CN112804993A

CN112804993A - Compositions containing clofazimine, combinations containing them, processes for their preparation, uses and processes containing them

Info

Publication number: CN112804993A
Application number: CN201980064766.7A
Authority: CN
Inventors: 托马斯·霍夫曼; 斯蒂芬·厄弗; 凯文·斯泰普勒顿
Original assignee: Mankind
Current assignee: Mankind
Priority date: 2018-08-23
Filing date: 2019-04-03
Publication date: 2021-05-14
Also published as: JP7377259B2; CA3109572A1; AU2019324330A1; JP2024012393A; JP2021535124A; WO2020040818A1; KR20210090607A; EP3840737A1

Abstract

The present invention relates to pharmaceutical compositions for inhalation comprising a therapeutically effective dose of clofazimine and processes for their preparation, wherein the clofazimine is provided in the form of a suspension. In addition, the present invention provides a pharmaceutical combination containing clofazimine in the form of an aerosol for pulmonary inhalation. The combinations and compositions provided by the present invention are useful for the treatment and/or prevention of pulmonary fungal infections and pulmonary infections caused by mycobacteria and other gram-positive bacteria.

Description

Compositions containing clofazimine, combinations containing them, processes for their preparation, uses and processes containing them

This application claims priority to U.S. provisional application No. 62/722,048 filed on 23/8/2018, and U.S. provisional application No. 62/796,322 filed on 25/1/2019, both of which are incorporated herein by reference.

Technical Field

The present invention relates to a pharmaceutical composition for inhalation comprising a therapeutically effective dose of clofazimine, wherein the clofazimine is provided in the form of a suspension; processes for their preparation; as well as uses and methods of treatment comprising them. Furthermore, the present invention provides a pharmaceutical combination containing clofazimine in the form of an aerosol for pulmonary inhalation.

The combinations (combinations) and compositions (combinations) provided by the present invention are useful for the treatment and/or prevention of pulmonary fungal infections and pulmonary infections caused by mycobacteria and other gram positive bacteria.

Background

Clofazimine is a very hydrophobic methanophenazine antibiotic with antimycobacterial and anti-inflammatory activity (Log P7.66) and was first described in 1957. The structural formula is as follows:

the exact mechanism by which clofazimine exerts its antimicrobial effect is not known. However, it is known to preferentially bind mycobacterial DNA, thereby inhibiting DNA replication and cell growth. Other proposed mechanisms of action include membrane damage/destabilization, production of membrane-unstable lysophospholipids, interference of potassium transport, and/or intracellular redox cycling. Although clomazemine has significant activity in vitro against Mycobacterium Tuberculosis (MTB), including multidrug resistant strains, until recently it was generally considered ineffective in treating tuberculosis (see, e.g., Cholo M et al, J Antimicrob chemie, 2012Feb,67(2): 290-8).

Clofazimine is one of the three major drugs recommended by the world health organization for the treatment of leprosy caused by Mycobacterium leprae (Mycobacterium leprae), and has been increasingly used in recent years for the treatment of other mycobacterial infections, such as resistant tuberculosis and infections caused by non-tuberculous mycobacteria (NTM).

Clofazimine is almost insoluble in water and exhibits high membrane permeability, and has therefore been classified as a Biopharmaceutical Classification System (BCS) class II drug.

To overcome the problems associated with poor oral absorption and poor bioavailability of drugs, various strategies such as micronization, nanocrystallization, supercritical fluid recrystallization, spray freeze-drying into liquids, solid dispersions and solutions have been employed in the optimization of oral dosage forms.

Being classified as a BCS class II drug, clofazimine is generally considered an ideal drug candidate for formulating formulations as solid dispersions to enhance oral bioavailability (see, e.g., bhushure et al.ijrpc 2014,4(4), 906-918).

In line with this, clofazimine is usually administered as a microcrystalline suspension in an oily wax matrix to improve oral absorption due to its lipophilic nature. Absorption in humans after oral administration varies widely (45-62%). The adverse effects of clofazimine are dose-dependent and affect mainly the skin, eyes, gastrointestinal tract, and QT-prolonging side effects include reddish-brown discoloration of the skin and conjunctiva, which is gradually reversible after withdrawal. They are the result of chronic systemic accumulation.

Mycobacterium (Mycobacterium) is a genus of actinomycetes, which belongs to the genus Mycobacteria, and a genus of Mycobacteriaceae.

Mycobacteria (Mycobacteria) have a characteristic rod-like shape and a waxy outer shell.

Thus, mycobacteria can be divided into three groups:

mycobacterium tuberculosis (Mycobacterium tuberculosis) complex-the causative agent of tuberculosis

Mycobacterium leprae (Mycobacterium leprae) -causative agent of leprosy

Nontuberculous mycobacteria (NTM), other mycobacteria encompassing all nontuberculous mycobacteria or M.leprae, including Mycobacterium abscessus (Mycobacterium abscessus) complex (MABSC), Mycobacterium avium (Mycobacterium avium) complex (MAC).

Tuberculosis (TB) is an infectious disease caused by bacteria of the mycobacterium tuberculosis complex. TB remains a significant cause of death and morbidity worldwide as one of the most well documented human infectious pathogens, with 1040 new cases of Tuberculosis infection estimated in 2015, and 140 million people dying from active TB (see, e.g., World Health Organization (WHO) Global Tuberculosis Report 2016). In addition to the high prevalence and mortality, the incidence of multidrug-resistant tuberculosis (MDR-TB) is also of increasing concern, with 580000 patients presenting with drug-resistant TB infections now existing in 2015. Complications such as Human Immunodeficiency Virus (HIV) complicate treatment and lead to 120 tens of thousands of TB cases in 2015.

To treat multidrug resistance (MDR) infections, the WHO recommends a 9 to 12 month treatment regimen with a second-line anti-TB drug. These regimens (such as the 9 to 12 month Bangladesh regimen), together with a combination of gatifloxacin, ethambutol, pyrazinamide and clofazimine, treat MDR-TB, which results in a relapse-free cure in 87.9% of patients (see, e.g., Sotgiu, G, et al, "Applicability of the short 'Bangladesh region' in high multidrug-resistant tuboculocations settings", International Journal of Infectious Diseases (2017) 56190-.

