JP7670778B2 - Compositions of clofazimine, combinations containing same, processes for preparing same, uses and methods containing same - Google Patents

Description

This application claims priority to U.S. Provisional Application No. 62/722,048, filed August 23, 2018, and also claims priority to U.S. Provisional Application No. 62/796,322, filed January 25, 2019, the contents of both of which are incorporated herein by reference.

The present invention provides a therapeutically effective dose
The present invention further relates to a pharmaceutical composition for inhalation comprising clofazimine (Oxoacetate), wherein the clofazimine is provided in the form of a suspension, a process for preparing the same, and methods of use and treatment comprising the same.The present invention further provides a pharmaceutical combination comprising clofazimine in the form of an aerosol for pulmonary inhalation.

The combinations and compositions provided by the present invention can be used for the treatment and/or prevention of pulmonary infections caused by mycobacteria and other gram-positive bacteria, as well as pulmonary fungal infections.

Clofazimine is a very hydrophobic (Log P=7.66) antibiotic riminophenazine with antimycobacterial and anti-inflammatory activity that was first described in 1957. The structural formula is:

The exact mechanism by which clofazimine exerts its antibacterial action is unknown. However, it is known to preferentially bind to mycobacterial DNA, thereby inhibiting DNA replication and cell proliferation. Other proposed mechanisms of action include membrane damage/destabilization, generation of membrane-destabilizing lysophospholipids, potassium transport and/or interference with the intracellular redox cycle. In vitro, clofazimine has been shown to inhibit Mycobacterium tuberculosis (including multidrug-resistant strains) and its antibacterial properties.
While it has been shown to have excellent activity against mycobacterial tuberculosis (MTB), until recently it was generally believed that it was not effective in treating pulmonary tuberculosis (e.g., Cholo M et al., J.
Antimicrob Chemother, 2012 Feb, 67(2):290
See -8).

Clofazimine is an antibacterial agent that inhibits the leprosy bacteria Mycobacterium leprae.
It is one of the three main drugs recommended by the World Health Organization for the treatment of leprosy, caused by Mycobacterium leprae, and in recent years has been increasingly used to treat other mycobacterial infections, such as drug-resistant tuberculosis, caused by nontuberculous mycobacteria (NTM).

Clofazimine is practically insoluble in water and exhibits high membrane permeability, and is classified as a drug in the Biopharmaceutics Classification System (BSC).
It is classified as a Class II drug in the BCS (Bilirubin Syndrome).

In order to overcome the problems associated with poor oral absorption and poor bioavailability of drugs, micronization and nanoparticles have been used to optimize oral dosage forms.
recrystallization, supercritical fluid recrystallization
re-crystallization, spray freeze drying in liquid (spray freeze drying
Various strategies have been used, such as dry ice drying into liquid, solid dispersions, and solid solutions.

Since clofazimine is classified as a BCS Class II drug, it is generally considered an ideal option to formulate it into a solid dispersion to improve its oral bioavailability (see, e.g., Bhusnure et al. IJRPC 2014, 4).
(4), 906-918).

Accordingly, clofazimine, being lipophilic, is usually administered as a microcrystalline suspension in an oil-wax base in order to improve its oral absorption. It is absorbed by the human body after oral administration to a very variable extent (45-62%). Adverse effects of clofazimine are dose-related and mainly affect the skin, eyes and gastrointestinal tract.
The QT prolongation side effect is accompanied by the development of reddish-brown discoloration of the skin and conjunctiva, which is reversible and slowly resolves upon discontinuation of the drug, due to long-term systemic retention.

The genus Mycobacterium is a member of the Actinobacteria phylum.
It is a genus of Mycobacteria and belongs to the family Mycobacteriaceae.
Mycobacteria are a distinct family (Mycobacteriae) that are rod-shaped and have a waxy outer membrane.

As such, mycobacteria can be divided into three groups:
Mycobacterium tuberculosis (Mycobacterium tuberculosis)
Mycobacterium leprae (Mycobacterium leprae), the causative agent of tuberculosis
)) - Nontuberculous mycobacteria (NT), the causative agent of leprosy
M) - Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium leprae (
Mycobacterium abscessus complex includes all mycobacteria except M. leprae.
Mycobacterium sus complex (MABSC), Mycobacterium avium complex (MAC), etc.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis
TB is an infectious disease caused by the TB virus.
It is one of the oldest documented infectious human pathogens and remains a significant cause of mortality and morbidity worldwide. In 2015, 10 new cases of TB infection were reported.
It is estimated that 400,000 people are infected with TB, and 1.4 million people die from active TB (see, for example, the World Health Organization (WHO) Global Tuberculosis Report).
In addition to the high morbidity and mortality rates, there is growing concern about the emergence of multidrug-resistant tuberculosis (MDR-TB), and it has been reported that 580,000 patients were infected with drug-resistant TB in 2015.
Co-morbidities such as TB (including TB-associated pulmonary tuberculosis (TB) and V) complicate treatment and were responsible for 1.2 million TB cases in 2015.

To treat multidrug-resistant (MDR) infections, the WHO has recommended administering a 9-12 month treatment regimen with second-line anti-TB drugs. Such regimens, such as the 9-12 month Bangladesh regimen, treat MDR-TB with a combination of gatifloxacin, ethambutol, pyrazinamide, and clofazimine, with 87.9% of patients achieving relapse-free cure (e.g.,
Sotgiu, G. et al. , “Applicability of the sh
alter'Bangladesh regimen'in high multidr
ug-resistant tuberculosis settings”,Inte
National Journal of Infectious Diseases
(2017) 56 190-193).

Other studies investigating shortening the duration of TB treatment have shown that clofazimine has no clinical benefit after 2 weeks of oral administration (e.g., Diacon, A.H., et al., J. Pharmacol. 2011, 113:131–135).
tal. , “Bactericidal Activity of Pyrazina
mide and Clofazimine Alone and in Combin
ations with Pretomanid and Bedaquiline”,
American Journal of Respiratory and Crit
(See Medical Care Medicine (2015), 191(8), 943-953). This lack of efficacy was attributed to poor bioavailability of the drug, as it is theorized that the drug binds with high affinity to serum proteins in the circulating blood. Clofazimine has been shown to be effective in treating MDR-TB and extensively drug-resistant TB (XDR-TB).
Although empirically demonstrated to be effective in the treatment of rheumatoid arthritis (B), short-term treatment appears to have limited biological activity due to poor bioavailability after systemic administration (e.g.,
Swanson, R. V. , et al. , “Pharmacokinetics an
d Pharmacodynamics of Clofazimine in a M
ose Model of Tuberculosis”, Antimicrobia
l Agents and Chemotherapy (2015), 59(6), 30
(See No. 42-3051).

Treatment of pulmonary infections with inhaled antibiotics results in higher drug concentrations in the lungs and reduced side effects compared to systemic delivery (e.g., Touw, D.J., et al.,
“Inhalation of antibiotics in cystic fib
European Respiratory Journal (1995
), 8, 1594-1604), which is known to result in enhanced biological activity and efficacy (see, for example, Hickey, A. J., "Inhaled drug therapy with cyclosporine,
eatment for tuberculosis: Past progress a
nd future prospects”, Journal of Control
ed Release, (2016), 240, 127-134). Aerosolized clofazimine administered to a TB infection model has been shown to significantly improve bacilli clearance after only 28 days of treatment compared to oral clofazimine (see, e.g., Verma et al., J. Immunol. 1999, 144:127–134).
,R. K. , et al. , “Inhaled microparticles con
taining clofazimine are effective in t
Reaction of experimental tuberculosis in
Mice”, Antimicrobial Agents and Chemothe
(See Rapy (2013), 57(2), 1050-1052.) This improved short-term efficacy is likely due to higher clofazimine concentrations in pulmonary macrophages of tuberculous granulomas as a result of direct delivery of clofazimine to the site of infection in the lungs.

Therefore, administration of aerosolized clofazimine to patients infected with MDR-TB or XDR-TB should further improve patient outcomes and potentially shorten the duration of current treatment regimens.

The group of nontuberculous mycobacteria (NTM), formerly called atypical or ubiquitous mycobacteria, includes more than 150 species. NTM are widespread and diverse in the natural environment. They can be found in soil, ground, and drinking water, as well as in foods such as pasteurized milk and cheese. NTM are generally considered to have low pathogenicity. However, they can cause significant disease in humans, especially those with weakened immune systems or those already suffering from lung disease. Currently, NTM are classified according to their growth rate and are divided into slow-growing (SGM) and rapid-growing (RGM) mycobacteria.

Slow-growing Mycobacterium avium complex (Mycobacterium
Mycobacterium avium complex (MAC) includes Mycobacterium avium (Mycobacterium avium)
Mycobacterium avium, Mycobacterium chimera
cterium chimaera), and Mycobacterium intracellulare (
Mycobacterium kansasii (Mycobacterium intracellulare) species, which are the most important and most common pathogenic NTM.
Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium xenopi, Mycobacterium simiae
obacterium. simiae, Mycobacterium abscessus
bacterium abscessus, Mycobacterium gordonii
bacterium gordonae), Mycobacterium fortuitum (M
Most of these also cause pulmonary infections, as do Mycobacterium fortuitum, and Mycobacterium chelonae.
Aquarium arinum is involved in skin and soft tissue infections such as aquarium granuloma.

In particular, RGM causes severe, chronic, life-threatening lung disease and is associated with disseminated, often fatal, infections. Infections are usually acquired through the exposure to contaminated material, usually catheters.
The causes of NTM are invasive procedures, non-sterile surgical procedures or injections, and foreign body implants. Contact with shower heads and Jacuzzis has also been reported to pose a risk of infection. NTMs usually cause opportunistic infections in patients with chronic lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) and other immune-compromised patients.

