected from MAC pulmonary disease and nontuberculous infections in patients with cystic fibrosis, chronic obstructive pulmonary disease, or acquired immunodeficiency syndrome. In another embodiment, the infection is an opportunistic nontuberculous mycobacterial infection in a patient with cystic fibrosis. In another embodiment, the combination for use is used to administer a composition of the present invention before, simultaneously with, or following administration of a drug selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof. In another embodiment, the combination for use is used to administer a composition of the present invention before, simultaneously with, or following administration of a drug selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof. In another embodiment, the combination for use is used to administer a composition of the present invention before, simultaneously with, or following administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.

[0099] In another embodiment, a system is provided for use in providing antibiotic activity in treating or providing prophylaxis against pulmonary infections caused by mycobacteria or other gram-positive bacteria, the system comprising: 1) A nebulized pharmaceutical combination, (a) a therapeutically effective dose of clofazimine; (b) a nonionic surfactant having a hydrophilic-lipophilic balance value greater than 10; (c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline, and aqueous electrolyte solutions; an atomized pharmaceutical combination comprising: 2) A nebulizer, The clofazimine is present in the form of a suspension, The system is equipped with a nebulizer such that the aerosol particles generated by the system have a mass median aerodynamic diameter of 1 to 5 μm.

[0100] In a further embodiment, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein for use in the treatment and / or prevention of a pulmonary fungal infection or Clostridium difficile, or a combination thereof. In another embodiment, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein for use in the treatment and / or prevention of a pulmonary fungal infection. In a further embodiment, the pulmonary fungal infection is Candida albicans or Aspergillus fumigatus, or a combination thereof.

[0101] In further embodiments, there is provided a pharmaceutical combination according to any one of the combination embodiments described herein for use in the treatment and / or prevention of a pulmonary fungal infection or Clostridium difficile, or a combination thereof. There is provided a pharmaceutical combination according to any one of the combination embodiments described herein for use in the treatment and / or prevention of a pulmonary fungal infection. In further embodiments, the pulmonary fungal infection is Candida albicans or Aspergillus fumigatus, or a combination thereof.

[0102] In another embodiment, a method of treating or preventing a pulmonary infection is provided, comprising administering by inhalation to a patient in need thereof a composition according to any one of the composition embodiments described herein. In another embodiment, the infection is caused by a Mycobacterium species selected from nontuberculous mycobacteria and the Mycobacterium tuberculosis complex, and combinations thereof. In a further embodiment, the nontuberculous mycobacteria is selected from Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium leprae, and combinations thereof. In a further embodiment, the infection is an opportunistic infection selected from MAC pulmonary disease and nontuberculous infections in patients with cystic fibrosis, chronic obstructive pulmonary disease, or acquired immune deficiency syndrome. In another embodiment, the infection is an opportunistic nontuberculous mycobacterial infection in a patient with cystic fibrosis.

[0103] In a further embodiment, there is provided a method of treating or preventing a pulmonary infection caused by mycobacteria or other gram-positive bacteria in a patient in need thereof, comprising administering by inhalation a composition according to any one of the composition embodiments described herein before, simultaneously with, or following administration of a drug selected from bedaquiline, or a pharmaceutically acceptable salt of a derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof. In another embodiment, the drug is bedaquiline or amikacin. In a further embodiment, the drug is bedaquiline.

[0104] Particle size and distribution The therapeutic effect of aerosolized therapy depends on the deposited dose and its distribution. Aerosol particle size is one of the key variables in defining the deposited dose and distribution of drug aerosol in the lungs.

[0105] In general, inhaled aerosol particles undergo deposition by one of two mechanisms: impaction, which typically predominates for larger aerosol particles, and sedimentation, which is more common for smaller aerosol particles. Impaction occurs when the momentum of an inhaled aerosol particle is large enough that the particle does not follow the airflow and encounters a physiological surface. In contrast, sedimentation occurs primarily in the lower lung when very small aerosol particles traveling with the inhaled airflow encounter a physiological surface as a result of gravitational settling.

[0106] Drug delivery to the lungs can be achieved by inhalation of aerosols through the mouth and throat. Aerosol particles with an aerodynamic diameter greater than approximately 5 μm generally do not reach the lungs. Instead, they tend to impact the back of the throat, where they are swallowed and, in some cases, absorbed orally. Aerosol particles with diameters of approximately 3 to 5 μm are small enough to reach the upper to middle regions of the lungs (the conducting airways) but are too large to reach the alveoli. Smaller aerosol particles, i.e., approximately 0.5 to 3 μm, can reach the alveolar region. Aerosol particles with diameters smaller than approximately 0.5 μm tend to be exhaled during tidal breathing but can also be deposited in the alveolar region by breath-holding.

[0107] Aerosols used for pulmonary drug delivery are composed of a wide range of aerosol particle sizes, so statistical descriptors are used. Aerosols used for pulmonary drug delivery are typically described by their mass median diameter (MMD), i.e., half of the mass is contained in aerosol particles larger than the MMD and half of the mass is contained in aerosol particles smaller than the MMD. For particles with uniform density, the volume median diameter (VMD) can be used interchangeably with the MMD. VMD and MMD determinations are performed by laser diffraction. The width of the distribution is expressed as the geometric standard deviation (GSD). However, aerosol particle deposition in the respiratory tract is more accurately described by the particle's aerodynamic diameter, and therefore the mass median diameter is typically used. MMAD determinations are performed by inertial impaction or time-of-flight measurements. For aqueous particles, the VMD, MMD, and MMAD should be the same. However, if humidity is not controlled as the aerosol passes through the impactor, the MMAD determination will be smaller than the MMD and VMD due to dehydration. For the purposes of this description, VMD, MMD, and MMAD measurements are considered to be under controlled conditions such that the descriptions of VMD, MMD, and MMAD are equivalent.

[0108] Nevertheless, for purposes of illustration, the aerosol particle size of the aerosol particles is given as the MMAD determined by measuring at room temperature using a Next Generation Impactor (NGI) according to the United States Pharmacopeial Convention (USP). Also disclosed in Jolyon Mitchell and Mark Nagel, "Particle Size Analysis of Aerosols from Medicinal Inhalers," KONA Powder and Particle Journal (2004), Volume 22, pages 32-65, ongoing revision. <601> Aerosols, Nasal Sprays, Metered-Dose Inhalers, and Dry Powder Inhalers, Pharmacopeial Forum (2003), Volume Number 29, pages 1176-1210.

[0109] According to the present invention, the aerosol particle size is optimized to maximize clofazimine deposition at the site of infection and maximize tolerability. Aerosol particle size can be expressed as mass median aerodynamic diameter (MMAD). Large particles (e.g., MMAD > 5 μm) are too large to navigate the bends of the airways and tend to deposit in the extrathoracic and upper airways. Intolerance (e.g., coughing and bronchospasm) can result from upper airway deposition of large particles.

[0110] Therefore, according to a preferred embodiment, the MMAD of the aerosol should be less than about 5 μm, preferably about 1-5 μm, and more preferably less than 3 μm (<3 μm).

[0111] However, guided breathing maneuvers can be used to allow larger particles to pass through the extrathoracic and upper airways and enter deeper into the lungs than during tidal breathing, thereby increasing aerosol deposition in the middle and lower lungs. Guided breathing maneuvers can be as slow as 100 ml / min. Therefore, when used with guided breathing maneuvers, the preferred MMAD of the aerosol should be less than about 10 μm.

[0112] Another equally important factor (in addition to aerosol particle size) is the particle size and size distribution of the solid particles, in this case clofazimine particle size and distribution. The size of the solid particles in a given aerosol particle must be smaller than the aerosol particles it contains. Larger aerosol particles may contain one or more solid particles. Furthermore, when dealing with dilute suspensions, the majority of the aerosol particles may not contain solid particles.

[0113] For this reason, it is desirable to have solid drug particles that are significantly smaller than the MMAD of the aerosol particles.

[0114] Another consideration is that, for example, when using a vibrating mesh nebulizer, the formulation is pumped through orifices in a plate, which breaks the suspension into droplets, and therefore solid particles must be smaller than these orifices in order to pass through.

[0115] The particle size of solids in a suspension can be given by the average size of the particles and by the distribution of the particles: the D90 value indicates that 90% of the particles in the suspension are of the average size or smaller.