Other studies that have investigated the reduction in the duration of TB treatment have shown that there is no clinical benefit after 2 weeks of oral clotrimine (see, e.g., Diacon, A.H., et al, "bacterial Activity of Pyrazinamide and Clofazimine acetone and in compositions with pretomanidine and Bedaquiline," American Journal of research and clinical Care Medicine (2015),191(8), 943) 953). The lack of activity is attributed to the low bioavailability of the drug, as it is theorized that the binding affinity of the drug to circulating serum proteins is high. Although Clofazimine has been empirically demonstrated to be effective in the treatment of MDR-TB and widely-resistant Tuberculosis (XDR-TB), its low bioavailability following systemic administration appears to limit its biological activity in short-term treatments (see, e.g., Swanson, R.V., et al, "Pharmacokinetics and Pharmacodynmics of Clofazimine in a Motor Model of Tuberculosis," Antimicrobial Agents and chemotherapeutics (2015),59 (3056), 3042-.

It is known that treatment of lung infections with Inhaled antibiotics causes higher drug concentrations and reduced adverse effects in the lung compared to systemic delivery (see, e.g., Touw, d.j., et al., "inflammation of infectious in circulatory fibrosis", European Respiratory Journal (1995),8, 1594-. In vivo mouse models have demonstrated that aerosol (aerosolized) administration of clofazimine shows a significant improvement in bacillus (bacillus) clearance in TB infection models compared to oral administration of clofazimine alone for 28 days after treatment initiation alone (see, e.g., Verma, r.k., et al, "inert microbial contamination of microbial bacteria in vitro in rice," Antimicrobial Agents and Chemotherapy (2013),57(2), 1050-. The effect of this short-term improvement may be due to direct delivery of clofazimine to the site of infection in the lungs, resulting in higher concentrations of clofazimine in lung macrophages within the tuberculous granulomas.

Thus, the use of clofazimine aerosol administration in MDR-TB or XDR-TB infected patients should further improve the patient's treatment outcome and possibly shorten the duration of the current treatment regimen.

The group of nontuberculous mycobacteria (NTM), previously known as atypical or ubiquitous mycobacteria, comprises over 150 species. NTM is ubiquitous in nature and exhibits a wide variety. They can be detected in soil, ground, drinking water and food like pasteurized milk or cheese. In general, NTM disease is considered to be less pathogenic. However, they can cause severe disease in humans, especially in humans with compromised immune systems or who have previously suffered from pulmonary disease. Currently, NTM is classified according to its growth rate and divided into Slow Growing (SGM) and fast growing (RGM) mycobacteria.

The slow growing Mycobacterium Avium Complex (MAC) includes the following species: mycobacterium avium, chimeric Mycobacterium (Mycobacterium chimaera) and Mycobacterium intracellulare (Mycobacterium intracellulare), all of which are the most important and most common pathogenic NTM. Like Mycobacterium kansasii (Mycobacterium kansasii), Mycobacterium morganii (Mycobacterium malmoense), Mycobacterium bufonis (Mycobacterium xenopi), Mycobacterium similis (Mycobacterium. simiae), Mycobacterium abscessus, Mycobacterium gordonii (Mycobacterium gordonae), Mycobacterium fortuitum (Mycobacterium fortuitum) and Mycobacterium chelonii (Mycobacterium chelona), they mainly cause pulmonary infections. Marine mycobacteria (Mycobacterium marinum) cause skin and soft tissue infections such as aquarium granuloma (aquaria).

In particular, RGM causes severe, life-threatening chronic lung disease and causes disseminated and often fatal infection. Infections are often caused by contaminated materials and invasive procedures involving catheters, non-sterile surgery, or injection and implantation of foreign bodies. Contact with showerheads and whirlpools also has a report of risk of infection. NTM often leads to opportunistic infections in patients with chronic lung diseases such as Chronic Obstructive Pulmonary Disease (COPD), Cystic Fibrosis (CF) and other immune function compromised patients.

In recent years, the following subspecies have been included: fast growing (RGM) Mycobacterium abscessus group strains (Mycobacterium abscessus complex, MABSC) of Mycobacterium abscessus subspecies (m.a. abscessus), Mycobacterium abscessus and Mycobacterium abscessus are becoming important human pathogens and are associated with significantly higher mortality rates than any other RGM disease.

Mycobacterium abscessus infection in CF patients is particularly problematic because it leads to increased lung destruction and is often untreatable with failure rates as high as 60-66% (see, e.g., Obreg)on-Henao A et al,Antimicrobial Agents and Chemotherapy,November 2015,Vol59,No11,p.6904-6912；Qvist,T.,Pressler,T.,

and Katzenstein,TL.,“Shifting paradigms of nontuberculous mycobacteria in cystic fibrosis",Respiratory Research(2014),15(1):pp.41-47)。

Human NTM infection is more associated with the occurrence of the prevalence of human acquired immunodeficiency syndrome. Mycobacteria from the Mycobacterium Avium Complex (MAC) are identified as the major cause of opportunistic infections in patients infected with Human Immunodeficiency Virus (HIV).

Several NTM species are known to form biofilms. Biofilms are small bacterial colonies embedded in an extracellular matrix that provide stability and resistance to human immune mechanisms. In recent years, some species of NTM have been shown to form biofilms, enhancing resistance to disinfection and antimicrobial agents. The assembly of biofilms goes through several stages, including reversible attachment, irreversible attachment, formation of biofilms by bacterial aggregation, tissue and signaling, and finally diffusion. In this process, bacteria form a matrix containing Extracellular Polymeric Substances (EPS), such as polysaccharides, lipids and nucleic acids, to form a complex three-dimensional structure (see, e.g., Sousa s.et al, International Journal of bacteriology 4(2015), 36-43). In particular, mycobacterial EPS is qualitatively different from other biofilms because mycobacteria do not produce exopolysaccharides (see, e.g., Zambrano MM, Kolter R.Mycobacterium bifolims: a great way to hold it to get. cell.2005). Mycobacterial biofilms vary from species to species but may contain mycolic acids, glycopeptide lipids, mycobactyl-diacylglycerols, lipooligosaccharides, lipopeptides and Extracellular DNA (reviewed and originally studied from: Rose SJ, Babraak LM, Bermudz LE (2015) Mycobacterium vacuums strains Exceller DNA that is to Biofilm Format, Structural Integrity, and toll to biology. PLoS ONE). Assembly in biofilms is known to enhance resistance to antimicrobials (see, e.g., Faria s.et al, Journal of Pathogens, Vol 2015, Article ID 809014).

Delivery of aerosolized liposomal amikacin/inhaled amikacin solutions nebulized by a jet nebulizer is suggested as a new method of treating NTM lung infections (Rose s.et al,2014, PLoS ONE, Volume 9, Issue 9, e108703, and Olivier k.et al, Ann Am Thorac Soc Vol 11, No 1, pp.30-35) and Inhalation of anti-TB drug dry powder microparticles is suggested for pulmonary delivery (Cholo M et al, J Antimicrob chemicother.2012feb; 67(2):290-8 and fourier b.and Nettey o.,2015Inhalation Magazine, Verma 2013Antimicrob Agents chemicother).