In recent years, rapid growing type (RGM) Mycobacterium abscessus
Mycobacterium abscessus group bacteria (subspecies Mycobacterium abscessus subsp. abscessus)
Mycobacterium abscessus boleti (Mycobacterium abscessus) (M. a. abscessus (M. a. abscessus)), Mycobacterium abscessus boleti (Mycobacterium abscessus
bolletii.), and Mycobacterium abscessus massiliense (My
Mycobacterium abscessus complex (Mycobacterium abscessus massiliense)
The importance of Mycobacterium essus complex (MABSC) as a human pathogen is becoming evident, with a much higher mortality rate than any other RGM.

CF patients are infected with Mycobacterium abscessus
cessus) is particularly problematic because it causes extensive lung destruction and is often untreatable, with a high treatment failure rate of 60-66%.
nao A et al, Antimicrobial Agents and Che
mothertherapy, November 2015, Vol 59, No 11, p.
6904-6912; Qvist, T. , Pressler, T. , Hoiby, N. a
nd Katzenstein, T.L. , “Shifting paradigms o
f nontuberculous mycobacteria in cystic
fibrosis”, Respiratory Research (2014), 15 (
1):pp. 41-47).

Human infections with NTM have been increasingly linked to outbreaks of human acquired immune deficiency syndrome.
Mycobacteria belonging to the Mycobacterium vium complex (MAC) have been identified as the major causative agent of opportunistic infections in patients infected with the human immunodeficiency virus (HIV).

Some NTM species are known to form biofilms, which are bacterial microcolonies encapsulated in an extracellular matrix that confer stability and resistance to the human immune system. Recently, it has been shown that some NTM species form biofilms that enhance resistance to disinfectants and antibacterial agents. Biofilm assembly progresses through several stages, including reversible attachment, irreversible attachment, biofilm formation by bacterial aggregation, organization, and signaling, and finally dispersal. During this process, bacteria build a matrix that contains extracellular polymeric substances (EPS), such as polysaccharides, lipids, and nucleic acids, forming a complex three-dimensional structure (e.g., Sousa S. et al., 2003).
, International Journal of Mycobacteriolo
gy 4 (2015), 36-43). Specifically, mycobacteria do not produce exopolysaccharides, so the EPS of mycobacteria has different properties from other biofilms (
For example, Zambrano MM, Kolter R. Mycobacterial b
iofilms: a greasy way to hold it together
Mycobacterial biofilms vary between species but can contain mycolic acids, glycopeptide lipids, mycolyl-diacylglycerol, lipooligosaccharides, lipopeptides, and extracellular DNA (Rose SJ, Babrak LM, Bermud et al., J. Immunol. 1999, 144:131-132).
dez LE (2015) Mycobacterium avium Possesse
s Extracellular DNA that Contributes to
Biofilm Formation, Structural Integrity, a
nd Tolerance to Antibiotics. Review and original research from PLoS ONE). Aggregation within biofilms is known to increase resistance to antibacterial agents (e.g., Faria S. et al., Journal of Pathogenesis, 1999, 144: 1117-1122, 2001).
(See J. Org. 2015, Article ID 809014).

Aerosolized liposomal amikacin delivery/inhalation of amikacin solution nebulized by a jet nebulizer (Rose et al., 2003) as a novel approach to the treatment of NTM pulmonary infections.
S. et al, 2014, PLoS ONE, Volume 9, Issue 9, e
108703 and Olivier K. et al., Ann Am Thorac Soc.
c Vol 11, No 1, pp. 30-35), inhalation of dry powder particles for pulmonary delivery of anti-TB drugs (Cholo M et al., J Antimicrob Che
mother. 2012 Feb; 67(2):290-8 and Fourie B
．． and Nettie O. ,2015 Inhalation Magazine
, Verma 2013 Antimicrob Agents Chemother
) has been proposed.

In the treatment of NTM lung disease, initial treatment with parenteral aminoglycosides, tigecycline, and other promising oral antibiotics such as linezolid, delamanid, and bedaquiline, as well as surgical intervention in selected cases, followed by a multidrug therapy regimen with inhaled amikacin has shown promise (Lu Ryu et al., 2013).
al., Tuberc Respir Dis 2016;79:74-84). However, the increasing incidence and prevalence of NTM infections, especially NTM pulmonary disease, and the limited treatment options necessitate the development of novel dosage forms/pharmaceutical formulations that improve the bioavailability of currently used antibacterial agents, such as clofazimine. Inhalation may increase efficacy and reduce side effects compared to oral and parenteral treatments.

The combination of clofazimine and amikacin inhibits Mycobacterium abscessus (M
Mycobacterium abscessus) and Mycobacterium avium (
It has been shown that it acts synergistically against both Mycobacterium avium and
It has been shown in vitro (e.g., van Ingen, J., et al., "In
Vitro Synergy between Clofazimine and A
mikacin in Treatment of Nontuberculous M
ycobacterial Disease”, Antimicrobial Agent
TS and Chemotherapy 56(12), 6324-6327(201
2). In addition, Mycobacterium tuberculosis (Mycobacterium tuberculosis)
Synergistic effects of clofazimine and bedaquiline in combination against E. cerium tuberculosis have also been demonstrated (see, for example, Cokol, M. et al., "E.
efficient measurement and factorization o
f high-order drug interactions in Mycoba
cterium tuberculosis”, Sciences Advances
2017:3:e170881, 11 October 2017). In addition, a synergistic effect of clofazimine/bedaquiline combination has been shown against Mycobacterium abscessus, a nontuberculous acid-fast bacterium (Ruth, M.M. et al., "A B
edaquiline/Clofazimine Combination Regim
en Might Add Activity to the Treatment o
f Clinically Relevant Non-Tuberculous My
cobacteria”, Journal of Antimicrobial Che
mothertherapy (2019), doi. org/10.1093/jac/dky5
26).

Pathogenic fungi are emerging as a major cause of human mortality. Current estimates suggest that deaths due to deep mycoses are comparable to those of better known infectious diseases such as tuberculosis.
ns), Cryptococcus neoformans
mans), and Aspergillus fumigatus
Fungal pathogens such as Clofazimine and Clostridium tus are the most common pathogenic fungal infections in humans. Each of these species is responsible for hundreds of thousands of infections annually, but poor diagnostic methods and limited treatment options have led to unacceptably high mortality rates. Clofazimine has been shown to be effective against multiple fungi as a combination drug (e.g., Robbins, N., et al., 2003).
al. , “An Antifungal Combination Matrix
Identifications a Rich Pool of Adjuvant Molec
ules that Enhance Drug Activity again
Diverse Fungal Pathogens”, Cell Reports 1
3, 1481-1492, November 17, 2015). Fungi can act as commensals, colonizers, and/or pathogens in cystic fibrosis (e.g., C.
hotirmall, S. H. and McElvaney, N. G. , “Fungi
in the cystic fibrosis lung: Bystanders o
r pathogens? ”,The International Journal
of Biochemistry & Cell Biology 52 (2014),
See 161-173).

Clofazimine has low water solubility, resulting in low oral bioavailability and high microbial resistance.
A nebulizer is used to deposit aerosol particles onto the
Special techniques are also required to solubilize and stabilize drugs for formulation in liquid aqueous carriers for aerosolization by, for example, liquid suction.

According to one embodiment of the present invention:
(a) a therapeutically effective dose of clofazimine or a pharma- ceutically acceptable derivative or salt thereof;
(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;
A pharmaceutical composition comprising:
The clofazimine or a pharma- ceutically acceptable derivative or salt thereof is provided in the form of particles in a suspension;
and wherein the particles of clofazimine or a pharma- ceutical acceptable derivative or salt thereof have a median diameter of less than 5 μm and a D90 of less than 6 μm.

According to another embodiment of the invention, the particles of clofazimine or a pharma- ceutically acceptable derivative or salt thereof have a mean diameter 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;
A pharmaceutical composition comprising:
The clofazimine is provided in the form of particles in a suspension,
and the clofazimine particles have a median diameter of less than 5 μm and a D90 of less than 6 μm.
Pharmaceutical compositions are provided.

In another embodiment of the invention, the particles of clofazimine have a median diameter of less than 2 μm;
And D90 is less than 3 μm.

The compositions of the present invention may be aerosolized with a suitable nebulizer to allow for the aerosolized clofazimine to reach the lower respiratory system (i.e., the bronchi, bronchioles, and mid- and peripheral lungs).
The delivery of the active ingredient to the pulmonary arteries (lungs, pulmonary arteries and lower peripheral lungs) is greatly improved, thereby substantially improving the therapeutic effect.

More preferably, the inhalation device should further be adapted to deliver aerosols locally to the lungs with an optimal particle size distribution for uniform deposition in the lower respiratory system.

The present invention thus provides aerosols with particle sizes that facilitate delivery to the alveoli and bronchioles. The preferred aerodynamic particle size for targeting the alveoli and bronchioles is between 1 and 5 μm. Larger particles are preferentially deposited in the upper lung, i.e., the bronchi and trachea, and the mouth and pharynx, i.e., the oropharyngeal region. Thus, the inhalation device is adapted to generate aerosols with mass median aerodynamic diameters (MMAD) in the range of about 1 to about 5 μm, preferably in the range of about 1 to about 3 μm. In a further embodiment, the particle size distribution is narrow, with a geometric standard deviation (GSD) of less than about 2.5.

The present invention is based on the unexpected discovery that aerosol administration of clofazimine in the form of a suspension to the lungs can deposit the active agent in the lower (i.e., deep) respiratory system, thereby increasing the bioavailability of highly hydrophobic BCS Class II agents, resulting in significantly improved therapeutic efficacy and reduced systemic side effects.