[0116] Nebulizers: For aqueous and other non-pressurized liquid systems, various nebulizers (including small-volume nebulizers) are available for aerosolizing formulations. Compressor-driven nebulizers incorporate jet technology and use compressed air to generate liquid aerosols. Such devices are commercially available from, for example, Healthdyne Technologies, Inc.; Invacare, Inc.; Mountain Medical Equipment, Inc.; Pari Respiratory, Inc.; Mada Medical, Inc.; Puritan-Bennet; Schuco, Inc., DeVilbiss Health Care, Inc.; and Hospitalak, Inc. Ultrasonic nebulizers rely on mechanical energy in the form of vibrations of piezoelectric crystals to generate respirable droplets and are commercially available from, for example, Omron Healthcare, Inc. and DeVilbiss Health Care, Inc. Vibrating mesh nebulizers rely on either piezoelectric or mechanical pulses to generate respirable droplets.Other examples of nebulizers for use with clofazimine described herein are disclosed in U.S. Patent Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911; 4,510,929; 4,624,251; 5,164,740; 5,586,550; 5,758,637; 6,644,304; 6,338,443; 5,906,202; 5,934,272; 5,960,792; 5,971,951; 6,070,575; 6,199 Nos. 2,876; 6,230,706; 6,349,719; 6,367,470; 6,543,442; 6,584,971; 6,601,581; 4,263,907; 5,709,202; 5,823,179; 6,192,876; 6,644,304; 5,549,102; 6,083,922; 6,161,536; 6,264,922; 6,557,549; and 6,612,303 (all of which are incorporated by reference in their entireties). Commercially available examples of nebulizers that can be used with the clofazimine compositions described herein include Respirgard II®, Aeroneb®, Aeroneb® Pro, and Aeroneb® Go manufactured by Aerogen; AERx® and AERx Essence™ manufactured by Aradigm; Porta-Neb®, Freeway Freedom™, Sidestream, Ventstream, and I-neb manufactured by Respironics, Inc.; and PARI LCPlus®, PARI LC-Star®, and e-Flow7m manufactured by PARI, GmbH. Further non-limiting examples are disclosed in U.S. Patent No. 6,196,219.

[0117] In one embodiment, the pharmaceutical composition can be aerosolized using a nebulizing device, preferably selected from ultrasonic nebulizers, electrospray nebulizers, vibrating membrane nebulizers, jet nebulizers, or mechanical soft mist inhalers. The device preferably controls the patient's inhalation flow rate by either electrical or mechanical processes. In a more preferred embodiment, the aerosol generation by the device is triggered by the patient's inhalation, such as with an AKITA device.

[0118] Preferred (commercially available) examples of such nebulizers / devices to be used according to the present invention are Vectura fox, Pari eFlow, Pari Trek S, Philips Innospire mini, Philips InnoSpire Go, Medspray devices, Aeroneb Go, Aerogen Ultra, Respironics Aeroneb, Akita, Medspray Ecomyst and Respimat.

[0119] Use in treatment and / or prophylaxis The pharmaceutical compositions and pharmaceutical combinations (aerosols, aerosolized formulations) and systems according to the invention are intended for use in the treatment and / or prevention of pulmonary infections caused by mycobacteria or other clofazimine-susceptible bacteria, such as Staphylococcus aureus (including methicillin-resistant and vancomycin-intermediate-resistant strains), Streptococcus pneumoniae, and Enterococcus spp. The pharmaceutical compositions and pharmaceutical combinations of the invention may also be used to treat and / or prevent pulmonary fungal infections.

[0120] Administration of clofazimine In one embodiment, the pharmaceutical composition is delivered by nebulization in about 1-5 ml, preferably 1-2 ml, of the pharmaceutical composition of the present invention, with a target load dose of about 1-5 ml corresponding to 20-100 mg of clofazimine, based on a clofazimine concentration in the pharmaceutical composition of about 20 mg / ml.

[0121] The daily lung dose (i.e., the dose deposited in the lung) of clofazimine administered according to the present invention is approximately 5-10 mg, which corresponds to a nominal dose (device dose) of 15-30 mg for M. abscessus infections.

[0122] It will be appreciated that those skilled in the art will routinely adjust the administered pulmonary dose of clofazimine (and therefore the loaded / nominal dose / nebulized volume) based on the minimum inhibitory concentration (MIC) of clofazimine for each bacterial strain, which is well established in the art.

[0123] Depending on the frequency of administration, once or twice a day, the daily pulmonary dose is divided accordingly.

[0124] In one embodiment, clofazimine is administered once or twice daily, resulting in a total daily pulmonary dose of about 5-10 mg.

[0125] Mucolytic / biofilm modifiers To reduce the viscosity of sputum during aerosol treatment and to disrupt existing biofilms, treatment and / or prevention according to the present invention may include the additional administration of a mucolytic agent and / or a biofilm disrupting agent.

[0126] These agents may be prepared in a fixed combination or may be administered simultaneously with or subsequent to a pharmaceutical composition / aerosol combination comprising clofazimine according to the present invention.

[0127] The agent for biofilm dispersal / disruption, mucolytic and / or mucosally active agent and / or agent that reduces biofilm formation used in accordance with the present invention is selected from nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth thiol, ethylenediaminetetraacetic acid, gentamicin-loaded phosphatidylcholine-decorated gold nanoparticles, chelating agents, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides.

[0128] Additionally, other pharmaceutically active agents may be used in combination with the pharmaceutical composition / aerosol combination according to the present invention, such active agents may be selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylates, and mixtures thereof.

[0129] These agents may be prepared in a fixed combination or may be administered before, simultaneously with, or after a pharmaceutical composition / aerosol combination containing clofazimine according to the present invention. [Example]

[0130] Example The following examples will serve to more fully describe the manner of using the invention as described above, as well as to illustrate the best modes contemplated for carrying out various aspects of the invention. Examples according to the invention are included within the scope of the claims appended hereto. The following exemplary compositions and combinations have been prepared according to the processes described herein.

[0131] Example 1 Preparation of clofazimine composition: 200 mg of clofazimine (as triclinic Form I), 90 mg of sodium chloride, and 9.5 ml of water were mixed twice for 5 minutes at 10,000 rpm using a high-shear homogenizer. 0.5 ml of polysorbate 80 (NOF Hx2) was added. The mixture was treated with an ultrasonic probe seven times for 3 minutes each at 70% amplitude. The volume was adjusted to 10 ml with water. The suspension was filtered through filter paper to obtain the composition of Example 1. The composition of Example 1 had a median clofazimine particle size of 3.9 μm and a D90 of 6.7 μm. The clofazimine concentration was calibrated with a 1 mg / ml stock solution of clofazimine diluted in mobile phase and determined to be 7.16 mg / ml by UV-visible spectroscopy at 280 nm.

[0132] The composition of Example 1 is shown in Table 1: [Table 1]

[0133] Preparation of orthorhombic form III of clofazimine: A slurry of clofazimine (10 g) in toluene (20 ml) was stirred using a magnetic stirrer at 800 rpm for 72 hours in an oil bath at 40°C. The solid portion of the slurry was collected by crucible filtration and dried under vacuum in an oven at a maximum temperature of 40°C. This yielded 8.64 g of clofazimine as substantially pure (≥98%) orthorhombic Form III.

[0134] Example 2 Preparation of clofazimine suspension: A suspension containing 6 g of orthorhombic Form III clofazimine in 100 ml of water containing 0.5% polysorbate 80 and 0.6% sodium chloride was homogenized using a high-shear mixer at 10,000 rpm for approximately 40 seconds. A preformulation was prepared by adding 0.6% aqueous sodium chloride solution to obtain a volume of 300 ml. This 300 ml suspension was wet-milled for 15 minutes using a microfluidizer by circulating the suspension at 5,000 psi. The suspension was further homogenized at 25,000 psi for 23 minutes. Particle size analysis was performed using a HORIBA LA950, showing a median particle size of 0.83 μm and a D90 value of approximately 1.2 μm. The concentration of 16.05 mg / ml of clofazimine was determined by UV / visible spectroscopy at 280 nm, calibrated with a 1 mg / ml stock solution of clofazimine diluted with mobile phase.