Initial treatment with parenteral aminoglycosides, tigecycline, and other promising oral antibiotics such as linezolid, delaminanil, and bedaquiline, followed by various combination regimens of inhaled amikacin, and surgical intervention in selected cases, has shown promising results in the treatment of NTM lung disease (Lu Ryu et al. However, the increasing incidence and prevalence of NTM infection, particularly NTM pulmonary disease, and the limited treatment regimens necessitate the development of new dosage forms/pharmaceutical formulations to improve the bioavailability of currently used antibiotics (such as clofazimine). Inhalation can improve efficacy and reduce adverse reactions compared to oral and parenteral treatments.

The combination of Clofazimine and amikacin has shown Synergy against both mycobacterium abscessus and mycobacterium avium In Vitro (see, e.g., van Ingen, j., et al, "In Vitro Synergy between Clofazimine and Am ikacin In Treatment of nonuberculus mycobacteriol Disease," antibiotic Agents and chemitherapy 56(12), 6324. sup. 6327 (2012)). In addition, the synergistic effect of clofazimine and bedaquiline combinations has been shown to be against M.tuberculosis (see, e.g., Cokol, M.et., effective Measurement and differentiation of high-order drug interactions in Mycobacterium tuberculosis, Sciences Advances 2017:3: e170881,11October 2017). The synergistic effect of Clofazimine/Bedaquiline combinations against nontuberculous Mycobacteria abscesses is also shown (Ruth, M.M.et. al., "A Bedaquiline/Clofazimine Combination culture Medium Activity to the Treatment of clinical Reynant Non-Tuberculous Mycobacteria", Journal of antibiotic chemistry (2019), doi.org/10.1093/jac/dky).

Fungal pathogens have become a leading cause of human death. Current evaluations indicate that death due to invasive fungal infections is comparable to more widely known infectious diseases such as tuberculosis. Candida albicans (Candida albicans), Cryptococcus neoformans (Cryptococcus neoformans) and Aspergillus fumigatus (Aspergillus fumigatus) are the most common fungal pathogens in humans. Each of these species causes hundreds of thousands of infections each year, with unacceptably high mortality rates due to poor diagnosis and limited treatment regimens. Chloramphenicol has been shown to be effective against a variety of fungi as a combined agent (see, e.g., Robbins, N., et al, "An antibacterial Combination Matrix ingredients a Ring Pool of An additive organisms at which enzyme Activity against enzyme fungi intermediates Pathologens", Cell Reports 13,1481-1492, November 17,2015). Fungi also function as commensals, colonizers and/or pathogens in cystic fibrosis (see, e.g., Choterimal, S.H.and McElvaney, N.G., "Fungi in The cystic fibrosis lung: Bystans or pathogens.

The low solubility of clofazimine in water results in low oral bioavailability and high microbial resistance, and specific techniques are also needed to dissolve and stabilize drugs formulated in liquid aqueous carriers, such as for aerosolization by a nebulizer, so that aerosol particles achieve more downward lung deposition.

Disclosure of Invention

In one embodiment of the present invention, there is provided a pharmaceutical composition comprising:

(a) a therapeutically effective dose of clofazimine or a pharmaceutically acceptable derivative or salt thereof;

(b) a nonionic surfactant having a hydrophilic-lipophilic balance value of greater than 10; and

(c) an aqueous liquid carrier selected from the group consisting of water, isotonic saline solution, buffered saline solution, and aqueous electrolyte solution,

wherein the clofazimine or a pharmaceutically acceptable derivative or salt thereof is provided in the form of particles in suspension, and

wherein the median particle size of the clofazimine or the pharmaceutically acceptable derivative or salt thereof is less than 5 μm and the D90 is less than 6 μm.

In another embodiment of the invention, the particles of clofazimine or a pharmaceutically acceptable derivative or salt thereof have an average size of less than 2 μm and D90 is less than 3 μm.

In another embodiment of the present invention, there is provided a pharmaceutical composition comprising:

(a) a therapeutically effective dose of clofazimine;

wherein the clofazimine is provided in the form of particles in suspension,

and is

Wherein the particles of clofazimine have a median size of less than 5 μm and a D90 of less than 6 μm.

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

Aerosolization of the compositions of the present invention by a suitable nebulizer provides significantly increased delivery of aerosolized clofazimine into the lower lung (i.e., bronchi, bronchioles and alveoli to the central and lower peripheral lungs), thereby significantly enhancing the therapeutic effect.

Furthermore, the inhalation device should preferably be further adapted for local pulmonary delivery of an aerosol with an optimal particle size distribution for uniform deposition in the lower lung.

Accordingly, the present invention provides an aerosol having aerosol particles of a size that facilitates delivery to the alveoli and bronchioles. Suitable aerodynamic particle sizes for the alveoli and bronchioles are between 1 and 5 μm. Particles larger than this selectively deposit in the upper lungs (i.e. bronchi and trachea) and the mouth and throat (i.e. oropharyngeal region). Thus, the inhalation device is adapted to produce an aerosol having a Mass Median Aerodynamic Diameter (MMAD) in the range of from about 1 to about 5 μm, and preferably in the range of from about 1 to about 3 μm. In another embodiment, the particle size distribution is narrow and the Geometric Standard Deviation (GSD) is less than about 2.5.

Detailed Description

The present invention is based on the unexpected discovery that by pulmonary aerosol administration of clofazimine in suspension, more downward (i.e., deeper) pulmonary deposition of the active agent can be achieved, thereby significantly increasing the bioavailability of the extremely hydrophobic BCS class II agent, which results in significant increased therapeutic efficacy with reduced systemic side effects.

In another aspect, the discovery results in providing improved antibiotic treatment for infections caused by mycobacteria and gram-positive bacteria, particularly NTM lung infections such as opportunistic infections in CF, COPD and immunodeficient patients such as HIV patients.

Furthermore, the present invention aims to overcome the systemic side effects of established oral treatment regimens against gram positive bacterial pulmonary infections, in particular pulmonary TB and NTM infections, as well as to reduce the dose and treatment duration of clofazimine.

It will be understood by those skilled in the art that the present application also discloses each and any combination of the various features disclosed herein.

Definition of

The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compounds of the present invention and which is not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts due to the presence of amino and/or carboxyl groups or similar groups. Pharmaceutically acceptable acid addition salts may 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, and the like. Organic acids from which salts can be derived include, for example, acetic, propionic, naphthoic, oleic, palmitic, pamoic (embonic), stearic, glycolic, pyruvic, oxalic, maleic, malonic, succinic, fumaric, tartaric, citric, ascorbic, glucoheptac, glucuronic, lactic, lactobionic, tartaric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic, and the like.