In another aspect, this discovery improves the antibiotic treatment of infections caused by mycobacteria and gram-positive bacteria, particularly pulmonary infections due to NTM, such as CF, COPD, and opportunistic infections in immunocompromised patients (such as HIV patients).

Furthermore, the present invention aims to reduce the dose and duration of clofazimine treatment as well as eliminate the systemic side effects of established oral treatment regimens for pulmonary infections due to Gram-positive bacteria, particularly pulmonary TB and NTM infections.

It will be understood by one of skill in the art that the present application also discloses any and all combinations of the individual features disclosed herein.

Definitions The term "pharmaceutical acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of the present invention and which are not biologically or otherwise undesirable.
The compounds of the present invention can form 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 from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, naphthoic acid, oleic acid, palmitic acid, pamoic (embonic) acid, stearic acid, glycolic acid, pyruvic acid,
Examples of the acidic acid include 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, and salicylic acid.

Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc.; ammonium, potassium, sodium, calcium, and magnesium salts are particularly preferred. Organic bases from which salts can be derived include, for example, primary, secondary,
and tertiary amines, naturally occurring substituted amines and other substituted amines, cyclic amines, basic ion exchange resins, and the like. Specific examples of such amines include 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.

In addition to the free base, it is preferred according to the invention to use the methanesulfonic acid, maleic acid, isonicotinic acid, nicotinic acid, malonic acid and salicylates, especially clofazimine methanesulfonate.

The term "pharmaceutical acceptable derivatives" as used herein refers to compounds disclosed, for example, in U.S. Pat. No. 9,540,336, U.S. Pat.
The disclosure of No. 40,336 is incorporated herein by reference in its entirety. In addition, the meaning of derivatives is as set forth in Lu, Y., Zhen, M., Wang, B., Fu,
,L. , Zhao, W. , Li, P. , Xu, J. , Zhu, H. , Jin, H. , Yi
n,D. , Huang, H. , Upton, AM. and Ma, Z. , “Clofaz
imine Analogs with efficiency against expe
rimental Tuberculosis and reduced Potent
ial for Accumulation"Antimicrobial Agent
S and Chemotherapy (2011), 55 (11): pp. 5185-
5193. Furthermore, a "pharmaceutically acceptable derivative" of a compound is, for example, a prodrug of the compound. In general, a prodrug is a derivative of the compound that is capable of providing an active form of the compound after administration. Such derivatives can be, for example, esters or amides of a carboxyl group, carboxyl esters of a hydroxyl group, or phosphate esters of a hydroxyl group.

"Therapeutically effective amount
"therapeutically effective dose"
e) or "pharmaceutical effective amount"
"Amount" refers to the amount of clofazimine or a pharma- ceutically acceptable salt or derivative thereof disclosed herein that is therapeutically effective. A therapeutically effective amount is a therapeutically effective amount. Thus, as used herein, a therapeutically effective amount refers to the amount of clofazimine that produces the desired therapeutic effect as determined from clinical trial results and/or infection studies using animal models.

The amount and daily dose of clofazimine can be routinely determined by one skilled in the art and will vary depending on several factors, such as the specific microbial strain involved. The amount may also depend on the patient's height, weight, sex, age, and medical history. A therapeutically effective amount in the case of prophylactic treatment is an amount that will be effective in preventing microbial infection.

A "therapeutic effect" is a relief to some extent of one or more symptoms of an infection, including curing the infection. "Cure" means that the symptoms of an active infection are eliminated, including the complete or substantial disappearance of the excess of viable bacteria involved in the infection below the detection threshold by conventional measurements. However, certain long-term or permanent effects of the infection may exist even after cure is achieved (such as extensive tissue damage). As used herein, a "therapeutic effect" is defined as a statistically significant reduction in the host's bacterial load, development of resistance, or improvement in the symptoms of infection as determined by human clinical outcomes or animal studies.

As used herein, "treat" and "treatment" refer to
") or "treating" refers to the administration of a pharmaceutical composition/combination for prophylactic and/or therapeutic purposes.

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

Unless otherwise specified herein, the term "inhalation" means pulmonary inhalation.

As used herein, unless otherwise specified, the term "infection" is meant to refer to a pulmonary infection.

Unless otherwise specified, the term "substantially" when used to refer to the purity of a compound means
This indicates that the purity of the compound is greater than 95%.

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

Unless otherwise specified, the term "appropriate concentration" refers to the concentration of ingredients in a composition or combination that provides a pharma- ceutical acceptable composition or combination.

Pharmaceutical Compositions and Combination Drugs The following water grades are particularly suitable for the present invention: sterile purified water, sterile water for injection, sterile water for irrigation.
, sterile water for inhalation (US
P) and equivalent water quality grades according to, for example, the European Pharmacopoeia or the National Formulary.

The aqueous electrolyte solution used as the aqueous liquid carrier according to the present invention may further comprise sodium chloride,
It may include potassium chloride, lithium chloride, magnesium chloride, calcium chloride, or mixtures thereof.

The aqueous liquid carrier is preferably isotonic saline (0.9% NaCl, which corresponds to about 150 mM NaCl, preferably 154 mM NaCl).

Clofazimine has been shown to exist in at least four polymorphic forms (e.g., B
annigan, et al. , “Investigation into the S
Solid and Solution Properties of Known an
d Novel Polymorphs of the Antimicrobial
"Molecule Clofazimine",Cryst.Growth Des.2
016, 16(12), pp. 7240-7250. Clofazimine can exist in triclinic form FI, monoclinic form FII, and orthorhombic form FIII. Another form, FIV, has also been found only at elevated temperatures.

Thus, in a further embodiment of the 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;
A pharmaceutical composition comprising:
The clofazimine is provided in the form of particles in a suspension,
and the particles of clofazimine have a median diameter of less than 5 μm and a D90 of less than 6 μm, preferably a median diameter of less than 2 μm and a D90 of less than 3 μm, and the clofazimine is provided in one or more polymorphs selected from triclinic Form FI, monoclinic Form FII, and orthorhombic Form FIII, and mixtures of such forms. In another embodiment, the clofazimine is provided substantially in orthorhombic Form FIII.

In a further embodiment of the invention there is provided a pharmaceutical composition according to any of the composition embodiments described herein, wherein the non-ionic surfactant is polysorbate 20 (e.g., T
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), polyethylene glycol derivatives of hydrogenated castor oil having a hydrophilic-lipophilic balance value of 15 to 17 (e.g., Cremophor® RH50),
mophor® RH60), sorbitan monolaurate (e.g., Span (
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® 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., Brij® 93),
Polyoxyethylene (2) cetyl ether (e.g., Brij® S2), caprylocaproyl polyoxyl-8 glyceride (e.g., Labrasol®)
, polyethylene glycol (20) stearate (e.g., Myrj™ 49), polyethylene glycol (40) stearate (e.g., Myrj™ S40), polyethylene glycol (100) stearate (e.g., Myrj™ S100), polyethylene glycol (8) stearate (e.g., Myrj™ S8), and polyoxyl 40 stearate (e.g., Myrj™ 52), and mixtures thereof.

In another embodiment of the invention, a pharmaceutical composition is provided 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, a pharmaceutical composition is provided, wherein the hypertonic saline is 1% to 7% (w/v) sodium chloride. In a further embodiment of the invention, a pharmaceutical composition is provided, wherein the non-ionic surfactant is ultra-pure polysorbate 80 (e.g., NOF Corporation Po
lysorbate 80 (Hx2)) and the aqueous liquid carrier is isotonic saline.

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 composition has an osmolality of 200-700 mOsm/kg
In a further embodiment, the osmolality of the composition is in the range of 300 to 400 m
in the range of Osm/kg.

In a further embodiment of the present invention there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the non-ionic surfactant is in an amount ranging from 0.001% to 5% of the total composition.
% (v/v) of the total composition, and the amount of clofazimine ranges from 0.1% to 20% (w
/v).

In another embodiment of the present invention, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, comprising the steps of:
(1) homogenizing a suspension of clofazimine, a non-ionic surfactant, and water to obtain a suspension containing clofazimine of suitable particle size;
(2) adjusting the pH of the suspension obtained from (1) to a pH between pH 5.5 and pH 7.5;
(3) adjusting the sodium chloride concentration to an appropriate concentration;
(4) adjusting the osmolality to an appropriate level;
It is prepared by a process comprising:

In a further embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is 154 mM
The homogenization 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 the clofazimine is carried out by multi-stage homogenization. In another embodiment, the clofazimine of suitable particle size is particles having a mean diameter of less than 5 μm and a D90 of less than 6 μm. In a further embodiment, the clofazimine of suitable particle size is particles having a mean diameter of less than 2 μm and a D90 of less than 3 μm.

In a further embodiment of the present invention there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, comprising the steps of:
(1) homogenizing a suspension of clofazimine and a non-aqueous liquid to obtain a suspension containing clofazimine of suitable particle size;
(2) isolating clofazimine; and
(3) adding clofazimine to a non-ionic 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;
It is prepared by a process comprising:

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 the clofazimine is carried out by multi-stage homogenization. In another embodiment, the clofazimine having a suitable particle size is a particle having a mean diameter of less than 5 μm and a D90 of less than 6 μm. In a further embodiment, the clofazimine having a suitable particle size is a particle having a mean diameter of less than 2 μm and a D90 of less than 3 μm.
The particles are smaller than 1 μm.