[0135] The composition of Example 2 is shown in Table 2. [Table 2]

[0136] Example 3 The composition of Example 3 was produced by processing a suspension of clofazimine (orthorhombic crystalline modification Form III) in a solution of water, sodium chloride, and polysorbate 80 using a Microfluidizer® (Microfluidics, Westwood, Massachusetts, USA) processor operated at a pressure of 28,250 psi for 30 minutes to produce the composition of Example 3, wherein the resulting particles of clofazimine had a median particle size of 1.28 μm and a D90 of less than 2 μm.

[0137] The composition of Example 3 is shown in Table 3. [Table 3]

[0138] Viscosity measurement: The viscosity of the composition of Example 3 was tested using a STRESSTECH rheometer in stress-controlled mode. A double-gap configuration was utilized, with the spindle rotating continuously to ensure that the microparticles remained suspended between temperature points. Viscosity was measured over stresses of 0.01, 0.05, and 0.1 Pa at 20°C, 25°C, and 30°C, respectively. Two separate loads were performed to obtain the average viscosities shown in Table 4 below. [Table 4]

[0139] Animal models and efficacy studies: To obtain preliminary data to establish clofazimine concentration levels in lung tissue after direct respiratory delivery as opposed to systemic administration, the compositions of the present invention were tested for their ability to inhibit the growth of clinical NTM species in an acute in vivo mouse model of pulmonary infection. Two separate mouse models are used to examine pulmonary NTM infection, depending on the bacterial species of interest. For testing, Mycobacterium avium 2285 and Mycobacterium abscessus 103 bacterial strains were used (strain details can be found in "Phylogenetic analysis of mycobacterial species using whole genome sequences," Hazbon MH, Riojas MA, Damon AM, Alalade R, Cantwell BJ, Monaco A, King S, Sohrabi A. (SEP-2014) submitted to the EMBL / GenBank / DDBJ database). These two species have been used previously in the literature as models for NTM infection (Obregon-Henao et al. 2015 Antimicrob Agents Chemother; and Chan et al. Animal Models of Non-Tuberculous Mycobacterial Infections, Mycobacter Dis 2016).

[0140] In vivo safety study in Balb / C mice: For in vivo safety and tolerability, 6-8 week old Balb / C female mice were obtained from Charles River. Mice were allowed to rest for one week before administration. For each dose of clofazimine, three healthy mice were administered a total of three doses every other day. Mice were administered 10.0, 5.01, and 2.51 mg / kg of clofazimine in the composition of Example 1. The compound was administered to three healthy mice by Microsprayer® aerosol intratracheal administration every other day for a total of three doses.

[0141] Clofazimine was found to be safe at 20 mg / kg (gavage, 200 μl). The composition of Example 1 showed no toxicity at the highest dose tested (10.0 mg / kg; 0.2506 mg / dose (35 μl, intratracheal)). Therefore, the composition of Formula I was considered safe and well-tolerated at 10.0 mg / kg.

[0142] Determination of minimum inhibitory concentrations Minimum inhibitory concentration (MIC) testing was performed by microbroth dilution using Mueller Hinton (MH) broth (cation-adjusted) to the calcium and magnesium ion concentrations recommended in CLSI standard M7-A7 (Becton Dickinson). MIC testing was also performed by microbroth dilution using 7H9 broth (Sigma-Aldrich). The justification for using both MH and 7H9 broth for compound screening is that antimycobacterial compounds have been shown to exhibit different MIC activity depending on the broth used in the MIC assay. M. abscessus was grown on 7H11 agar plates (Sigma-Aldrich) in ambient air at 35–37°C for 3 days (depending on the bacterial strain), and M. avium was grown on agar 7H11 plates (Sigma-Aldrich) in ambient air at 37°C for 21–30 days.

[0143] Colony-forming units (CFUs) were harvested from the agar plates and placed in either MH or 7H9 broth containing 0.05% Tween-80. They were grown at 35–37 °C in ambient air until the optical density (OD) absorbance (OD) obtained after 3 days (M. abscessus) or 12 days (M. avium) of growth reached 0.08–0.1 (0.5 McFarland standard). Bacterial cell suspensions were then prepared in saline to confirm an OD of 0.08–0.1 (0.5 McFarland standard). Compound stock solutions were made by suspending compounds in DMSO at a concentration of 1.28 mg / ml and immediately used for the test range of 64–0.062 μg / ml. Following this, 180 μl of broth (either MH or 7H9) was added to the first column of a 96-well plate, and 100 μl of broth was added to the remaining columns of the 96-well plate. 20 μl of compound stock solution was added to the first row of wells and serially diluted. Finally, 100 μl of NTM cell suspension was added to all wells except for the media-only control well. Specific QC agents for each organism were added: 1) bacteria-only negative control; 2) media-only negative control; and 3) clarithromycin positive drug control.

[0144] M. abscessus OD was assayed on day 3, and M. avium OD was assayed on day 12. After these measurements, plates were assayed using the resazurin microtiter assay plate method. Briefly, this method involves the addition of resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) to 96-well plates. Resazurin is a blue dye that is weakly fluorescent by itself but is irreversibly reduced to the pink, highly red-fluorescent resorufin. It is used as a redox indicator to determine bacterial cell viability in MIC assays.

[0145] Assays were performed in triplicate: Assay No. 1 was performed after storing the composition of Example 1 at 4° C. for 2 months, Assay No. 2 was performed at 4 months, and Assay No. 3 was performed at 5 months.

[0146] Minimum inhibitory concentrations in the presence and absence of CF sputum Minimum inhibitory concentration assays were performed as described above. To examine the effect of sputum from cystic fibrosis (CF) patients on the antibacterial activity of clofazimine (CFZ) and the composition of Example 1, sputum was collected from patients who had not received antibiotics for the past 48 hours, and the sputum was sterilized by exposure to UV light to remove endogenous bacteria. After sterilization, M. abscessus, M. avium, M. intracellulare, and M. chimaera were incubated in 10% CF sputum before undergoing MIC testing. The MIC of the composition of Example 1 was measured in the presence and absence of sputum from CF patients according to the same CLSI protocol described above. All studies were performed in duplicate.

[0147] The MIC values ​​for clofazimine and the composition of Example 1 in the presence and absence of sputum are shown in Table 5. [Table 5]

[0148] The results, shown in Table 5, demonstrate consistent MICs for both clofazimine and the composition of Example 1 against a range of nontuberculous mycobacterial species.

[0149] These data demonstrate that the composition of Example 1 exhibits potent in vitro activity against both M. abscessus and M. avium and is stable over at least this period.

[0150] Mouse model of M. abscessus in SCID mice Six- to eight-week-old SCID female mice were ordered from Charles River. Mice were rested for 1 week before infection.

[0151] A working stock of M. abscessus strain 103 was frozen in 1 ml aliquots and stored at −80°C prior to use. For infection, aliquots were thawed, disrupted 20 times with a 1 ml Luer-Lok syringe fitted with a 26 g needle, and diluted with sterile 1x PBS.

[0152] The acute SCID mouse model was performed using 1 x 10 6 They underwent non-invasive intratracheal instillation pulmonary infection with 10 CFU / mouse (M. abscessus strain 103).

[0153] Three mice were sacrificed on day 1 post-infection to measure bacterial uptake. Whole lungs, spleens, and livers were extracted and homogenized in 4.5 ml of 1x PBS. The homogenates were serially diluted 1:10 and plated on 7H11 agar plates (0-1-2-3-4-5-6-7). Plates were placed in a 32°C dry-air incubator (strain dependent) for 7 days.

[0154] 10.0 mg / kg of the composition of Example 1 was administered via the pulmonary route by Microsprayer® (35 μl), clofazimine (gavage), amikacin (subcutaneous) in a volume of 200 μl per mouse, starting on day 2 post-infection and continuing every other day for 8 consecutive days.

[0155] The mice were sacrificed two days after the administration of the last dose of compound. Six mice from each group (untreated control, clofazimine (gavage), composition of Example 1, and amikacin-treated mice) were sacrificed to determine bacterial load. Plating of lung homogenates from 0-1-2-3-4-5-6-7, spleens from 0-1-2-3-4-5-6-7, and livers from 0-1-2-3-4-5-6-7.