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, and the like; particularly preferred are ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, histidine, arginine, lysine, benzylaminopyrimidine, N-methyl-glucamine, and ethanolamine, among others. Other acids include dodecyl sulfuric acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, and saccharin.

According to the invention, in addition to the free base, preference is given to using the salts of methanesulfonic acid, maleic acid, isonicotinic acid, nicotinic acid, malonic acid and salicylic acid, particularly preferably clofazimine mesylate.

The term "pharmaceutically acceptable derivative" as used herein, for example, refers to the compounds disclosed in US9,540,336, the disclosure of US9,540,336 being incorporated herein in its entirety. Furthermore, derivatives are described in Lu, Y., Zhen, M., Wang, B., Fu, L., ZHao, W., Li, P., Xu, J., Zhu, H., Jin, H., Yin, D.Huang, H., Upton, AM.AndMa, Z., "clone Analogs with electronic acquisition experience experimental batteries and reduced position for access" Antimicrobial Agents and chemistry (2011),55(11): pp.5185-5193. Furthermore, the term "pharmaceutically acceptable derivative" of a compound is, for example, a prodrug of said compound. Generally, a prodrug is a derivative of a compound that, when administered, provides the active form of the compound. These derivatives may be, for example, esters or amides of carboxyl groups, esters of carboxyl groups of hydroxyl groups, or phosphate esters of hydroxyl groups.

"therapeutically effective amount", "therapeutically effective dose", or "pharmaceutically effective amount" refers to the amount of clofazimine or a pharmaceutically acceptable salt or derivative thereof disclosed herein that has a therapeutic effect. The dose of clofazimine that can be used in the treatment is a therapeutically effective amount. Thus, as used herein, therapeutically effective amounts refer to those amounts of clofazimine that are judged by clinical trial results and/or model animal infection studies to produce the desired therapeutic effect.

The amount and daily dosage of clofazimine can be routinely determined by one of skill in the art and will vary depending on several factors, such as the particular microbial strain involved. The amount may also depend on the patient's height, weight, sex, age and medical history. For prophylactic treatment, a therapeutically effective amount is an amount effective to prevent a microbial infection.

A "therapeutic effect" alleviates one or more symptoms of an infection to some extent and includes curing the infection. By "curing" is meant that the symptoms of active (active) infection are eliminated, and excess members of viable microorganisms, including those involved in the infection, are completely or substantially eliminated, at or below the detection threshold of traditional measurements. However, even after healing, the infection may have some long-term or permanent effects (such as extensive tissue damage). As used herein, "therapeutic effect" is defined as a statistically significant reduction in bacterial load, the appearance of drug resistance, or an improvement in the symptoms of infection in a host as measured by human clinical outcome or animal studies.

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

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

Unless otherwise indicated herein, the term "inhalation" refers to pulmonary inhalation.

The term "infection" as used herein refers to a pulmonary infection, unless otherwise indicated herein.

Unless otherwise indicated, the term "substantially" when used in reference to the purity of a compound means 95% purity or greater of the compound.

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

The term "suitable concentration" refers to the concentration of a component in a composition or combination that provides a pharmaceutically acceptable composition or combination, unless otherwise indicated.

Pharmaceutical compositions and combinations

The following grades of water are particularly suitable for use in the present invention: sterile purified water, sterile water for injection, sterile water for irrigation, sterile water for inhalation (USP) and corresponding grades of water according to e.g. the european pharmacopoeia or the national formulary.

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

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

Chlorine-derived Zincin has been demonstrated to exist in at least four polymorphic forms (see, e.g., Bannigan, et al, "Investigation into the Solution and Solution Properties of Known and Novel Polymorphs of the Antimicrobial biological Molecule Clofazimine", Crystal. growth Des.2016, (16) (12), pp.7240-7250). Clofazimine may exist in the triclinic form FI, the monoclinic form FII, and the orthorhombic form FIII. Another form of FIV is visible only at high temperatures.

Thus, in a further embodiment of the present invention, there is provided a pharmaceutical composition comprising:

(a) a therapeutically effective dose of clofazimine;

wherein the clofazimine is provided in the form of particles in suspension,

and is

Wherein the particles of clofazimine have a median size of less than 5 μm and a D90 of less than 6 μm, preferably a median size of less than 2 μm and a D90 of less than 3 μm, and wherein the clofazimine is provided in one or more polymorphic forms selected from the group consisting of triclinic FI, monoclinic FII and orthorhombic FIII, and mixtures of these forms. In another embodiment, clofazimine is provided substantially in the 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 selected from polysorbate 20 (e.g., polysorbate 20)

20) Polysorbate 60 (e.g. sodium chloride)

60) Polysorbate 80 (e.g. Polysorbate)

80) Stearyl alcohol, polyethylene glycol derivatives of hydrogenated castor oils having a hydrophilic-lipophilic balance of from 14 to 16 (for example

RH 40), polyethylene glycol derivatives of hydrogenated castor oil with a hydrophilic lipophilic balance of 15 to 17 (e.g. polyethylene glycol derivatives of hydrogenated castor oil with a hydrophilic lipophilic balance of 15 to 17

RH 60), sorbitol monolaurate (e.g. R60)

20) Sorbitol monopalmitate (e.g. sorbitol monopalmitate)

40) Sorbitol monostearate (e.g. sorbitol monostearate)

60) Polyoxyethylene (20) oleyl alcohol ether (e.g. methanol)

020) Polyoxyethylene (20) cetyl ether (e.g. polyethylene oxide)

58) Polyoxyethylene (10) cetyl ether (e.g. polyethylene oxide)

C10) Polyoxyethylene (10) oleyl alcohol ether (e.g. methanol)

O10), polyoxyethylene (100) stearyl ether (e.g., ethyl stearyl ether

S100), polyoxyethylene (10) stearyl ether (e.g.

S10), polyoxyethylene (20) stearyl ether (e.g. polyoxyethylene

S20), polyoxyethylene (4) lauryl ether (e.g. sodium lauryl ether)

L4), polyoxyethylene (20) cetyl ether (e.g. polyethylene oxide)

93) Polyoxyethylene (2) cetyl ether (e.g. polyethylene oxide)

S2), caprylocaproyl polyoxy-8 glycerides (e.g. caprylocaproyl polyoxy-8 glycerides)

) Polyethylene glycol (20) stearate (e.g. Myrj)^TM49) Polyethylene glycol (40) stearate (e.g. Myrj)^TMS40), polyethylene glycol (100) stearate (e.g. Myrj)^TMS100), polyethylene glycol (8) stearate (e.g. Myrj)^TMS8) and polyethylene glycol 40 stearate (e.g., Myrj)^TM52) And mixtures thereof.