In a further embodiment, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, comprising administering to said patient a pharmaceutical composition comprising the steps of:
(1) micronizing clofazimine to obtain clofazimine of a suitable particle size;
(2) adding clofazimine to a non-ionic surfactant and water;
(3) adjusting the pH of the suspension obtained from (2) to a pH between pH 5.5 and pH 7.5;
(4) adjusting the sodium chloride concentration to an appropriate concentration;
It is prepared by a process comprising:

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

In other embodiments, micronization of clofazimine is carried out by jet milling, spray drying, ball milling, or supercritical fluid processing. In other embodiments, micronization of clofazimine is carried out by multi-stage micronization. In other embodiments, clofazimine of suitable particle size is particles having a mean diameter of less than 5 μm and a D90 of less than 6 μm. In further embodiments, clofazimine of suitable particle size is particles having a mean diameter of less than 2 μm and a D90 of less than 3 μm.

In a further embodiment, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, prepared by a process comprising homogenizing a suspension of clofazimine in water comprising a non-ionic surfactant, a suitable concentration of sodium chloride, and having a pH adjusted to between pH 5.5 and pH 7.5, to obtain a suitable particle size of clofazimine. 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, homogenization is performed by high pressure homogenization, high shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes. In another embodiment, homogenization of clofazimine is performed by multi-stage homogenization. In another embodiment, the suitable particle size of clofazimine is particles having a mean diameter 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 a mean diameter of less than 2 μm and a D90 of less than 3 μm.

In other embodiments, a process for preparing a pharmaceutical composition according to any of the composition embodiments described herein comprises the steps of:
(1) homogenizing a suspension of clofazimine, a non-ionic surfactant, and water to obtain a suspension containing clofazimine of suitable particle size;
(2) adjusting the pH of the suspension resulting from (1) to a pH between pH 5.5 and dpH 7.5;
(3) adjusting the sodium chloride concentration to an appropriate concentration;
(4) adjusting the osmolality to an appropriate level;
A process is provided that includes:

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, wet milling, ultrasonic homogenization, or a combination of such processes. In a further embodiment, homogenization of the clofazimine is performed by multi-stage homogenization. In a further embodiment, clofazimine of suitable particle size is particles with a mean diameter of less than 5 μm and a D90 of less than 6 μm. In another embodiment, clofazimine of suitable particle size is particles with a mean diameter of less than 2 μm and a D90 of less than 6 μm.
m, and D90 is less than 3 μm.

In other embodiments, a process for preparing an embodiment of the pharmaceutical composition described herein comprises:
(1) homogenizing a suspension of clofazimine and a non-aqueous liquid to obtain a suspension containing clofazimine of suitable particle size;
(2) isolating clofazimine; and
(3) adding clofazimine to a non-ionic 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;
A process is provided that includes:

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, wet milling, ultrasonic homogenization, or a combination of such processes. In a further embodiment, homogenization of the clofazimine is performed by multi-stage homogenization. In a further embodiment, clofazimine of suitable particle size is particles with a mean diameter of less than 5 μm and a D90 of less than 6 μm. In another embodiment, clofazimine of suitable particle size is particles with a mean diameter of less than 2 μm and a D90 of less than 6 μm.
m and D90 is less than 3 μm.

In a further embodiment, a process for preparing a pharmaceutical composition according to any one of the pharmaceutical composition embodiments described herein, comprising the steps of:
(1) micronizing clofazimine to obtain clofazimine of a suitable particle size;
(2) adding clofazimine to a non-ionic surfactant and water;
(3) adjusting the pH of the suspension obtained from (2) to a pH between pH 5.5 and pH 7.5;
(4) adjusting the sodium chloride concentration to an appropriate concentration;
A process is provided that includes:

In another embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is 154 mM
Adjusted to sodium chloride. In a further embodiment, micronization of clofazimine is carried out by jet milling, spray drying, ball milling, or supercritical fluid processing. In a further embodiment, micronization of clofazimine is carried out by multi-stage micronization.
In a further embodiment, the suitable particle size of clofazimine is less than 5 μm in average diameter;
In another embodiment, the clofazimine of suitable particle size is particles having a mean diameter of 2 μm and a D90 of less than 3 μm.

In another embodiment, there is provided 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 an adjusted pH 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, homogenization is performed by high pressure homogenization, wet milling, ultrasonic homogenization, or a combination of such processes. In a further embodiment, homogenization of clofazimine is performed by multi-stage homogenization. In a further embodiment, clofazimine of a suitable particle size is particles having a mean diameter of less than 5 μm and a D90 of less than 6 μm. In another embodiment, clofazimine of a suitable particle size is particles having a mean diameter of less than 2 μm and a D90 of less than 3 μm.

In a further embodiment, a process for preparing a pharmaceutical composition according to any one of the composition embodiments described herein, comprising the steps of: (a) homogenizing a suspension of clofazimine, a non-ionic surfactant, and water to obtain a suspension containing clofazimine of suitable particle size; and (b) adjusting the pH of the resulting suspension to between pH 5.5 and pH 6.0.
(c) adjusting the sodium chloride concentration to an appropriate concentration; and (d) adjusting the osmolality to an appropriate level;
Steps (b), (c) and (d) are
(c), (b), (d); (c), (d), (b); (d), (b), (c); or (d), (c), (b).

In another embodiment, a process for preparing a pharmaceutical composition according to any one of the composition embodiments described herein, comprising the steps of: (a) homogenizing a suspension of clofazimine and a non-aqueous liquid to obtain a suspension containing clofazimine of suitable particle size; (b) isolating clofazimine; (c) adding clofazimine to a non-ionic surfactant and water; and (d) adjusting the pH of the resulting suspension to pH 7.0 or higher.
(d) adjusting the pH of the solution to between pH 5.5 and pH 7.5; and (e) adjusting the sodium chloride concentration to an appropriate concentration;
(e); or (e) and (d).

In another embodiment, a process for preparing a pharmaceutical composition according to any one of the composition embodiments described herein, comprising the steps of: (a) micronizing clofazimine to obtain a suitable particle size of clofazimine; and (b) dissolving the clofazimine in a 500 mL aqueous soluble glycerol solution containing a non-ionic surfactant, a suitable concentration of sodium chloride, and having a pH of 5.5.
and b. adding to water adjusted to between 7.5 and 7.5.

In another embodiment of the present invention, a pharmaceutical combination in the form of an aerosol for inhalation is provided, comprising a composition according to any one of the composition embodiments described herein, administered by an ultrasonic nebulizer, an electrospray nebulizer, a vibrating membrane nebulizer, or a combination nebulizer.
A nebulizer selected from a jet nebulizer and a mechanical soft mist metered dose inhaler (
The pharmaceutical combination is prepared by aerosolization with a nebulizing device, the aerosol particles produced by the nebulizing device having a mass median aerodynamic diameter of 1-5 μm. In a further embodiment, the inhalable aerosol is for deposition in the lower respiratory system. In another embodiment, the output velocity of the nebulizing device is 0.1-
In another embodiment, the total inhaled volume is between 1 ml and 5 ml.

In other embodiments, a pharmaceutical composition according to any one of the composition embodiments described herein comprises nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth-thiol, ethylenediaminetetraacetic acid, gentamicin-loaded phosphatidylcholine modified gold nanoparticles (
Gentamicin loaded phosphotidylcholine-de
coated gold nanoparticles, chelating agents, cis-2-decenoic acid, D-amino acids, peptides containing D-amino acid residues (D-enantiomers
c peptide), gallium mesoporphyrin IX, gallium protoporphyrin I
an agent for dispersing and/or disrupting a biofilm, a mucolytic agent and/or a mucolytic agent selected from X, 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 this use comprises bedaquiline or a pharmacoactive agent, cefoxitin (cefoxitin), or a mucoactive agent, and/or an agent that reduces biofilm formation.
foxitinine), amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylates, and mixtures thereof.

In other embodiments, a pharmaceutical combination according to any of the combination embodiments described herein may comprise any one of the following: nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth-thiol, ethylenediaminetetraacetic acid, gentamicin-loaded phosphatidylcholine modified gold nanoparticles, chelating agents, cis-2-decenoic acid, D-amino acids, peptides containing D-amino acid residues, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid,
A pharmaceutical combination is provided for use in combination with an agent for dispersing and/or disrupting biofilms, a mucolytic and/or mucoactive agent, and/or an agent for reducing biofilm formation selected from fatty acids, host defense peptides, and antimicrobial peptides. In another embodiment, the combination for this use is used to administer the composition of the invention before, simultaneously, or after administering an agent selected from bedaquiline or a pharma- ceutically 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 after administering an agent selected from bedaquiline or a pharma- ceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof. In a further embodiment, the composition is administered before, simultaneously, or after administering bedaquiline or a pharma- ceutically acceptable salt or derivative thereof.

In other embodiments, a pharmaceutical composition according to any one of the composition 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 further embodiments, the infection is caused by nontuberculous mycobacteria and tuberculosis (Mycobacterium tuberculosis).
In a further embodiment, the nontuberculous mycobacterium is caused by a species of the genus mycobacterium selected from the group Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium tuberculosis, and combinations thereof.
and Mycobacterium leprae
In another embodiment, the infection is selected from:
The opportunistic infection is selected from pulmonary MAC disease and nontuberculous infection in patients with cystic fibrosis, chronic obstructive pulmonary disease, or acquired immune deficiency syndrome. In another embodiment, the infection is an opportunistic infection caused by nontuberculous mycobacteria in a cystic fibrosis patient. In another embodiment, the composition for this use comprises bedaquiline or a pharmacologic acceptable salt or derivative thereof, cefoxitin (cefoxitin), or a combination thereof.
oxitine), amikacin, clarithromycin, pyrazinamide, rifampin,
The composition is administered before, simultaneously, or after administration of an agent selected from moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof. In other embodiments, the composition is administered before, simultaneously, or after administration of an agent selected from bedaquiline or a pharma- ceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof. In further embodiments, the composition is administered before, simultaneously, or after administration of bedaquiline or a pharma- ceutically acceptable salt or derivative thereof.