[0156] A Log10 protection value of at least 0.60 indicates statistically significant activity. Statistical analysis was performed by first converting CFU to logarithms, which were then evaluated by one-way ANOVA, followed by multiple comparison analysis of variance with one-way Tukey's test (GraphPad Prism analysis software). Differences were considered significant at the 95% confidence level.

[0157] Table 6 shows the mean Log 10CFU data and standard error of the mean (SEM) are shown, where "n" is the total number of animals in the group at the time of sacrifice. [Table 6]

[0158] The data in Table 6 show that treatment with the composition of Example 1 resulted in the greatest reduction in bacterial recovery in the lungs and spleens of animals infected with M. abscessus. This bacterial reduction was statistically improved over treatment with amikacin or oral clofazimine.

[0159] A mouse model of M. avium infection in Beige mice For this study, 6- to 8-week-old beige female mice were ordered from Charles River. Mice were rested for 1 week before infection.

[0160] The acute Beige mouse model was performed using 1 x 10 8 Noninvasive aerosol lung infection was performed using M. avium strain 2285rough at 100 colony-forming units (CFU) / ml. Working stocks of M. avium strain 2285rough were frozen in 1 ml aliquots and stored at -80°C prior to use. For infection, aliquots were thawed, disrupted 20 times with a 1 ml Luer-lock syringe fitted with a 26 g needle, and diluted with sterile 1x phosphate-buffered saline (PBS).

[0161] Three mice were sacrificed on days 1 and 7 post-infection to measure bacterial uptake. Whole lungs, spleens, and livers were extracted, homogenized in 4.5 ml of 1x PBS, and diluted 1:10. Dilutions (0-1-2-3-4-5-6-7) were plated onto 7H11 / OADC, TSA, and charcoal agar plates and incubated at 32°C in a dry air incubator (strain dependent) for 30 days.

[0162] 10.0 mg / kg of the composition of Example 1 was administered via the pulmonary route with clofazimine (gavage) by Microsprayer® (35 μl) in a volume of 200 μl per mouse, beginning on day 7 post-infection and continuing every other day for 10 consecutive days.

[0163] Mice were sacrificed 5 days after the administration of the last dose of compound. Six mice from each group (untreated control, clofazimine (gavage), and the composition of Example 1) were sacrificed and bacterial loads were determined. Plating of lung homogenates from 0-1-2-3-4-5-6-7, spleens from 0-1-2-3-4-5-6-7, and livers from 0-1-2-3-4-5-6-7.

[0164] A Log10 protection value of at least 0.60 indicates statistically significant activity. Statistical analysis was performed by first converting CFU to logarithms, which were then evaluated by one-way ANOVA, followed by multiple comparison analysis of variance with a one-way Tukey test (SigmaStat software program). Differences were considered significant at the 95% confidence level.

[0165] Table 7 shows the mean Log 10 CFU data are shown. [Table 7-1] [Table 7-2]

[0166] The data in Table 7 show that treatment with the composition of Example 1 resulted in a greater reduction in bacterial recovery in the lungs and spleens of animals infected with M. avium.

[0167] Chronic Beige Mouse Model In this experiment, 6- to 8-week-old Beige mice were rested for 1 week before infection. Mice were infected with 1 × 10 8Mice were infected with 2285 CFU of M. avium 2. Three mice were sacrificed on day 1 and six mice on day 27 to determine bacterial uptake and pretreatment bacterial load. Whole lungs, spleens, and livers were extracted, homogenized in 4.5 ml of 1x PBS, and plated on 7H11 and charcoal agar plates at dilutions of 0-1-2-3-4-5-6-7. Plates were placed in a 37°C dry-air incubator for 25–30 days.

[0168] The remaining infected Beige mice were treated every other day for a total of 14 treatments starting on day 28. Animals received one of the following treatments: saline (Microsprayer®, 35 μl); clofazimine (oral gavage, 20 mg / kg, 200 μl); or the composition of Example 1 (IT, Microsprayer®, 10 mg / kg, 35 μl).

[0169] Mice were sacrificed on day 57, two days after the final treatment. The plates were placed in a dry air incubator at 37°C for 30 days.

[0170] Statistical analysis was performed by first transforming CFU into logarithms, which were then evaluated by one-way ANOVA, followed by multiple comparison analysis of variance with one-way Tukey's test. Differences were considered significant at the 95% confidence level.

[0171] Mean Log after chronic infection of Beige mice with M. avium 10 CFU data are shown in Table 8. [Table 8-1] [Table 8-2]

[0172] These data suggest that clofazimine has difficulty penetrating the granuloma-like structures formed by established "chronic" animal NTM infection models. The compositions of the present invention do not have the same problem and appear to be able to maintain antimycobacterial activity even after the infection is well established.

[0173] Effect of the composition of Example 3 on barrier integrity and inflammation after exposure to lung epithelial cells in vitro Cell viability: Lung epithelial cell viability was assessed using three different cell types under two in vitro conditions: Calu-3 cells, A549 cells, and hAELVi cells. Cells were treated either in "immersion conditions" (i.e., in cell culture medium on Transwell™ plates) or in "air-liquid interface" mimicking conditions (ALI), where the cell culture medium was removed from the apical side of the cells. In "immersion conditions," Calu-3 cells were exposed to three doses (10%, 50%, or 100%) of the composition of Example 3 for 4 hours. To estimate cell viability, cells were stained using acridine orange / propidium iodide (AO / PI) staining to distinguish between live and dead cells. Red fluorescence indicated cell death.

[0174] Macrophage uptake: THP-1 cells were differentiated into macrophage-like cells after incubation with 124 ng / ml phorbol 12-myristate 13-acetate (PMA) for 3 days. Once mature, the cells were exposed to the composition of Example 3 (diluted 1:200 in Hank's buffered salt solution (HBSS)) for 4 hours. Cells were stained with AO / PI as described above to determine cell viability after exposure.

[0175] Transepithelial Electrical Resistance (TEER) measurement Calu-3 cells were plated in a Transwell™ 3460 at 1 × 10 5Calu-3 cells were seeded at 1000 x g / well and allowed to grow to confluence for 12 days. TEER measurements were performed using an EVOM2 (World Precision Instruments, Friedberg, Germany) according to the manufacturer's instructions. After seeding, Calu-3 cells were exposed to either saline (negative control) or the composition of Example 3 (concentrations: 20 mg / ml, 10 mg / ml, or 2 mg / ml). Cells were exposed for 2-4 hours before TEER measurements.

[0176] Inflammatory cytokine production Differentiated THP-1 cells (dTHP-1) were exposed for 4 or 24 hours (1:200 HBSS dilution) to the composition of Example 3. HBSS exposure alone was used as a negative control, and lipopolysaccharide (LPS) (100 ng / ml) was administered as a positive control.

[0177] After incubation, supernatants were removed from the cells (t = 4 h or 24 h) and pooled. Enzyme-linked immunosorbent assays (ELISAs) were performed on pooled supernatant samples. Individual ELISA kits for TNF-α, IL-6, IL-8, and IL-10 were used according to the manufacturer's instructions. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. Statistical significance was determined at a probability value of <0.05.

[0178] Results: Under "immersion" conditions, the composition of Example 3 did not result in a visible decrease in cell viability over a 4 hour incubation period at any administered concentration.

[0179] Under "ALI" conditions, three different cell types (Calu-3, A549, and HAELVi cells) were investigated over three different time points (5 hours, 2 days, and 7 days). Little or no cytotoxicity was observed in either cell type at 4 hours or in Calu-3 cells at day 2. Some toxicity was seen in A549 cells at days 2 and 7, and in Calu-3 cells at day 7. Technical limitations did not allow for quantification of cell death.

[0180] For macrophage uptake, differentiated THP-1 cells were incubated with 1:200 HBSS for 4 hours to determine macrophage cell viability after exposure. The composition of Example 3 did not induce cell death but demonstrated clofazimine uptake by macrophages.

[0181] For TEER measurements, Calu-3 cells were exposed to HBSS or three concentrations of the composition of Example 3 for 4 hours, and TEER measurements were sampled at various time points throughout the exposure. A decrease in TEER of 50% or more compared to the control at any given time point was considered a significant loss of barrier integrity.

[0182] Exposure of Calu-3 cells to the composition of Example 3 did not affect barrier integrity after 1 hour of exposure. Exposure at 20 mg / ml resulted in a significant (i.e., 50% or greater) reduction after 2 hours. The 10 mg / ml concentration showed a slight reduction (i.e., 25-35%) at all time points after 2 hours. Exposure at 2 mg / ml did not show a reduction in barrier function over the entire study period.