In another embodiment of the present invention, there is provided a pharmaceutical composition according to any of the composition embodiments described herein, wherein the non-ionic surfactant is polysorbate 80 and wherein the aqueous liquid carrier is distilled water, a hypertonic salt solution, or an isotonic salt solution. In another embodiment of the present invention, a pharmaceutical composition is provided wherein the hypertonic saline solution is 1% to 7% (w/v) sodium chloride. In a further embodiment of the invention, there is provided a pharmaceutical composition wherein the non-ionic surfactant is ultra pure polysorbate 80 (e.g. polysorbate 80(Hx2) by NOF) and wherein the aqueous liquid carrier is an isotonic saline solution.

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 osmolality of the composition is in the range of 200-700 mOsm/kg. In another embodiment, the osmolality of the composition is in the range of 300-400 mOsm/kg.

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

In another embodiment of the present invention, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the pharmaceutical composition is prepared by a process comprising the steps of:

(1) homogenizing a suspension of clofazimine, a nonionic surfactant and water to obtain a suspension containing clofazimine of a suitable particle size,

(2) adjusting the pH of the suspension obtained in (1) to a pH between pH 5.5 and pH 7.5, and

(3) adjusting the sodium chloride concentration to a suitable concentration, an

(4) Osmolality was adjusted to the appropriate level.

In a further embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154mM sodium chloride. In another embodiment, the homogenization in step (1) is performed by high pressure homogenization, high shear homogenization, wet milling, ultrasonic homogenization, or a combination of these methods. In another aspect, the homogenization of clofazimine is performed in multiple homogenization steps. In another embodiment, clofazimine of a suitable particle size is a particle having an average size of less than 5 μm and D90 of less than 6 μm. In a further embodiment, clofazimine of a suitable particle size is a particle having an average size of less than 2 μm and 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, wherein the pharmaceutical composition is prepared by a process comprising the steps of:

(1) homogenizing the suspension of clofazimine and non-aqueous liquid to obtain a suspension containing clofazimine of suitable particle size,

(2) separating the clofazimine to obtain the clofazimine,

(3) adding clofazimine into a nonionic surfactant and water,

(4) adjusting the pH of the suspension obtained in (3) to a pH between pH 5.5 and pH 7.5, and

(5) the sodium chloride concentration was adjusted to a suitable concentration.

In a further embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154mM sodium chloride. In a further embodiment, the homogenization in step (1) is performed by high pressure homogenization, high shear homogenization, wet milling, ultrasonic homogenization or a combination of these methods. In another embodiment, the homogenization of clofazimine is performed in multiple homogenization steps. In another embodiment, clofazimine of a suitable particle size is a particle having an average size of less than 5 μm and D90 of less than 6 μm. In a further embodiment, clofazimine of a suitable particle size is a particle having an average size of less than 2 μm and D90 of less than 3 μm.

In a further embodiment, there is provided a pharmaceutical composition according to any of the composition embodiments described herein, wherein the composition is prepared by a process comprising the steps of:

(1) micronizing the clofazimine to obtain clofazimine with a proper particle size,

(2) adding clofazimine into a nonionic surfactant and water,

(3) adjusting the pH of the suspension obtained in (2) to a pH between pH 5.5 and pH 7.5, and

(4) the sodium chloride concentration was adjusted to a suitable concentration.

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

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

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

In another embodiment, there is provided a method of preparing a pharmaceutical composition according to any of the composition embodiments described herein, comprising the steps of:

(3) adjusting the sodium chloride concentration to a suitable concentration, an

(4) Osmolality was adjusted to the appropriate level.

In another embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154mM sodium chloride. In further embodiments, homogenization is performed by high pressure homogenization, wet milling, ultrasonic homogenization, or a combination of these methods. In a further embodiment, the homogenization of clofazimine is performed in multiple homogenization steps. In a further embodiment, clofazimine of a suitable particle size is a particle having an average size of less than 5 μm and D90 of less than 6 μm. In a further embodiment, clofazimine of a suitable particle size is a particle having an average size of less than 2 μm and D90 of less than 3 μm.

In another embodiment, a process for preparing any of the pharmaceutical composition embodiments described herein is provided, wherein,

(2) separating the clofazimine to obtain the clofazimine,

(3) adding clofazimine into a nonionic surfactant and water,

(5) the sodium chloride concentration was adjusted to a suitable concentration.

In a further embodiment, there is provided a method of preparing a pharmaceutical composition according to any of the pharmaceutical composition embodiments described herein, comprising the steps of:

(2) adding clofazimine into a nonionic surfactant and water,

(4) the sodium chloride concentration was adjusted to a suitable concentration.

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

In another embodiment, there is provided a method of preparing a pharmaceutical composition according to any of the composition embodiments described herein, comprising homogenizing a suspension of clofazimine in water containing a suitable concentration of sodium chloride, a non-ionic surfactant, and the pH of the suspension has been adjusted to a 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 a suitable concentration of sodium chloride is 154mM sodium chloride. In further embodiments, homogenization is performed by high pressure homogenization, wet milling, ultrasonic homogenization, or a combination of these methods. In a further embodiment, the homogenization of clofazimine is performed in multiple homogenization steps. In a further embodiment, clofazimine of a suitable particle size is a particle having an average size of less than 5 μm and D90 of less than 6 μm. In a further embodiment, clofazimine of a suitable particle size is a particle having an average size of less than 2 μm and D90 of less than 3 μm.

In a further embodiment, there is provided a method of preparing a pharmaceutical composition according to any of the composition embodiments described herein, comprising the steps of: (a) homogenizing a suspension of clofazimine, a nonionic surfactant and water to obtain a suspension containing clofazimine of a suitable particle size; (b) adjusting the pH of the resulting suspension to a pH between pH 5.5 and pH 7.5; (c) adjusting the sodium chloride concentration to a suitable concentration, and (d) adjusting the osmolality to a suitable level; and wherein, steps (b), (c) and (d), may be (b), (c), (d); (b) (d), (c); (c) (b), (d); (c) (d), (b); (d) (b), (c); or (d), (c) and (b) occur sequentially.

In another embodiment, there is provided a method of preparing a pharmaceutical composition according to any of the composition embodiments described herein, comprising the steps of: (a) homogenizing the suspension of clofazimine and non-aqueous liquid to obtain a suspension containing clofazimine of suitable particle size; (b) separating clofazimine; (c) adding clofazimine to a nonionic surfactant and water; (d) adjusting the pH of the resulting suspension to a pH between pH 5.5 and pH 7.5; and (e) adjusting the sodium chloride concentration to a suitable concentration; and wherein steps (d) and (e) may be (d), (e); or (e) and (d) occur sequentially.