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.
Nontuberculous acid-fast bacteria and tuberculosis (Mycobacterium tuberculosis)
In a further embodiment, the nontuberculous mycobacterium is caused by a species belonging to the genus mycobacterium 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 pulmonary MAC disease and nontuberculous mycobacterial disease in patients with cystic fibrosis, chronic obstructive pulmonary disease, or acquired immune deficiency syndrome. In another embodiment, the infection is an opportunistic infection caused by nontuberculous mycobacteria in patients with cystic fibrosis. In another embodiment, the combination for this use is used to administer the composition of the present invention before, simultaneously, or after administering an agent selected from bedaquiline or a pharma- ceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof. In another embodiment, the combination for this use is used to administer the composition of the present invention before, simultaneously, or after administering an agent selected from bedaquiline or a pharma- ceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof. In other embodiments, the combination for this use is used to administer a composition of the invention prior to, simultaneously with, or after administration of bedaquiline or a pharma- ceutically acceptable salt or derivative thereof.

In another embodiment, there is provided a system for use in providing antibacterial activity in treating or preventing pulmonary infections caused by mycobacteria or other gram positive bacteria, the system comprising:
1)
(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 aerosolized combination pharmaceutical comprising:
2) a sprayer;
Including,
The clofazimine is present in the form of a suspension,
The aerosol particles generated by this system have mass median aerodynamic diameters of 1-5 μm.

In further embodiments, the present invention relates to a pulmonary fungal infection or Clostridium difficile (
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 Clostridium difficile, or a combination thereof. In other embodiments, 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 further embodiments, the pulmonary fungal infection is Candida albicans.
da albicans or Aspergillus fumigatus
fumigatus), or a combination thereof.

In a further embodiment, 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 a further embodiment, the pulmonary fungal infection is Candida albicans (
candida albicans or Aspergillus fumigatus
gilus fumigatus), or a combination thereof.

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

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

Particle Size and Distribution The therapeutic effect of aerosol therapy depends on the dose deposited and its distribution. Aerosol particle size is one of the important variables that determine the dose and distribution of drug aerosols in the lungs.

In general, inhaled aerosol particles tend to be deposited by one of two mechanisms: impaction (usually dominating larger aerosol particles) and settling (predominant for smaller aerosol particles). Impaction occurs when the momentum of an inhaled aerosol particle is great enough that the particle does not follow the airflow and falls on a physiological surface.
In contrast, deposition occurs when very small aerosol particles carried in the inspiratory flow end up on physiological surfaces as a result of gravity settling, primarily in the lower respiratory system.

Drug delivery to the lungs can be achieved by inhaling an aerosol through the oropharynx. Aerosol particles with an aerodynamic diameter greater than about 5 μm generally do not reach the lungs. Instead, they tend to impact the back of the throat and may be swallowed and orally absorbed. Aerosol particles with a diameter of about 3 to about 5 μm are more likely to reach the upper-to-middle airway regions.
Aerosol particles that are smaller than this, i.e., about 0.5 to about 3 μm, can reach the alveolar region. Aerosol particles smaller than about 0.5 μm in diameter tend to be exhaled during quiet breathing, but can also be deposited in the alveolar region during breath-holding.

Aerosols used for pulmonary drug delivery consist of a wide range of aerosol particle sizes, and therefore statistical descriptors are used. Aerosols used for pulmonary drug delivery are usually described by their mass median diameter (MMD), i.e., half of the mass contained in the aerosol particle is within the MMD.
D and half of the mass contained in the aerosol particles is smaller than the MMD. When the particles have a uniform density, the volume median diameter (VMD) can be used interchangeably with the MMD. The determination of VMD and MMD is performed using laser diffraction. The width of the distribution is expressed as the geometric standard deviation (GSD). On the other hand, the deposition of aerosol particles in the respiratory tract is more accurately represented by the aerodynamic diameter of the particles, so the mass median aerodynamic diameter is usually used. MMA
D measurements are made by inertial impaction or time-of-flight measurements. For water particles, VMD, MMD
However, if humidity is not controlled while the aerosol passes through the impactor, dehydration will occur and the measured MMAD will differ from the MMD and VM.
To use this notation, VMD, MMD, and MM
Measurements of AD are comparable in description of VMD, MMD, and MMAD.
) is deemed to be carried out under controlled conditions.

However, for purposes of illustration, the aerosol particle size of the aerosol particles will be given as the MMAD as determined by measuring at room temperature with a Next Generation Impactor (NGI) in accordance with the US Pharmacopeial Convention (In Pr
ocess Revision <601> Aerosols, Nasal Spra
ys, Metered-Dose Inhalers, and Dry Powder
Inhalers, Pharmacopeial Forum (2003), Vol.
me Number 29, pages 1176-1210, Jolyon Mitc
hell, Mark Nagel“Particle Size Analysis
of Aerosols from Medical Inhalers”, KON
A Powder and Particle Journal (2004), Vol.
me 22, pages 32-65).

According to the present invention, the aerosol particle size is optimized to maximize deposition of clofazimine at the site of infection while maximizing tolerability. Aerosol particle size can be expressed in terms of mass median aerodynamic diameter (MMAD). Large particles (e.g., MMAD > 5μ) are considered to be large.
m) are too large to follow the bends of the airways and therefore tend to deposit outside the thoracic cavity and in the upper airways, where deposition of large particles in the upper airways can result in intolerance symptoms (e.g. coughing and bronchospasm).

Therefore, according to a preferred embodiment, the MMAD of the aerosol should be less than about 5 μm, preferably between about 1 and 5 μm, and more preferably less than 3 μm (<3 μm).
It is.

However, guided breathing maneuvers
) would allow larger particles to pass through the extrathoracic and upper airways and penetrate deeper into the lungs than quiet breathing, thereby increasing deposition of the aerosol in the middle and lower respiratory system. Breathing could be slowed to 100 ml/min with instruction. Thus, when using instructional breathing, the preferred MMAD of the aerosol should be less than about 10 μm.

Another factor (besides the aerosol particle size) that is equally important is the size and size distribution of the solid particles, in this case the clofazimine. The size of the solid particles of a given aerosol particle must be smaller than the aerosol particles that contain it. 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.

It is therefore desirable to use solid drug particles that are significantly smaller than the MMAD of the aerosol particles - for example, if the MMAD of the aerosol particles is 3 μm, it would be desirable for the solid particles to be 1 μm or smaller.

Another consideration is, for example, when using a vibrating mesh nebulizer, where the formulation is pumped through holes in a plate that breaks the suspension into droplets, and for this reason the solid particles also need to be smaller than the holes in order to pass through.

The particle size of the solids in suspension can be given by the mean diameter of the particles, or it can be given by the distribution of the particles: the D90 value indicates that 90% of the particles in the suspension are equal to or smaller than the mean diameter.

Nebulizers For aqueous and other non-pressurized liquid systems, a variety of nebulizers, including small volume nebulizers, are available for aerosolizing the formulation. Compressor-driven nebulizers incorporate jet nebulization technology and use compressed air to generate an aerosol of the liquid. Devices of this type are available from, for example, Healthdyne Technologies, Inc.; Invacare, Illinois.
nc. ;Mountain Medical Equipment, Inc. ;Pari
Respiratory, Inc. ; Mada Medical, Inc.; ;Puri
tan-Bennet; Schuco, Inc. , DeVilbiss Health
Ultrasonic nebulizers are commercially available from, for example, Omron Heathcare, Inc. and DeVil Heathcare, Inc.; and Hospitak, Inc. Ultrasonic nebulizers use mechanical energy in the form of vibrating piezoelectric crystals to generate inhalable droplets, and are commercially available from, for example, Omron Heathcare, Inc. and DeVil Heathcare, Inc.
Vibrating mesh nebulizers are commercially available from Biss Health Care, Inc. Vibrating mesh nebulizers utilize either piezoelectric or mechanical pulses to generate inhalable droplets. Other examples of nebulizers for use with clofazimine as described herein include:
U.S. Pat. Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911; 4,510,929; U.S. Pat.
No. 5,164,740; U.S. Pat. No. 5,5
No. 86,550; U.S. Pat. No. 5,758,637; U.S. Pat. No. 6,644
,304; U.S. Pat. No. 6,338,443; U.S. Pat. No. 5,906,2
No. 5,934,272; U.S. Pat. No. 5,960,792
Nos. 5,971,951; 6,070,575; 6,192,876; 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,
No. 192,876; U.S. Pat. No. 6,644,304; U.S. Pat. No. 5,54
Nos. 6,161,910, 6,083,922, and 6,161,
536; U.S. Pat. No. 6,264,922; U.S. Pat. No. 6,557,54
No. 6,612,303; and U.S. Pat. No. 6,612,303, all of which are incorporated herein by reference in their entirety. Examples of commercially available nebulizers that can be used with the clofazimine compositions described herein include the Aerosol nebulizer and the Clofazimine nebulizer.
Respirgard II (registered trademark), Aeroneb (registered trademark), Aegen
Aeroneb® Pro, and Aeroneb® Go; Aradigm
AERx® and AERx Essence™ manufactured by I-neb Resp
Porta-Neb (registered trademark), Freeway Free, manufactured by ironics, Inc.
edom™, Sidestream, Ventstream; and PARI, G
mbH PARI LCPlus (registered trademark), PARI LC-Star (registered trademark)
Other non-limiting examples include U.S. Pat.
This is disclosed in specification No. 9.

In accordance with the present invention, the pharmaceutical composition may be aerosolized using a nebulizing device, preferably selected from an ultrasonic nebulizer, an electrostatically charged spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer, or a mechanical soft mist metered dose inhaler.

Preferably the device controls the patient's inhalation rate either electrically or mechanically.