[0183] Inflammatory cytokine production In this model, the positive control LPS behaved as expected. The composition of Example 3 did not show significant changes in cytokines at any of the time points examined. The results are shown in Table 9. [Table 9] Cytokine production after dTHP-1 cell exposure

[0184] In vivo safety and tolerability In this study, 6-8 week-old Balb / C female mice were administered a total of three doses every other day. Using the composition of Example 1, mice were administered 10.0, 5.01, and 2.51 mg / kg. The composition was administered via intratracheal (IT) administration with a Microsprayer® aerosol in a volume of 35 μl per mouse. Following instillation, mice were observed 10 minutes, 1, 2, and 4 hours after administration, and daily thereafter.

[0185] Table 10 shows the macroscopic observations after administration. "BAR" indicates that the animals were bright, active and responsive. [Table 10]

[0186] Table 11 shows the body weights of the animals over the three days studied. [Table 11]

[0187] These data show that there was no statistically significant change in body weight over the three treatment days. These results indicate that the compositions of the present invention were well tolerated at the doses tested.

[0188] Example 4 A toxicokinetic study using clofazimine inhalation suspension (CIS) for the treatment of pulmonary NTM infection in a canine model. This study evaluated clofazimine inhalation suspension in dogs to determine toxicity over a 28-day once-daily dosing regimen. Animals were studied for a period of 84 days. Using recommended good laboratory procedures (GLP) guidance, a repeat-dose study evaluated low, medium, and high doses of clofazimine (2.72 mg / kg and 2.95 mg / kg (low); 5.45 mg / kg and 5.91 mg / kg (medium); and 10.87 mg / kg and 10.07 mg / kg (high)) in average males versus females.

[0189] Nebulized clofazimine inhalation suspension (CIS) The clofazimine inhalation suspension (CIS) formulation consists of clofazimine particles (20 mg / mL) suspended in 0.9% saline with polysorbate 80 (0.5% v / v) to stabilize the suspension. The drug product is stable for 1 year at room temperature. The vehicle consisted of sterile 0.5% polysorbate 80 (Hx2) in 0.9% saline. To reduce animal testing, the formulation was optimized and tested in vitro at PharmBioTec (data not shown).

[0190] The aerosolized / nebulized clofazimine formulations prepared above were administered to animals using a jet nebulizer for 30, 60, and 120 minutes, and samples were collected for evaluation and analysis as described below.

[0191] Research Plan All GLP canine research activities were conducted by the Lovelace Biomedical Research Institute (LBRI) under an IACUC-approved protocol. Five groups of beagle dogs were divided into study groups (Table 1) and exposed once daily via facemask inhalation to filtered air (Group 1), vehicle (Group 2 - 0.5% polysorbate in saline), or CIS (Groups 3-5) for 28 consecutive days. Low, medium, and high doses (Groups 3, 4, and 5) were exposed to a mean aerosol concentration of 0.209 mg / L for 30, 60, and 120 minutes, respectively. The 28-day exposure period was followed by a 28-day and 56-day exposure-free recovery period (recovery study, study days (SD) 56 and 84, respectively). Blood was collected for toxicokinetic (TK) analysis from animals on both the first day of dose administration (Day 1) (0.5 hours (±5 minutes), 6 hours (±30 minutes), 12 hours (±30 minutes), and 24 hours (±1 hour)), the final day of dose administration (SD28), and after SD41, SD56, and SD84 as shown in Table 1. The main study animals were necropsied 1 day after the final exposure (SD29), and two male and two female recovery study animals were necropsied at SD56 and SD84, respectively. [Table 12]

[0192] Aerosol administration Aerosolized CIS was delivered to each dog via a six-port aerosol exposure system using three Micro Mist compressed air jet nebulizers (Supplementary Figure S1). All animals were acclimated to the face mask and restraint system prior to the study. The exposure system and aerosol characteristics of the API and vehicle, including target aerosol concentration, concentration uniformity, concentration reproducibility, and aerosol size distribution, were confirmed by the LBRI prior to the study. Each dog's dosing was measured for every dose delivered throughout the study and calculated relative to individual dog body weight.

[0193] Tissue and plasma analysis Blood samples were either processed immediately or kept on wet ice for no more than 2 hours (h) and then processed to plasma by centrifugation (1300 g, 2–8°C, ≥10 min), and plasma was aliquoted into appropriately labeled vials and stored frozen (−70 to −90°C) until CFZ analysis.

[0194] The main study animals (3M / 3F / time point) had blood samples collected at the following time points after exposure on SD1 and SD28: 0.5 hours (±5 minutes), 6.0 hours (±30 minutes), 12 hours (±30 minutes), and 24 hours (±1 hour). Recovery animals from each group (2M / 2F per recovery time point) had blood collected on SD42 and before euthanasia on SD56 or SD84. Sample sizes for each group, sex, and time point are listed in Table 1. Actual collection times were recorded, and all animals in this study were subjected to scheduled sampling at each time point.

[0195] Determination of CIS by LC-MS Clofazimine was extracted from dog plasma by protein precipitation. Clofazimine was extracted from tissues by first homogenizing the tissue with a bead filter during the extraction process. Reverse-phase HPLC separation was achieved using a Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm) column on a Shimadzu Nexera X2 UHPLC system. MS / MS detection (Sciex Triple Quad 5500) was then set in positive mode to mass transitions of m / z 473.2 → 431.1 for clofazimine and 480.2 → 432.1 for clofazimine-d7, respectively. Retention times and peak areas were determined using Analyst® Data Acquisition / Processing Software (version 1.6.3). Analyte concentrations were obtained from calibration curves constructed using Analyst by plotting peak areas against nominal concentrations.

[0196] pathology Tissues were collected, examined, and weighed as necessary, with representative samples preserved for histopathology. Eyes with optic nerves, testes, and epididymis were fixed in modified Davidson's solution, while other tissues were fixed in 10% neutral buffered formalin (NBF). Lung lobes were instilled with NBF via the main airway(s) (to approximate physiological total lung volume at 25 cm hydrostatic pressure). The main airway(s) used for instillation were then closed, and the lungs / lobes were immersed in NBF for fixation.

[0197] Tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin for microscopic examination. Histopathological examination was performed in a "read-down" format: all tissues and gross lesions were examined for animals exposed to filtered air control, vehicle control, or CIS via facemask inhalation at the high dose. In low- and mid-dose animals, only respiratory tissues (lungs, tracheobronchial lymph nodes, pharynx, larynx, trachea, and nose / turbinates) and gross lesions were examined.

[0198] Visceral adipose tissue was evaluated for discoloration during necropsy for each animal. Skin discoloration was noted by visual inspection by the attending pathologist during necropsy. Several skin samples were taken from the groin for evaluation. All findings for a given tissue were subjectively and semiquantitatively graded by a single pathologist on a scale of 1 to 5 (1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe).

[0199] analysis- Clinical findings were descriptive, and numerical assessments (mean and standard deviation) were used for all other parameters whenever possible. Plasma toxicokinetic parameters were estimated using Phoenix WinNonlin version 8.3 software (Certara LP) using noncompartmental analysis (NCA) consistent with aerosol administration (extravascular model) for each subject at each time point. NCA was performed only if there were quantifiable concentrations at two consecutive time points. Concentration values ​​below the lower limit of quantification of 2.00 ng / mL for plasma were labeled as below the lower limit of quantification (BQL). These BQL values ​​were treated as missing and excluded from the calculation of descriptive statistics and toxicokinetic analysis.