In another embodiment, there is provided a method of preparing a pharmaceutical composition according to any of the composition embodiments described herein, comprising the steps of: (a) micronizing clofazimine to obtain clofazimine of suitable particle size, and (b) adding clofazimine to a nonionic surfactant, water containing a suitable concentration of sodium chloride, and adjusted to a pH between pH 5.5 and 7.5.

In another embodiment of the present invention, there is provided a pharmaceutical combination in aerosol form for inhalation prepared by nebulizing a composition according to any of the composition embodiments described herein using a nebulizing device selected from the group consisting of an ultrasonic nebulizer, an electrospray nebulizer, a vibrating membrane nebulizer, a jet nebulizer, and a mechanical soft mist inhaler, and

wherein the mass median aerodynamic diameter of the aerosol particles produced by the nebulisation means is from 1 to 5 μm. In a further embodiment, the aerosol for inhalation is for lower lung deposition. In another embodiment, the atomizing device exhibits an output rate of 0.1 to 1.0 ml/min. In another embodiment, the total inspired volume is between 1ml to 5 ml.

In another embodiment, there is provided a pharmaceutical composition according to any of the composition embodiments described herein for use in combination with an agent that disperses and/or disrupts biofilm, a mucolytic and/or mucoactive agent, and/or an agent that reduces biofilm formation selected from the group consisting of: nebulized 4-7% hypertonic saline solution, metaperiodate, sodium lauryl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth thiols (bismuths), ethylenediaminetetraacetic acid, gentamicin-loaded phosphatidylcholine-modified gold nanoparticles, chelating agents, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides. In another embodiment, the composition for use is administered before, simultaneously with or after the administration of an agent selected from the group consisting of bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and p-aminosalicylate and mixtures thereof.

In another embodiment, there is provided a pharmaceutical combination according to any of the combination embodiments described herein for use in combination with an agent for dispersing and/or disrupting biofilm, a mucolytic and/or mucoactive agent, and/or an agent for reducing biofilm formation selected from the group consisting of: nebulized 4-7% hypertonic saline solution, metaperiodate, sodium lauryl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth mercaptane, ethylene diamine tetraacetic acid, gentamicin-loaded phosphatidylcholine-modified gold nanoparticles, chelating agents, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides. In another embodiment, the combination for said use is for administration of a combination of the compositions of the invention before, simultaneously with or after administration of an agent selected from the group consisting of bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampicin, moxifloxacin, levofloxacin and p-aminosalicylate and mixtures thereof. In another embodiment, the composition is administered prior to, simultaneously with, or after administration of an agent selected from the group consisting of bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and acamicin, and mixtures thereof. In further embodiments, the composition is administered prior to, simultaneously with, or after the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.

In another embodiment, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein for use in the treatment and/or prevention of a pulmonary infection caused by mycobacteria or other gram-positive bacteria. In a further embodiment, the infection is caused by a species of mycobacterium selected from the group consisting of nontuberculous mycobacteria and Mycobacterium tuberculosis complex and combinations thereof. In a further embodiment, the non-tuberculous mycobacterium is selected from the group consisting of mycobacterium avium, mycobacterium intracellulare, mycobacterium abscessus, and mycobacterium leprae and combinations thereof. In another embodiment, the infection is an opportunistic infection selected from the group consisting of MAC lung disease and non-tuberculous infection in a patient with cystic fibrosis, chronic obstructive pulmonary disease, or acquired immunodeficiency syndrome. In another embodiment, the infection is an opportunistic nontuberculous mycobacterial infection in cystic fibrosis patients. In another embodiment, the composition for use is administered prior to, simultaneously with, or subsequent to the administration of an agent of bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and p-aminosalicylate, and mixtures thereof. In another embodiment, the composition is administered prior to, simultaneously with, or after administration of an agent selected from the group consisting of bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and acamicin, and mixtures thereof. In further embodiments, the composition is administered prior to, simultaneously with, or after the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.

In another embodiment, a pharmaceutical combination according to any of the combination embodiments described herein is provided for the treatment and/or prevention of a pulmonary infection caused by mycobacteria or other gram-positive bacteria. In a further embodiment, the infection is caused by a species of mycobacterium selected from the group consisting of nontuberculous mycobacteria and Mycobacterium tuberculosis complex and combinations thereof. In a further embodiment, the non-tuberculous mycobacterium is selected from the group consisting of mycobacterium avium, mycobacterium intracellulare, mycobacterium abscessus, and mycobacterium leprae and combinations thereof. In another embodiment, the infection is an opportunistic infection selected from the group consisting of MAC lung disease and non-tuberculous infection in a patient with cystic fibrosis, chronic obstructive pulmonary disease, or acquired immunodeficiency syndrome. In another embodiment, the infection is an opportunistic nontuberculous mycobacterial infection in cystic fibrosis patients. In another embodiment, the combination for said use is for administration of a composition of the invention before, simultaneously with or after administration of an agent selected from the group consisting of bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and p-aminosalicylate and mixtures thereof. In another embodiment, the combination for said use is for administering a composition of the invention before, simultaneously with or after the administration of an agent selected from the group consisting of bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and acamicin, and mixtures thereof. In another embodiment, the combination for said use is for administration of a composition of the invention before, simultaneously with or after the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.

In another embodiment, a system is provided for providing antibiotic activity in treating or providing prophylaxis against a pulmonary infection caused by a mycobacterium or other gram-positive bacterium, wherein the system comprises:

1) an aerosolized pharmaceutical combination comprising:

(a) a therapeutically effective dose of clofazimine;

and

2) an atomizer is arranged on the bottom of the water tank,

wherein the clofazimine is provided in the form of a suspension,

and is

Wherein the mass median aerodynamic diameter of the aerosol particles produced by the nebulisation means is from 1 to 5 μm.

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

In a further embodiment, a pharmaceutical combination according to any one of the combination embodiments described herein is provided for use in the treatment and/or prevention of a pulmonary fungal infection or clostridium difficile or a combination thereof. There is provided a combination of drugs 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 or aspergillus fumigatus or a combination thereof.