In a further preferred embodiment, the generation of aerosol by the device is activated by the inhalation action of the patient, such as the AKITA device.

A preferred (commercially available) example of the above-mentioned sprayer/instrument for use in accordance with the present invention is the Vectu
ra fox, Pari eFlow, Pari Trek S, Philips In
nospire mini, Philips InnoSpire Go, Medspr
ay device, Aeroneb Go, Aerogen Ultra, Respi
ronics Aeroneb, Akita, Medspray Ecomyst, and Respimat.

Use in Treatment and/or Prevention The pharmaceutical compositions and combination pharmaceuticals (aerosols, aerosolized formulations) and systems according to the invention are useful for treating mycobacteria or Staphylococcus aureus (Staphylococcus aureus).
Phylococcus aureus (including methicillin-resistant and vancomycin-intermediate resistant strains), Streptococcus pneumoniae (Streptococcus pneumoniae
Enterococcus pneumoniae), and Enterococcus faecalis
The pharmaceutical compositions and pharmaceutical combinations of the present invention are also intended for use in the treatment and/or prevention of pulmonary infections caused by other clofazimine-susceptible bacteria, such as Clofazimine-sensitive bacteria, Clostridium perfringens, Clostridium coccus spp. The pharmaceutical compositions and pharmaceutical combinations of the present invention can also be used for the treatment and/or prevention of pulmonary fungal infections.

Dosing of Clofazimine According to the present invention, the pharmaceutical composition of the present invention is administered in an amount of about 1 to 5 ml, preferably 1 to 2 ml.
Delivered by nebulization of 2 ml.

Thus, based on a concentration of clofazimine in the pharmaceutical composition of about 20 mg/ml, the target fill dose is about 1-5 ml, which corresponds to 20-100 mg of clofazimine.

Daily lung dose of clofazimine administered according to the present invention
The mean dose (ie, the amount of drug deposited in the lungs) was approximately 5-10 mg for M. abscessus infections, which corresponds to a nominal dose (the amount of drug administered by the device) of 15-30 mg.

It will be appreciated by those skilled in the art that the pulmonary dose of clofazimine to be administered (and thus the load volume/nominal dose/nebulized volume) will be routinely adjusted based on the minimum inhibitory concentration (MIC) of clofazimine for each bacterial strain, which is well established in the art.

The daily pulmonary dose will therefore be divided according to the frequency of administration, either once or twice daily.

According to the present invention, clofazimine is administered once or twice daily resulting in a total daily pulmonary dose of about 5-10 mg.

It will be apparent to one skilled in the art that the amounts stated above relate to the free base of clofazimine and that dosages of derivatives and salts must be adjusted based on the MIC of each compound and strain.

Mucolytic/biofilm modifying agents
)
In order to reduce the viscosity of sputum during aerosol treatment and to disrupt existing biofilms, treatment and/or prophylaxis according to the present invention may include the additional administration of a mucolytic agent and/or a biofilm disrupting agent.

These agents may be formulated as a fixed combination or
Or it may be administered simultaneously or subsequently with a pharmaceutical composition/aerosol combination containing clofazimine according to the present invention.

Agents for dispersing/disrupting biofilms, mucolytic agents and/or
or a mucoactive agent and/or an agent that reduces biofilm formation is administered by aerosolized 4
~7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth-thiol, ethylenediaminetetraacetic acid, gentamicin-loaded phosphatidylcholine modified gold nanoparticles, chelating agent, cis-2-decenoic acid,
D-amino acids, peptides containing D-amino acid residues, 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.

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

These agents may be formulated as a combination drug or may be administered before, simultaneously with, or after the clofazimine-containing pharmaceutical composition/aerosol combination according to the present invention.

The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. Embodiments in accordance with the invention are encompassed by the claims appended hereto.

Testing The following exemplary compositions and combinations were prepared according to the processes described herein.

Example 1
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 in an Ultra-Turrax homogenizer. 0.5 ml of polysorbate 80 (NOF Hx2) was added. The mixture was subjected to ultrasonic vibration using an ultrasonic probe (Bandelin S).
Branson Digital with Onoplus Probe MS73
The suspension was treated 7 times (3 min each) with a Sonifier™ 250D at 70% amplitude. The volume was adjusted to 10 ml with water. The suspension was filtered with a VWR folded qualitative filter paper (303, particle retention capacity 5-13 μm, size: 150 mm) to obtain the composition of Example 1.
The median particle size of clofazimine in the composition of Example 1 is 3.9 μm and the D90 is 6.7 μm.
The concentration of clofazimine was determined by UV/Vis spectroscopy at 280 nm and was found to be 7.16 mg/mL by calibration with a 1 mg/mL stock solution of clofazimine diluted in mobile phase.
/ml.

The composition of Example 1 is shown in Table 1.

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

Example 2
A suspension of 6 g of clofazimine in the orthorhombic form III in 100 ml of water containing 0.5% polysorbate 80 (NOF Hx2) and 0.6% sodium chloride was added to the Ultra-T
The suspension was pre-micronized using an Urrax® at 10,000 rpm for approximately 40 seconds. A preformulation was prepared by adding water containing 0.6% sodium chloride to a volume of 300 ml. 300 ml of this suspension was homogenized using a homogenizer, M-110EH-30 m
icrofluidizer (Microfluidics, Westwood, MA,
A pre-homogenization step was performed by feeding the suspension into the inlet of a H30Z chamber (Horton, NY, USA) and circulating the suspension through the H30Z chamber at 5,000 psi for 15 minutes. A second H10Z chamber was then attached in series to the first and the suspension was homogenized at 25,000 psi for an additional 23 minutes. Particle size analysis was performed using a HORIBA LA 950, revealing a median particle size of 0.01 mm.
The particle diameter was shown to be 0.83 μm and the D90 value was 1.17 μm. The concentration of clofazimine was determined to be 16.05 mg/ml by UV/Vis spectroscopy at 280 nm and calibrated with a 1 mg/ml stock solution of clofazimine diluted in mobile phase.

The composition of Example 2 is shown in Table 2.

Example 3
Clofazimine (crystal modification: orthorhombic Form III) was suspended in a solution of water, sodium chloride, and polysorbate 80, and the suspension was then immersed in M-110EH-30 Microfluidic.
The composition of Example 3 was produced using a dizer® Processor (chambers: H30Z and G10Z) in H30Z-G10Z configuration, operating at a pressure of 28,250 psi for 30 minutes. The resulting clofazimine particles had a median particle size of 1.28μ.
m and D90 was less than 2 μm.

The composition of Example 3 is shown in Table 3.

Viscosity Measurement The viscosity of the composition of Example 3 was measured using a STRESSTECH Rheometer in stress control mode. A hollow cylindrical fixture (double gap geometry) was used, and the spindle was rotated continuously to measure the viscosity at each temperature point (during temperature
The viscosity was measured at 0.05 V and 0.05 V for 10 min at 0.05 V. ...
The viscosity values were measured at 20° C., 25° C., and 30° C., respectively, at pressures of 0.01, 0.05, and 0.1 Pa. The average viscosity values obtained by performing two separate sample loadings are given in Table 4 below.

Animal Models and Efficacy Studies A clinical isolate of clofazimine was studied to obtain preliminary data to establish lung tissue concentration levels following direct respiratory delivery rather than systemic administration.
al) The ability of the compositions of the present invention to inhibit the growth of NTM species in an in vivo mouse model of acute lung infection was tested. Two separate mouse models were used to investigate NTM lung infection for each species of interest. To carry out the study, Mycobacterium avium (M
The following strains were used: Mycobacterium avium strain 2285 and Mycobacterium abscessus strain 103 (
The details of the strain are registered in the EMBL/GenBank/DDBJ database (September 2014).
"Phylogenetic analysis of Mycobacterium tuberculosis"
terial species using whole genome sequence
ces”. Hazbon M.H., Riojas M.A., Damon A.M.,
Alalade R. , Cantwell B. J. , Monaco A. ,King
These two species have already been used as models of NTM infection in the literature (Obregon-Henao et al., 2003; Sohrabi A., 2003).
l. 2015 Antimicrob Agents Chemother; and C
Han et al. Animal Models of Non-Tuberculo
us Mycobacterial Infections, Mycobact Dis
(2016).

In vivo safety study in Balb/C mice:
To evaluate the in vivo safety and tolerability, 6- to 8-week-old Balb/C female mice were cultured in
The compositions were obtained from Harles River. Mice were allowed to rest for one week before dosing. Three healthy mice were provided for each dose of clofazimine, and were dosed three times, every other day. Mice were dosed with 10.0, 5.01, and 2.51 mg/kg of clofazimine in the composition of Example 1. Three healthy mice were dosed with an aerosol of the compound intratracheally using a Microsprayer®, every other day, for a total of three doses.

The safety of clofazimine has been confirmed at 20 mg/kg (oral gavage, 200 μl). The composition of Example 1 showed no toxicity even at the maximum dose tested (10.0 mg
/kg; 0.2506 mg/dose in 35 μl, intratracheal). Thus, the composition of formula I
0.0 mg/kg is considered safe and well tolerated.

Determination of Minimum Inhibitory Concentration The minimum inhibitory concentration (MIC) test was performed using Mueller-Hinton (MH) broth (cation adjusted) (calcium and magnesium ion concentrations recommended by CLSI standard M7-A7) (Becton Dickinson) by the broth microdilution method (microbroth).
MIC testing was performed by the th dilution method. MIC testing was also performed by the broth microdilution method using 7H9 broth (Sigma-Aldrich). The rationale for using both MH and 7H9 broths for compound screening is that antimycobacterial compounds have been shown to exhibit different MIC activities depending on the broth used for the MIC testing.