[0200] Concentrations were used with full precision to three significant figures received from the bioanalytical data. Individual concentrations of clofazimine in plasma were collected separately for males and females per subject and time point. The area under the concentration versus time curve (AUC0-24h) for each subject at SD1 and SD28 from time 0 to the time the last quantifiable concentration was observed was calculated using a linear up, log-down interpolation method. Parameters were also estimated for the time of maximum observed concentration (Tmax), dose-normalized AUClast, maximum observed concentration (Cmax), and dose-normalized Cmax. At least the last three observed concentration values ​​were used to identify the terminal elimination phase of each concentration versus time curve. The slope of the terminal elimination phase was determined using logarithmic regression with uniform weighting. Parameters derived from the terminal elimination phase were reported if they passed the reporting criteria: coefficient of determination (R2) greater than or equal to 0.8 and extrapolation of the AUC to infinity less than or equal to 20% of the total area. Mean doses by group and sex were transcribed from the aerosol reports, assuming a 25% deposition fraction. Additionally, data were averaged for each sex and group to include time points sampled from the recovery periods of SD42, 56, and 84. Because blood was collected from different animals for the main study (SD1 and SD28) and recovery time points, individual NCA parameters could not be calculated. Instead, sex / group average concentrations were used to include both data sets.

[0201] statistics Arithmetic means, standard deviations (SDs), and sample sizes of plasma clofazimine concentrations were calculated for each group and Phoenix time point separately for males and females. Coefficients of variance (CV%) were calculated for NCA parameters, and geometric means and geometric SDs were calculated for Phoenix accumulation rates. Sex ratios were calculated by dividing the dose-normalized AUC and C for male animals by the same parameters for female animals in Phoenix. Accumulation rates were calculated by dividing the dose-normalized AUC and C after 28 consecutive daily doses (Day 28) by the same parameters after a single dose (Day 1) in Phoenix for male and female animals. Dose proportionality was calculated by comparing dose-normalized AUC and C after 28 consecutive daily doses (SD28) or a single dose (SD1) for male and female animals, paired by dose group.

[0202] The dose dependence of Cmax and AUC0-24 was assessed as a power law (i.e., Cmax, AUC0-24~Dosen) and evaluated for pooled data. The effects of sex, treatment day, and the interaction between sex and treatment day were estimated by multiple linear regression on log-transformed coordinates. A full model was run to identify significant terms (p<0.05), and then a reduced model was run to obtain parameter estimates. Regressions were performed in Excel.

[0203] Pulmonary function and clinical observations No CIS-related changes in tidal volume, respiratory frequency, or minute output were statistically significant at any dose level. Exposure to API produced results similar to those of the vehicle or air control. Differences between doses were within the expected range of variation in beagle dogs undergoing sporadic, transient, or similar study procedures and were therefore not considered test article related.

[0204] No abnormal clinical findings were reported for any animal at any of the study endpoints. Body weight trends were analyzed separately for male and female dogs. No specific trends in weight gain or loss were observed by gender. Body weight during the 28-day treatment period showed no specific trends for either male or female dog with any of the dose regimens, and generally, treated dogs maintained stable weights. Body weight changes during the recovery period were not correlated with drug administration, as both air and vehicle control weights fluctuated at least as much as the weights of dogs in either treatment group over the recovery period.

[0205] Ophthalmologic examination of the dogs before and after the 28-day treatment period was unremarkable, and no treatment-related problems were identified. Electrocardiogram results showed no changes in heart rate, PR, QRS, or Qt intervals. There were no obvious API-related abnormalities in rhythm or waveform morphology at any dose level compared with the vehicle group and the pre-treatment period.

[0206] Hematology and Clinical Chemistry Hematology, serum chemistry, and urinalysis parameters were obtained at necropsy on SD29 for animals designated for the main study, SD56 for animals designated for the 28-day recovery, and SD84 for animals designated for the 56-day recovery. There were three animals per sex / group for all measurements up to SD29, and two animals per sex / group for recovery measurements at SD56 and SD84.

[0207] All notable hematology, clinical chemistry, and urinalysis were unremarkable, incidental, or not considered related to the administration of CIS. None of the parameters indicated toxicity of any kind or followed any dose relationship with CIS.

[0208] clinical pathology At each designated necropsy (SD29, SD56, SD84), tissues were collected, weighed as necessary, and preserved for histopathological examination. Generally, gross examination of visceral adipose tissue revealed discoloration only in test article-treated animals at SD29. Macroscopic observations of the test article at necropsy in SD29 (main study) animals consisted of mild to moderate diffuse yellowing of adipose tissue in all high-dose animals, two mid-dose males and all females, and one low-dose female, with no correlating microscopic findings to explain the discoloration. No skin discoloration was observed in any animal. Organ weights were collected and analyzed relative to air and vehicle controls as absolute organ weights, organ-to-body weight ratios, and organ-to-brain weight ratios. Several organ weight differences were statistically significant in males (lungs, adrenals, heart, epididymis, testes) and females (adrenals, liver, and spleen), but no correlating microscopic findings to explain the differences. Residual organ weights were generally not significantly different from air and / or vehicle controls. Changes were typically small, often inconsistent across sex or ratio scales. Furthermore, there were no test article-related observations in any of the tissues examined.

[0209] Histopathological examination of tissues revealed no significant findings, and any reported lung or lymph node involvement was reported to be mostly mild or minimal in nature.

[0210] Plasma toxicokinetics There were no quantifiable clofazimine plasma concentrations in males or females in the Air or vehicle control groups (Groups 1 and 2) at any time point in the study. Male and female animals in the CIS low-, medium-, and high-dose groups had quantifiable clofazimine concentrations at all time points sampled on SD1 and SD28 (see Table 13). By SD42 (14-day recovery from exposure), measurable concentrations were reported for all animals except one, which was at BQL. By SD56, only one animal had a reportable CFZ concentration; all other animals were at BQL. All samples were at BQL by SD84. [Table 13]

[0211] After exposure on day 1, the maximum concentration or T max The time points for T were 0.5 or 6.0 hours for males and females at the low, medium, and high doses (Groups 3, 4, and 5, respectively). After 28 consecutive daily doses in SD28, T max were at 0.5, 6.0, or 12.0 hours for female and male animals in all dose groups.

[0212] Mean peak clofazimine concentration (C max ) were 16.2 ng / mL, 43.1 ng / mL, and 112 ng / mL in males from the low, medium, and high dose groups, and 22.9 ng / mL, 33.2 ng / mL, and 139 ng / mL in females from the low, medium, and high dose groups. After 28 consecutive daily doses of CIS, the mean C max The mean values ​​were 27.5 ng / mL, 93.5 ng / mL, and 271 ng / mL in males at the low, medium, and high doses, and 67.1 ng / mL, 183 ng / mL, and 241 ng / mL in females at the low, medium, and high doses (Figure 1).

[0213] Terminal elimination parameters could only be estimated for some animals at SD1 and could not be estimated for any animals at SD28. For animals with reportable terminal elimination parameters at SD1, the mean terminal elimination half-lives of clofazimine were 7.19, 8.95, and 7.89 hours for males at the low, medium, and high doses and 8.99, 9.43, and 7.23 hours for females at the low, medium, and high doses.

[0214] Terminal elimination parameters were also calculated at recovery time points of SD 42, SD 56, and SD 84. These terminal half-life values ​​were NR, 106 h, and 98.1 h for males in the low-, medium-, and high-dose groups, respectively, and 83.4 h, 78.2 h, and 115 h for females in the low-, medium-, and high-dose groups, respectively, corresponding to a range of 3 to 5 days for males and females. [Table 14]

[0215] In multiple linear regressions on log-transformed variables, treatment with SD28 had the effect of increasing the intercept of the model (p<0.003), but did not affect the slope. Animal sex (and its interaction) did not significantly affect the intercept or slope of the best-fit model. C max (1.44±0.14) and AUC 0-24 The mean ± SE index of (1.47 ± 0.16) is inconsistent with the assumed slope of 1 (p < 0.007), indicating a super-proportional dose response. max and AUC 0-24 The geometric mean ratios for α, β, and β were 2.56 (15% CV) and 3.76 (17% CV), respectively (Figure 2).

[0216] CIS DN AUC 0-24hr The mean accumulation ratios between SD28 (after 28 consecutive daily doses) and SD1 (after a single dose) of DN C were 5.39, 3.96, and 3.86 for males in the low-, medium-, and high-dose groups, and 3.80, 6.92, and 2.90 for females in the low-, medium-, and high-dose groups. max The accumulation ratios of MNKD-101 were 2.27, 2.54, and 2.44 in males in the low-, medium-, and high-dose groups, and 3.08, 5.43, and 1.98 in females in the low-, medium-, and high-dose groups. All ratios indicate a more than two-fold accumulation in males and females after 28 consecutive daily doses. Plasma levels of MNKD-101 rapidly decreased from the maximum level (SD28) to BQL by SD56 for almost all animals (Figure 3).