In another embodiment, there is provided a method of treating or preventing a pulmonary infection in a patient in need thereof, comprising administering by inhalation a composition according to any of the composition embodiments described herein. In another embodiment, the infection is caused by a species of mycobacterium selected from the group consisting of nontuberculous mycobacteria and Mycobacterium tuberculosis complex and combinations thereof. In another embodiment, the non-tuberculous mycobacterium is selected from the group consisting of mycobacterium avium, mycobacterium intracellulare, mycobacterium abscessus, and mycobacterium leprae and combinations thereof. In a further embodiment, the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient with cystic fibrosis, chronic obstructive pulmonary disease or acquired immunodeficiency syndrome. In another embodiment, the infection is an opportunistic nontuberculous mycobacterial infection in cystic fibrosis patients.

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

Particle size and distribution

The therapeutic effect of aerosolized therapy depends on the deposited dose and its distribution. Aerosol particle size is one of the important variables that define the lung deposition dose and the aerosol distribution of the drug.

Generally, inhaled aerosol particles deposit by one of two mechanisms: impaction (usually dominating larger aerosol particles) and sedimentation (dominating smaller aerosol particles). Collisions occur when the momentum of inhaled aerosol particles is large enough that the particles do not follow the airflow and encounter physiological surfaces. In contrast, when very small aerosol particles traveling with the inhaled air stream sink due to gravity to encounter physiological surfaces, settling occurs primarily in the lower lung.

Pulmonary drug delivery can be achieved by inhalation of aerosols through the mouth and throat. Aerosol particles having an aerodynamic diameter greater than about 5 μm do not typically reach the lungs; instead, they tend to impact the back of the throat and are swallowed and may be absorbed orally. Aerosol particles of about 3 to about 5 μm in diameter are small enough to reach the upper to middle lung region (conducting airways), but too large to reach the alveoli. Smaller aerosol particles, i.e., about 0.5 to about 3 μm, are able to reach the alveolar region. Aerosol particles less than about 0.5 μm in diameter tend to be exhaled during tidal (tidal) breathing, but may also be deposited in the alveolar region by breath-holding.

The aerosols used in pulmonary drug delivery consist of aerosols of various particle sizes, and therefore statistical descriptors are used. Aerosols used in pulmonary drug delivery are generally described by their Mass Median Diameter (MMD), i.e., half of the mass is contained in aerosol particles larger than the MMD and half of the mass is contained in aerosol particles smaller than the MMD. For particles of uniform density, Volume Median Diameter (VMD) may be used interchangeably with MMD. VMD and MMD were determined by laser diffraction. The width of the distribution is described by the Geometric Standard Deviation (GSD). However, deposition of aerosol particles in the respiratory tract is more accurately described by the aerodynamic diameter of the particles, and therefore mass median aerodynamic diameters are typically used. MMAD determinations are made by inertial impaction or time-of-flight measurements. For aqueous particles, VMD, MMD and MMAD should be the same. However, if humidity is not controlled when the aerosol passes through the impactor, the MMAD measurement will be less than MMD and VMD due to dehydration. For purposes of description, the VMD, MMD and MMAD measurements are considered to be under controlled conditions so that the descriptions of VMD, MMD and MMAD will be comparable.

Nevertheless, for the purposes of description, the aerosol particle size of the aerosol particles will be given in MMAD as measured at room temperature with a Next Generation Impactor (NGI) according to the usp convention. Disclosed in Process review <601> Aerools, Nasal Sprays, used-Dose Inhalers, and Dry Powder Inhalers, Pharmacopeial Forum (2003), Volume Number 29, pages 1176-.

According to the invention, the particle size of the aerosol is optimized to maximize the deposition of clofazimine at the site of infection and to maximize tolerability. Aerosol particle size can be expressed in terms of Mass Median Aerodynamic Diameter (MMAD). Large particles (e.g., MMAD > 5 μm) tend to deposit in the extrathoracic and upper airways because they are too large to pass in bends in the airways. Intolerance (e.g., cough and bronchospasm) may be caused by large particle deposition in the upper airway.

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

However, a guided breathing strategy may be employed that allows larger particles to pass through the extrathoracic and upper airways and deeper into the lungs than when tidal breathing, which will increase aerosol deposition in the middle and lower lungs. The pilot breathing strategy may be as slow as 100 ml/min. Therefore, when used with a guided breathing strategy, the preferred MMAD of the aerosol should be less than about 10 μm.

Another factor of equal importance (in addition to aerosol particle size) is the particle size and size distribution of the solid particles, in this case clofazimine. The size of the solid particles in a given aerosol particle must be smaller than the aerosol particle in which it is contained. Larger aerosols may contain one or more solid particles. Furthermore, when handling dilute suspensions, a majority of the aerosol particles may not contain solid particles.

Thus, it is desirable that the solid drug particles be significantly smaller than the MMAD of the aerosol particles. For example, if the MMAD of the aerosol particles is 3 μm, then the desired solid particles are 1 μm or less.

Another consideration is that, for example, using a vibrating mesh nebulizer, the formulation is pumped through the holes in the plate, breaking the suspension into droplets. Thus, the solid particles must also be smaller than these pores to pass through.

The solid particle size in the suspension can be given by the average size of the particles and the distribution of the particles. The D90 value indicates that 90% of the particles in the suspension have an average size or less.

Atomizer

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 employ jet technology and use compressed air to produce a liquid aerosol. Such devices are available from, for example, health Technologies, inc; invacare, Inc.; mountain Medical Equipment, inc; pari rapidity, inc; mada Medical, inc; Puritan-Bennet; schuco, inc., DeVilbiss Health Care, inc.; and Hospitak, Inc. Ultrasonic nebulizers rely on mechanical energy in the form of piezoelectric crystal vibration to produce respirable droplets and are commercially available from, for example, Omron Heathcare, Inc. and DeVilbiss Health Care, Inc. Vibrating mesh nebulizers rely on piezoelectric or mechanical pulses to produce respirable droplets. Other examples of atomizers for use with the clofazimine described herein are described in U.S. Pat. Nos. 4,268,460; 4,253,468, respectively; 4,046,146, respectively; 3,826,255, respectively; 4,649,911, respectively; 4,510,929, respectively; 4,624,251, respectively; 5,164,740, respectively; 5,586,550, respectively; 5,758,637, respectively; 6,644,304, respectively; 6,338,443, respectively; 5,906,202, respectively; 5,934,272, respectively; 5,960,792, respectively; 5,971,951, respectively; 6,070,575, respectively; 6,192,876, respectively; 6,230,706, respectively; 6,349,719, respectively; 6,367,470, respectively; 6,543,442, respectively; 6,584,971, respectively; 6,601,581; 4,263,907, respectively; 5,709,202, respectively; 5,823,179, respectively; 6,192,876, respectively; 6,644,304, respectively; 5,549,102, respectively; 6,083,922, respectively; 6,161,536, respectively; 6,264,922; 6,557,549, respectively; and 6,612,303, which are incorporated herein by reference in their entirety. Commercial examples of atomizers that can be used with the clofazimine compositions described herein include Respirgard, produced by Aerogen

Pro and

go; produced by Aradigm

And AERx Essence^TM(ii) a Manufactured by Respironics, inc

Freeway Freedom ^TMSidesstream, Ventstream and I-neb; and PARI produced by PARI GmbH

PARI

And e-Flow7 m. A further non-limiting example is disclosed in US 6,196,219.