M. abscessus was cultured on 7H11 agar plates (Sigma-Aldrich).
drich) at 35-37°C in ambient air (depending on the bacterial strain) for 3 days, and M. avium was grown on 7H11 agar plates (
The cells were grown on a Sigma-Aldrich plate at 37° C. in air for 21-30 days.

Colony forming units (CFUs) were harvested from the agar plates and added to either MH broth or 7H9 broth supplemented with 0.05% tween-80.
Optical density (OD) was measured by absorbance after 12 days (M. abscessus) or 12 days (M. avium) and grown in air at 35-37°C until OD of 0.08-0.1 (McFarland standard #0.5). Cell suspensions were then confirmed in saline to match OD of 0.08-0.1 (McFarland standard #0.5). Compound stock solutions were prepared by suspending the compound in DMSO at a concentration of 1.28 mg/ml and used immediately with a test range of 64-0.062 μg/ml. 180 μl of broth (either MH or 7H9) was then added to the first row of a 96-well plate and 100 μl of broth was added to the remaining rows of the 96-well plate.
20 μl of compound stock solution was added to wells in row 1 and serially diluted. Finally, 100 μl of NTM cell suspension was added to all wells except the media only control well. QC reagents specific for each organism: 1) Bacteria only negative control 2) Media only negative control 3) Clarithromycin positive drug control.

The OD of M. abscessus was measured after 3 days and for M. avium after 12 days. Following these measurements, Resazol
Plates were assayed using the resazurin Microtiter Assay Plate method. Briefly, this method uses resazurin (7-hydroxy-3H-phenoxazine-3-
Resazurin is a blue pigment that is weakly fluorescent by itself but is irreversibly reduced to resorufin, which emits strong pink-red fluorescence. It has been used as a redox indicator to determine bacterial cell viability in MIC measurements.

Tests were performed in triplicate: Test #1 was performed after the composition of Example 1 had been stored at 4° C. for 2 months, Test #2 was performed after 4 months, and Test #3 was performed after 5 months.

Minimum inhibitory concentrations in the presence and absence of CF sputum. Minimum inhibitory concentration determinations were performed as described above.

To investigate 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 an antibacterial agent within 48 hours and the sputum was sterilized to remove any endogenous bacteria by exposure to UV light.
After sterilization, M. abscessus, M. avium
), M. intracellulare, and M. chimaera were incubated in 10% CF sputum and then subjected to MIC testing. The same CLSI protocol as described above was followed to determine the MIC of the composition of Example 1 in the presence and absence of sputum from cystic fibrosis patients. All tests were performed in duplicate.

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

The results shown in Table 5 demonstrate that both clofazimine and the composition of Example 1 exhibit stable MICs against a broad range of nontuberculous mycobacterial species.

These data show that the composition of Example 1 is effective against M. abscessus
) and M. avium, suggesting that it is stable for at least this period.

Mouse model of M. abscessus in SCID mice. Six to eight week old SCID female mice were purchased from Charles River. Mice were rested for one week prior to infection.

Test stock solution of M. abscessus 103 strain
A 1 ml aliquot of the 100 ml stock was taken and stored at -80°C until use. For infection, the aliquot was thawed and 20 ml of the aliquot was injected into a 1 ml Luer lock syringe fitted with a 26 g needle.
The mixture was dissolved in water and diluted with sterile 1×PBS.

In an acutely infected SCID mouse model, 1×10 ⁶ CFU/mouse (M. abscessus (M. ab)
Lung infection was induced by non-invasive intratracheal instillation of S. scessus strain 103.

Three mice were sacrificed on day 1 post-infection and bacterial uptake was determined.
The whole lung, spleen, and liver were excised and homogenized in 4.5 ml of 1×PBS. The homogenates were serially diluted at a dilution ratio of 1:10 and diluted with the dilutions (0-1-2-
The mixture of the above strains (3-4-5-6-7) was smeared on a 7H11 agar plate, and the plate was placed in a dry air incubator at 32°C (varies depending on the strain) for 7 days.

The composition of Example 1 was administered at 10.0 mg/kg via the pulmonary route using a Microsprayer (registered trademark) (35 μl), clofazimine (oral gavage) and amikacin (subcutaneous) in a volume of 200 μl per mouse. These were administered starting on day 2 post-infection and continued for 1
This was continued on alternate days for eight days.

Mice were sacrificed two days after the last dose of compound was administered. Six mice from every group (untreated control, clofazimine (gavage), composition of Example 1, and amikacin treated mice) were sacrificed and bacterial loads were determined. Lung homogenates were 0-1-2-3-4-5-6
-7, spleen 0-1-2-3-4-5-6-7, and liver 0-1-2-3-4-5-6
Plates were plated at -7.

The protection value expressed in Log10 is at least 0.
60 indicates that the activity is statistically significant.
FU was transformed into logarithms, which were then evaluated by one-way ANOVA, followed by one-way
The analysis was performed by multiple comparison analysis using the one-way Tukey test (G
raphPad Prism analysis software. Differences are considered significant at the 95% confidence level.

Table 6 shows the mean and standard error of the mean (SEM) of _Logio CFU data following infection of SCID mice with M. abscessus, where "n" is the total number of animals per group at the time of sacrifice.

The data in Table 6 show that treatment with the composition of Example 1 reduced the incidence of M. abscessus (M. ab
The results show that treatment with oral amikacin significantly reduced the bacterial load recovered from the lungs and spleens of animals infected with S. scessus, which was statistically improved compared to treatment with amikacin or oral clofazimine.

Mouse model of M. avium infection in Beige mice. Female Beige mice aged 6-8 weeks were purchased from Charles River. Mice were allowed to rest for 1 week before infection.

In an acutely infected Beige mouse model, 1×10 ⁸ colony forming units (CFU)/ml (M
The lungs were non-invasively infected by aerosol exposure to M. avium 2285 strain rough.
1 ml aliquots of the mold test stock were frozen and stored at -80°C until use. For infection, the aliquots were thawed, disintegrated 20 times using a 1 ml Luer-lock syringe fitted with a 26 g needle, and diluted with sterile 1x phosphate-buffered saline.

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

The composition of Example 1 was administered at 10.0 mg/kg via the pulmonary route using a Microsprayer® (35 μl) and clofazimine (oral gavage) was administered at a volume of 200 μl per mouse starting on day 7 post-infection and continued every other day for 10 days.

Mice were sacrificed 5 days after the administration of the final dose of compound. Six mice from each group (untreated control, clofazimine (gavage), and the composition of Example 1) were sacrificed and bacterial loads were measured. 0-1-2-3-4-5-6-7 for lung homogenates, 0-1-2-
3-4-5-6-7, and liver 0-1-2-3-4-5-6-7 were smeared onto plates.

A protection value expressed in Log10 of at least 0.60 indicates statistically significant activity. Statistical analysis was performed by first transforming CFUs into logarithms, which were then compared on
The evaluation was performed by e-way ANOVA followed by analysis of variance with multiple comparisons with one-way Tukey test (SigmaStat software program). Differences are considered significant at the 95% confidence level.

Table 7 shows the Log α-β expression levels after infection of Beige mice with M. avium.
The average of ₁₀ CFU data is shown.

The data in Table 7 show that treatment with the composition of Example 1 reduced the M. avium
These results show that the amount of bacteria recovered from the lungs and spleens of animals infected with V. cerevisiae (M) was significantly reduced.

Chronically infected Beige mouse model Six to eight week old Beige mice were rested for one week before infection.
Mice were infected with 1×10 ⁸ CFU of M. avium 2285 rough strain via the lungs.
Three mice were sacrificed on the 27th day and six mice on the 28th day to measure bacterial uptake and pre-treatment bacterial load. Whole lungs, spleens, and livers were removed and homogenized in 4.5 ml of 1x PBS, and dilutions (0-1-2-3-4-5-6-7) were spread onto 7H11 agar and charcoal agar plates. The plates were placed in a 37°C dry air incubator for 25-30 days.

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

Mice were sacrificed on day 57, 2 days after the last treatment. The plates were placed in a dry air incubator at 37° C. for 30 days.

Statistical analysis was performed by first transforming CFU into logarithms and then performing a one-way ANOVA.
After evaluation in A, analysis of variance with multiple comparisons was performed with a one-way Tukey test. Differences were considered significant at a confidence level of 95%.

Table 8 shows the L. avium expression levels after chronic infection of Beige mice with M. avium.
Mean values of og ₁₀ CFU data are shown.

These data suggest that clofazimine has poor penetration into the granuloma-like structures formed by established "chronic" NTM infection animal models. The compositions of the present invention do not have this problem and appear to be able to maintain antimycobacterial activity even after the infection has become well established.

Effect on Barrier Integrity and Inflammation after In Vitro Exposure of Pulmonary Epithelial Cells to the Composition of Example 3 Cell Viability Three different types of cells were used to evaluate the cell viability of pulmonary epithelial cells: Calu-3;
549; and hAELVi cells were used under two in vitro conditions. Cells were treated either in "submerged conditions" (i.e., in cell culture medium on Transwell™ plates) or in "air-liquid interface" simulated conditions (ALI) where cell culture medium was removed from the apical side of the cells. In the "submerged conditions," Calu-3 cells were treated with the composition of Example 3 at three doses (10%,
The cells were exposed to 0% or 100% CO for 4 hours. To assess cell viability, cells were stained with acridine orange/propidium iodide (AO/PI) stain to distinguish live/dead cells. Red fluorescence indicates dead cells.

Macrophage uptake THP-1 cells were cultured with phorbol 12-myristate 13 acetate (PMA) at 124 ng/mL.
The cells were incubated with 1:200 ml of the composition of Example 3 (diluted 1:200 with Hank's Balanced Salt Solution (HBSS)) for 3 days to differentiate into macrophage-like cells.
Cell viability after exposure was determined by staining the cells with AO/PI as described above.