[0217] Pulmonary tissue toxicokinetics The toxicokinetics of CIS in lung tissue was evaluated in male and female beagle dogs after 28 consecutive daily doses (SD29) and during recovery periods (SD56 and SD84). There were no quantifiable clofazimine concentrations in the air or vehicle control groups. In general, clofazimine lung concentrations were dose-dependent across males and females, with drug levels measured after the dosing regimen at all but one time point (Table 14). Female lung drug levels were higher than male levels at SD54 for the mid- and high-dose groups, but showed no specific trend at SD29 or SD84. The combined gender data clearly demonstrate clofazimine retention at all time points in a dose-dependent manner.

[0218] Compared with plasma levels, lung concentrations of clofazimine decreased more slowly from SD29 to SD84 and remained well above the mean MIC (minimum inhibitory concentration) for NTM infection (Figure 4). Terminal elimination half-lives were estimated only for low-dose females (10.9 days), mid-dose males (9.96 days), and high-dose males (19.8 days). Other groups did not meet reporting criteria. The ratio of males compared to females for DN AUClast was near unity in the low-dose group, and DN AUClast and DN Cmax were near unity in the mid-dose group, indicating no significant (more than two-fold) differences between the sexes after 28 consecutive daily doses from SD29 to SD84. However, Cmax in the low-dose group increased nearly two-fold, and both DN AUClast and Cmax increased more than two-fold in females compared to males in the high-dose group; therefore, averages across males and females for toxicokinetic parameters are not presented because males and females were not comparable at all dose levels. The dose-normalized (DN) AUClast and Cmax between dose groups were also significant. max Dose proportionality, as assessed by the ratio, showed a much greater than dose-proportional increase across dose groups for males and females. The ratios between dose groups for AUC last ranged from 1.57 to 3.44 for males and 2.86 to 8.27 for females. maxThe ratios between dose groups for ranged from 1.21 to 4.91 for males and from 2.11 to 5.76 for females.

[0219] As shown in the figures and tables, the experiments demonstrated that CIS formulations can provide improved retention time and concentration in target organs, the lungs, while reducing systemic accumulation and improving systemic toxicity. This GLP toxicokinetic study in beagle dogs provides evidence that CIS administration by inhalation not only reduces systemic clofazimine accumulation and non-target organ toxicity, but also results in superior deposition in the lungs at levels above the average MIC for NTM infection. Although lung clofazimine levels remained well above the NTM MIC even 56 days after administration, systemic exposure to clofazimine remained low, indicating that a reserve pool of drug was not derived from tissue accumulation. This is an important finding supporting the observation that there were no overt or measurable adverse effects from drug accumulation. For example, the fact that no animals were reported to have skin discoloration is promising in reducing skin discoloration, a major adverse effect of clofazimine administration in humans. Importantly, CIS clearance was also independent of the dose received, indicating that the clearance mechanism was not saturated by the dose level used. This is t 1 / 2 This is clearly demonstrated by the fact that the β did not trend in any direction across dose groups, indicating that higher doses of clofazimine may be possible without adverse effects.

[0220] The dose level used in this study was determined from previous studies in mice and rats, with a maximum dose of 3.45 mg / kg / day. The rat study also demonstrated very poor clofazimine deposition in the lung from oral administration. Interestingly, while the total lung fraction of clofazimine in the low-dose group after 28 days of administration appears to be primarily due to simple accumulation, the highest dose accumulation (exponential increase) indicates reduced transport of clofazimine from the lung, likely due to uptake by the lung-resident macrophage population. This is particularly relevant for patients with reduced lung function from comorbidities that may result in irregular deposition of CIS. Coupled with its very small inhaled particle size (1.5–2.5 mmAD) that reaches deep within the lung, CIS delivered through the lung has an increased residence time of clofazimine via macrophage uptake, which may be a factor balancing the possibility of irregular deposition from lung abnormalities. Clofazimine concentrations in plasma and lung tissue at necropsy demonstrate superproportional PK behavior (Figure 5).

[0221] Even at the highest levels of clofazimine delivered, no direct adverse physiology was observed in lung tissue, and few systemic adverse events were observed. At all doses, both the liver and spleen showed less than 0.1 micrograms of clofazimine per gram of tissue just 28 days after dosing, whereas adipose tissue showed a rapid linear decline to BQL by SD56, demonstrating rapid clearance of clofazimine from non-lung tissues.

[0222] In summary, all three dose levels demonstrated significant residual drug in lung tissue, demonstrating impressive lung burden and long lung residence times for clofazimine. Pulmonary drug concentrations remained well above the mean NTM MIC at all time points, with measurable clofazimine levels at 28 and 56 days post-dose. In contrast, clofazimine plasma levels were consistently measurable only through 14 days post-dose, with measurements below the limit of quantification at 56 days post-dose. The data demonstrate that clofazimine inhalation suspension provides an effective treatment for NTM infections through direct delivery of the antibiotic to the lungs, overcoming the systemic toxicity seen with oral clofazimine treatment for NTM.

[0223] Example 5 Safety, Tolerability, and Pharmacokinetic (PK) Study of Clofazimine Delivered by Nebulization in Healthy Subjects - This study design involved a first-in-human, randomized, double-blind, placebo-controlled inhalation study of clofazimine in healthy adult participants, involving single and repeated daily doses of up to 90 mg of clofazimine for 7 days. A clofazimine inhalation suspension prepared to a concentration of 20 mg / mL was used in this study. The clofazimine composition was provided as a red / orange micronized suspension containing polysorbate 80 (0.5% v / v), sodium chloride (0.9% wt / v), and water. For the placebo, subjects were treated with nebulized sterile isotonic saline solution consisting of 0.9% wt / v sodium chloride. Subjects were treated with a 30 mg or 60 mg suspension containing clofazimine as needed throughout the study, while being monitored for adverse events such as dysphonia, sore throat, and cough while taking the drug. Blood samples for laboratory evaluation were collected at various intervals throughout the study. To investigate tolerability and pharmacokinetics (PK) in healthy human subjects, doses were administered by inhalation using a Pari e-Flow nebulizer system. Subjects were monitored for life-threatening or serious adverse events, and subject blood samples were collected for analysis. Before each dose escalation, interim safety and PK assessments were conducted to consider both local (pulmonary) and systemic effects and to ensure systemic drug levels were below the preclinical no-observed-adverse-effect level (NOAEL). A 90 mg dose of clofazimine in suspension was also tested in healthy individuals.

[0224] In one portion of the study, 24 adults were enrolled in one of three cohorts (n = 8 per cohort) that received a single inhaled dose of 30 mg, 60 mg, or 90 mg of clofazimine. Participants remained in the clinical research unit until day 5 after dosing, during which time safety was assessed and samples were collected for PK evaluation. Participants returned on days 8 and 15 for additional safety assessments and sample collection. During the MAD portion of the study, 16 adults were enrolled in one of two cohorts (n = 8 per cohort) that received daily inhaled doses of 30 mg or 90 mg of clofazimine for 7 days. Participants remained in the clinical research unit until day 8 after dosing, during which time safety was assessed and samples were collected for PK evaluation. Participants returned on days 15 and 36 for additional safety assessments and sample collection.

[0225] Nebulized and inhaled clofazimine was well tolerated, with no serious adverse events (SAEs) and most adverse events being mild. Subjects had no abnormal, clinically significant electrocardiogram (ECG) results reported. Parameters affected by abnormal, clinically insignificant ECG results were the QRS interval and PR interval. Subjects treated with nebulized clofazimine had no suicidal thoughts or behaviors during the study period on the Columbia Suicide Severity Rating Scale (C-SSRS) and no evidence of skin discoloration.

[0226] The clofazimine doses studied and used, both single and multiple doses, showed a proportional increase in clofazimine plasma levels with increasing dose in human subjects. Figure 6. With single doses, peak plasma concentrations of clofazimine were reached within 4-8 hours, whereas with multiple doses, this was reached within 2-12 hours. Dose increases within a single dose of clofazimine resulted in a significant increase in AUC with a two-fold dose increase. 0-24 A 2.3-fold increase in AUC with a 1.5-fold dose increase 0-24The data showed that clofazimine exhibited a long plasma half-life of 290 hours after repeated dosing (Figure 7). Figure 7 also shows that accumulation after repeated clofazimine dosing was considered significant. Cough levels were associated with a longer t 1 / 2 There did not appear to be a plateau between days 1 and 6 that reflects

[0227] The safety and PK data profile presented herein indicates that clofazimine compositions for inhalation by nebulization are safe for use in humans for the treatment of NTM infections, and a Phase 2 / 3 efficacy study in NTM (nontuberculous mycobacteria) pulmonary infections is underway.