Transepithelial Electrical Resistance (TEER) Measurement Calu-3 cells were seeded in Transwell™ 3460 at 1×10 ⁵ cells/well and grown for 12 days until confluent.
World Precision Instruments, Friedberg, Ge
TEER measurements were performed using a Caulmany according to the manufacturer's instructions.
-3 cells were incubated with saline (negative control) or the composition of Example 3 (concentration: 20 mg/ml, 10
After 2-4 hours of exposure, cells were exposed to either 100 mg/ml or 2 mg/ml of erythrocytes.
The TEER was measured.

Inflammatory Cytokine Production Differentiated THP-1 cells (dTHP-1) were exposed for 4 or 24 hours to the composition of Example 3 (diluted 1:200 in HBSS). Exposure to HBSS alone was used as a negative control, and lipopolysaccharide (LPS) (100 ng/ml) was administered as a positive control.

After t=4 or 24 h incubation, supernatants were removed from the cells and pooled. Enzyme-linked immunosorbent assays (ELISAs) were performed on pooled supernatant samples. Separate ELISAs were performed for TNF-α, IL-6, IL-8, and IL-10 according to the manufacturer's instructions.
A LISA kit was used.

One-way analysis of variance (ANOVA) was performed, followed by Tukey post-hoc
Statistical analysis was performed using st. Probability values <0.05 were considered statistically significant.

Results After 4 hours of incubation under "immersion" conditions, the composition of Example 3 did not visually reduce cell viability at any of the administered concentrations.

Under “ALI” conditions, three different cell types (Calu-3, A549, and HA
ELVi cells) were examined three times at different time points (5 hours, 2 days, and 7 days). Little or no cytotoxicity was observed in all cells after 4 hours, and in the case of Calu-3 cells after 2 days. Some toxicity was observed in A549 cells after 2 and 7 days, and in Calu-3 cells after 7 days. Quantification of dead cells was not possible due to technical limitations.

Regarding uptake by macrophages, differentiated THP-1 cells were cultured at 1:200 HB
The macrophages were incubated with SS for 4 hours and the cell viability of the macrophages after exposure was determined. Although the composition of Example 3 did not induce cell death, it was clearly demonstrated that clofazimine was taken up by the macrophages.

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 extracted at various time points during exposure. A > 50% decrease in TEER compared to the control at any given time point was considered to indicate a significant decrease in barrier function integrity.

When Calu-3 cells were exposed to the composition of Example 3, there was no effect on the integrity of the barrier function even after 1 hour of exposure.
At the 10 mg/ml concentration, there was a slight decrease (i.e., 25-35%) at all time points after 2 hours. Exposure to the 2 mg/ml concentration did not result in a decrease in barrier function over the course of the study.

Inflammatory Cytokine Production The positive control, LPS, behaved as expected in this model. The composition of Example 3 did not result in significant changes in cytokines at any of the time points examined.

The results are shown in Table 9.

In vivo safety and tolerability: Balb/C female mice aged 6-8 weeks were dosed every other day for a total of three times. Mice were dosed with 10.0, 5.01, and 2.51 mg/kg of the composition of Example 1. The composition was administered intratracheally (IT) using a Microsprayer® aerosol at a volume of 35 μl/mouse. After injection, mice were observed at 10 minutes, 1, 2, and 4 hours after dosing.
Thereafter, observations were made daily.

Table 10 shows the overall observations after dosing. "BAR" means the animals were bright and
), active and responsive.

These data indicate that the 3 days of treatment did not result in a statistically significant change in body weight.

These results indicate that the compositions of the present invention are well tolerated at the doses tested.

Claims (21)

a) a therapeutically effective dose of clofazimine or a pharma- ceutical acceptable salt thereof;
b) a nonionic surfactant having a hydrophilic-lipophilic balance value greater than 10;
c) an aqueous liquid carrier selected from at least one of water, isotonic saline, buffered saline, and aqueous electrolyte solutions;
d) a mucolytic and/or biofilm disrupting agent;
A pharmaceutical composition comprising:
The clofazimine or pharma- ceutically acceptable salt thereof is provided in the form of particles in a suspension;
and wherein said particles of said clofazimine or a pharma- ceutical acceptable salt thereof have a median diameter of less than 5 μm and a D90 of less than 6 μm. 2. The pharmaceutical composition of claim 1, wherein the particles of clofazimine or a pharma- ceutically acceptable salt thereof have a mean diameter of less than 2 μm and a D90 of less than 3 μm. The pharmaceutical composition of claim 1, wherein the mucolytic and/or biofilm disrupting agent is selected from the group consisting of at least one of nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth-thiol, ethylenediaminetetraacetic acid, gentamicin-loaded phosphatidylcholine modified gold nanoparticles, chelating agents, cis-2-decenoic acid, D-amino acids, peptides containing D-amino acid residues, 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. 2. The pharmaceutical composition of claim 1, wherein the mucolytic and/or biofilm disrupting agent is an active agent selected from bedaquiline or a pharma- ceutically acceptable salt thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-aminosalicylate, and mixtures thereof. The nonionic surfactants include polysorbate 20, polysorbate 60, polysorbate 80, stearyl alcohol, polyethylene glycol derivatives of hydrogenated castor oil having a hydrophilic-lipophilic balance value of 14 to 16, polyethylene glycol derivatives of hydrogenated castor oil having a hydrophilic-lipophilic balance value of 15 to 17, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, polyoxyethylene (20) oleyl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (10) cetyl ether, polyoxyethylene (10) oleyl ether, polyoxyethylene (100 ) stearyl ether, polyoxyethylene (10) stearyl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (4) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (2) cetyl ether, caprylocaproyl polyoxyl-8 glyceride, polyethylene glycol (20) monostearate, polyethylene glycol (40) stearate, polyethylene glycol (100) stearate, polyethylene glycol (8) stearate, and polyoxyl 40 stearate, and mixtures thereof. The non-ionic surfactant is polysorbate 80,
2. The pharmaceutical composition of claim 1, wherein the aqueous liquid carrier is distilled water, hypertonic saline, or isotonic saline. The pharmaceutical composition according to claim 6, wherein the hypertonic saline is 1% to 7% (w/v) sodium chloride. The non-ionic surfactant is ultra-pure polysorbate 80;
7. The pharmaceutical composition of claim 6, wherein the aqueous liquid carrier is an isotonic saline solution. The pharmaceutical composition according to claim 1, wherein the osmolality of the composition is in the range of 200 to 700 mOsm/kg. The pharmaceutical composition according to claim 1, wherein the osmolality of the composition is in the range of 300 to 400 mOsm/kg. The non-ionic surfactant is present in the range of 0.001% to 5% (v/v) of the total composition; and
2. The pharmaceutical composition of claim 1, wherein the amount of clofazimine ranges from 0.1% to 20% (w/v) of the total composition. A pharmaceutical composition in the form of an inhalable aerosol, comprising:
10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is prepared by aerosolizing the pharmaceutical composition with a nebulizing device selected from an ultrasonic nebulizer, an electrostatically charged spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer, and a mechanical soft mist metered dose inhaler;
A pharmaceutical composition , wherein the aerosol particles produced by said nebulizer device have a mass median aerodynamic diameter of 1 to 5 μm. 1. A system for use in providing antibacterial activity in treating or preventing pulmonary infections caused by mycobacteria or other gram-positive bacteria, the system comprising:
1)
a therapeutically effective dose of clofazimine;
a nonionic surfactant having a hydrophilic-lipophilic balance value of greater than 10;
an aqueous liquid carrier selected from water, isotonic saline, buffered saline, and aqueous electrolyte solutions;
a mucolytic and/or biofilm disrupting agent;
A pharmaceutical composition for administration by inhalation comprising:
2) a nebulizer for atomizing the pharmaceutical composition for administration by inhalation ;
Including,
said clofazimine being present in the form of a suspension ;
The aerosol particles produced by the system have a mass median aerodynamic diameter of 1 to 5 μm; and
The treatment or prevention comprises inhaling the pharmaceutical composition sprayed by the nebulizer.
system. 2. The pharmaceutical composition of claim 1,
20. A pharmaceutical composition for use in a method for the treatment or prevention of a pulmonary infection in a patient in need thereof comprising administering said pharmaceutical composition by inhalation. The pharmaceutical composition according to claim 14 , wherein the infectious disease is caused by a species belonging to the genus Mycobacterium selected from the group consisting of nontuberculous mycobacteria and Mycobacterium tuberculosis and combinations thereof. 16. The pharmaceutical composition of claim 15, wherein the nontuberculous mycobacteria is selected from Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium leprae , and combinations thereof. 16. The pharmaceutical composition of claim 15 , wherein the infection is an opportunistic infection selected from pulmonary MAC disease and nontuberculous mycobacterial disease in patients with cystic fibrosis, chronic obstructive pulmonary disease, or acquired immune deficiency syndrome . 18. The pharmaceutical composition of claim 17 , wherein the infection is an opportunistic nontuberculous mycobacterial infection in a cystic fibrosis patient. 2. The pharmaceutical composition of claim 1,
1. A pharmaceutical composition for use in a method for the treatment or prevention of pulmonary infections caused by mycobacteria or other gram-positive bacteria in a patient in need thereof comprising administering said pharmaceutical composition by inhalation before, simultaneously with, or after administration of an agent selected from bedaquiline or a pharma- ceutical acceptable salt thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para- aminosalicylate, and mixtures thereof . 20. The pharmaceutical composition of claim 19 , wherein the agent is bedaquiline or amikacin. 21. The pharmaceutical composition of claim 20 , wherein the agent is bedaquiline.