[0228] Example 6 In this study, subjects diagnosed with NTM were treated with a nebulized clofazimine suspension or placebo using a Pari eFlow® nebulizer, and the safety of treatment was evaluated. A 20 mg / mL clofazimine inhalation suspension prepared as described above was used in this study. The clofazimine composition was provided as a red / orange micronized suspension containing polysorbate 80 (0.5% v / v), sodium chloride (0.9% wt / v), and water. For the placebo, subjects were treated with a nebulized sterile isotonic saline solution consisting of 0.9% wt / v sodium chloride. Subjects were treated with a 30 mg or 60 mg clofazimine suspension throughout the study period and were monitored for adverse events, such as dysphonia, sore throat, and cough, as needed while taking the drug. Blood samples for laboratory evaluation were collected at various intervals throughout the study. Subjects were prohibited from using any inhaled antibiotics active against NTM for 28 days prior to study initiation and were then treated once daily for 28 days. Patients were monitored for life-threatening or serious adverse events, and blood and sputum samples were microbiologically analyzed for the presence of bacteria.

[0229] A positive treatment response will be determined by sputum cultures of patient samples being negative for NTM bacteria at the end of the 6-month period after three consecutive sputum cultures, each separated by at least two weeks post-treatment.

[0230] Analysis of blood samples and sputum from animal studies indicates that all doses of clofazimine inhalation treatment were modeled as effective against NTM bacterial infections as described above, exceeding the minimum inhibitory concentration (MIC) at the highest therapeutic dose administered at 90 mg / day. The data indicate that nebulized clofazimine compositions were determined to be well tolerated in humans with reduced or minimal adverse effects.

[0231] Example 7 Effect of surfactants on cell viability studies: The effects of surfactants, such as polysorbate 80 (PS80) containing various fatty acid moieties, were used in this study to determine their effect on cell viability in the lungs upon administration of a pulmonary delivery composition. In vitro experiments were performed using three different human cell lines: human cell lines A549 (derived from type II lung epithelial cells), hAELVi (human alveolar epithelial cells), and Calu-3 (lung adenocarcinoma cells). Cell lines were obtained from ATCC (Bethesda, MD) and maintained according to the supplier's recommendations, and were cultured at 4 x 10 in RPMI 1640 medium (Gibco). 4Cells were seeded into 96-well plates containing 1000 μg / well and incubated for 24 hours. For the test, the aspirated medium was aspirated, the cells were washed twice with Hank's Balanced Salt Solution (HBSS) at pH 7.4, and 0.2 ml of the sample solution containing the surfactant was added to the cells. HBSS was used as a negative control, and HBSS containing 1% Triton X-100 was used as a positive control. Two surfactants were studied: ultra-purified grade PS80, which contains a mixture of various fatty acid esters, with approximately 70% to 85% oleic acid and other components including myristic acid, palmitic acid, palmitoleic acid, stearic acid, linoleic acid, and linolenic acid esters. This ultra-purified PS80 (Tween® 80, A), manufactured by Mallinckrodt Baker or Merck, and a second PS80 (B, NOF Corporation), containing approximately 99% oleic acid, were tested for in vitro IC. 50 In experiments to determine the value, after 4 hours of incubation at 37°C with shaking and washing with HBSS, the cell lines were exposed to equal concentrations of two surfactants and several dilutions of the test compound in the same HBSS solution. Cell viability was assessed based on absorbance measurements at 550 nm obtained using the MTT assay as described in Metz et al., J. ALTEX, January 29, 2020:V.2 doi:10.14573 / altex.1910231. Figures 8 and 9 and Table 16 show the data obtained from the experiment.

[0232] Figures 8 and 9 show graphs comparing the surfactants used in in vitro cell survival experiments for each cell line used. As shown in the graphs, cells treated with HBSS were 100% viable. Cells treated with HBSS containing 1% Triton X-100 lost all viability in the experiment. The effect of the tested surfactants on cell viability was less pronounced at lower surfactant concentrations in the medium and for the cell line type. However, the experiment showed that a surfactant with a higher oleic acid concentration (PS80) (Figure 9) had a better cell viability effect or was less toxic to cells than ultra-purified PS80 (Figure 8).

[0233] The data also demonstrate that Calu-3 cells are less sensitive to the concentrations of detergent used than the two other cell lines used, as these cells are tumor-derived and would be expected to behave differently. [Table 16]

[0234] Example 8 A study was conducted on clofazimine compositions of the present invention administered to healthy adults via a vibrating mesh nebulizer. Single and multiple ascending doses up to 90 mg were investigated. Results demonstrate rapid and deep lung delivery (8-15 minutes / treatment) with a high lung deposition rate of at least 30%. max and AUC 0-24 A dose-proportional increase in clofazimine was seen for single and multiple doses with meaningful dose-dependent accumulation rates. The half-life was observed to be prolonged in single and multiple dose studies. Calculations indicate that pulmonary clofazimine levels remained above the NTM-MIC (nontuberculous mycobacterial minimum inhibitory concentration) for at least 56 days after the treatment course.

[0235] While the above refers to certain preferred embodiments, it is understood that the disclosure is not so limited. It is understood that those skilled in the art may make various modifications to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention.

Claims

1. 1. An inhalable pharmaceutical composition comprising clofazimine, or a pharmaceutically acceptable derivative of clofazimine, a clofazimine salt, or a polymorph of clofazimine, or combinations thereof, and a pharmaceutically acceptable carrier and / or excipient, for use in the treatment or prevention of non-tuberculous bacterial infections of the lungs, wherein the clofazimine is present in an amount of 1 mg to 20 mg weight % of the composition, and wherein the inhalable pharmaceutical composition provides, upon inhalation, an effective daily dose of clofazimine of up to 90 mg.

2. 2. The inhalable pharmaceutical composition of claim 1, wherein the non-tuberculous bacterial infection of the lung that is treated or prophylactically suppressed is caused by a mycobacterium selected from the group consisting of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium leprae, and combinations thereof.

3. 2. The inhalable pharmaceutical composition of claim 1, wherein the nontuberculous bacterial infection is an opportunistic infection selected from the group consisting of Mycobacterium avium complex pulmonary disease and opportunistic nontuberculous infections associated with one or more of the group consisting of cystic fibrosis, chronic obstructive pulmonary disease or acquired immune deficiency syndrome, or a combination thereof.

4. 4. The inhalable pharmaceutical composition of claim 3, wherein the infection is an opportunistic nontuberculous mycobacterial infection in a patient with cystic fibrosis.

5. 10. The inhalable pharmaceutical composition of claim 1, wherein the infection being treated or prophylactically suppressed is caused by mycobacteria or other gram-positive bacteria and is administered by inhalation before, simultaneously with, or after administration of a drug selected from the group consisting of bedaquiline, or a pharmaceutically acceptable salt of a derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof.

6. 2. The inhalable pharmaceutical composition of claim 1, wherein the clofazimine is at least about 90% orthorhombic polymorph III.

7. 7. The inhalable pharmaceutical composition of claim 6, wherein such composition is used to treat nontuberculous bacterial infection of the lung or as a prophylaxis against nontuberculous mycobacteriosis of the lung.

8. 10. The inhalable pharmaceutical composition of claim 1, wherein the composition is delivered by inhalation for treatment or prophylaxis with a high lung deposition rate of at least about 30%.

9. 1. An inhalable pharmaceutical composition comprising clofazimine, or a pharmaceutically acceptable derivative of clofazimine, a clofazimine salt, or a polymorph of clofazimine, or combinations thereof, and a pharmaceutically acceptable carrier and / or excipient, for use in the treatment or prevention of non-tuberculous bacterial infections of the lung, wherein the clofazimine is present in an amount of 1 mg to 20 mg weight % in the composition, and the composition is delivered by an inhaler configured to cause a high lung deposition rate of at least 30%.

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