According to the present invention, the pharmaceutical composition may preferably be nebulized using a nebulizing device selected from the group consisting of an ultrasonic nebulizer, an electronic spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer or a mechanical soft mist inhaler.

Preferably, the device controls the patient's inspiratory flow rate through an electrical or mechanical process.

In another preferred embodiment, the device is triggered to generate an aerosol by inhalation by the patient, such as with an AKITA device.

Preferred (commercially available) examples of the above-described atomizers/devices for use in accordance with the present invention are Vectura fox, Pari eFlow, Pari Trek S, Philips Innospire mini, Philips Innospire Go, Medspray device, Aeroneb Go, Aerogen Ultra, Respironics aerobe, Akita, Medspray ecomlst, and Respimat.

For the treatment and/or prophylaxis of

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

Administration of clofazimine (dosing)

According to the invention, the pharmaceutical composition is delivered by nebulization of about 1-5ml, preferably 1-2ml, of the pharmaceutical composition of the invention.

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

The daily lung dose of clofazimine administered according to the invention, i.e. the dose deposited in the lungs, is about 5-10mg, which corresponds to a nominal dose (device dose) of 15-30mg in case of mycobacterium abscessus infection.

It will be appreciated that the skilled person will routinely adjust the pulmonary dose of clofazimine to be administered (and hence the fill/nominal dose/volume to be aerosolized) based on the Minimum Inhibitory Concentration (MIC) of clofazimine for the corresponding bacterial strain as is well known in the art.

Depending on the frequency of administration (once or twice daily), daily lung doses will be assigned accordingly.

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

It will be apparent to those skilled in the art that the above amounts relate to clofazimine free base and that the dosages of the derivatives and salts must be adjusted accordingly to the MIC of the corresponding compound and strain.

Mucolytic and/or biofilm modifying agents

In order to reduce sputum viscosity and disrupt existing biofilm during aerosol treatment, treatment and/or prevention according to the present invention may include additional administration of mucolytic and/or biofilm disrupting agents.

These agents may be prepared in fixed combination form or administered simultaneously or subsequently to the clofazimine-containing pharmaceutical composition/aerosol combination according to the invention.

The agent for dispersing/disrupting biofilm, mucolytic and/or mucoactive agent and/or agent that reduces biofilm formation used according to the invention is selected from: nebulized 4-7% hypertonic saline solution, metaperiodate, sodium lauryl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth mercaptane, ethylene diamine tetraacetic acid, gentamicin-loaded phosphatidylcholine-modified gold nanoparticles, chelating agents, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides.

In addition, other pharmaceutically active agents may also be used in combination with the pharmaceutical composition/aerosol combination of the present invention. Such active agents may be selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and p-aminosalicylate, and mixtures thereof.

These agents may be prepared in a fixed combination, or administered prior to, simultaneously with, or subsequently to the clofazimine-containing pharmaceutical composition/aerosol combination according to the invention.

Examples

The present invention is described and the best mode contemplated for carrying out various aspects of the present invention is set forth. Embodiments in accordance with the present invention are those which fall within the scope of the claims herein.

Experiment of

The following exemplary compositions and combinations are prepared according to the methods described herein.

Example 1

200mg of clofazimine (in triclinic form I), 90mg of sodium chloride and 9.5ml of water were mixed twice in an Ultra-Turrax homogenizer at a speed of 10000rpm, 5 minutes each time. 0.5ml of polysorbate 80(NOF Hx2) was added. An ultrasonic probe (Branson Digital Sonifier) was used^TM250D in conjunction with Bandelin Sonoplus Probe MS73) seven times with an amplitude of 70% for 3 minutes each. The volume was adjusted to 10ml with water. The suspension was filtered through VWR fold qualitative filter paper (303, particle retention 5-13 μm, size: 150mm) to give the composition of example 1. The median particle size of clofazimine for the composition of example 1 was 3.9 μm and D90 was 6.7 μm. The concentration of clofazimine was measured by 280nm uv/vis spectrophotometry, calibrated using a stock solution of clofazimine (stock) diluted in mobile phase at 1mg/ml, and found to be 7.16 mg/ml.

The compositions of example 1 are shown in Table 1

TABLE 1

Preparation of orthorhombic III-type clofazimine

A slurry of clofazimine (10g) in toluene (20ml) was stirred in an oil bath at 40 ℃ for 72 hours at 800rpm with a magnetic stirrer. The solid portion of the slurry was collected by crucible filtration and dried in a vacuum oven at a maximum temperature of 40 ℃. 8.64g of substantially pure (98%) clofazimine in orthorhombic form III was obtained.

Example 2

6g of clofazimine in orthorhombic form III was added in 100ml of water containing 0.5% polysorbate 80(NOF Hx2) and 0.6% sodium chloride and pre-micronized using Ultra-Turrax at 10000rpm for about 40 seconds. A pre-formulation was prepared by adding 0.6% sodium chloride to water to give a volume of 300 ml. 300ml of this suspension was added to the inlet of a homogenizer (M-110EH-30 microfluidizer (Microfluidics, Westwood, Mass., USA)) and a pre-homogenization step was performed by circulating the suspension through the H30Z chamber at 5000psi for 15 minutes. Subsequently, a second H10Z chamber was installed in series with the first chamber and the suspension was further homogenized for 23 minutes at 25000 psi. Particle size analysis using HORIBA LA 950 showed a median particle size of 0.83 μm and a D90 value of 1.17 μm. The concentration of the clofazimine is 16.05mg/ml through 280nm ultraviolet/visible spectrum measurement by using the calibration of a 1mg/ml clofazimine stock solution diluted by a mobile phase.

The compositions of example 2 are shown in Table 2

TABLE 2

Example 3

The composition of example 3 was produced in the H30Z-G10Z configuration using a suspension of clofazimine (crystal modified orthorhombic form III) in water, sodium chloride and polysorbate 80 solution treated with M-110EH-30 microfluidizer processor (chambers: H30Z and G10Z) operating at 28250psi for 30 minutes, resulting in clofazimine particles with a median particle size of 1.28 μ M and D90 below 2 μ M.

The composition of example 3 is shown in Table 3

TABLE 3

Viscosity measurement

The viscosity of the composition of example 3 was tested using an STRESSTECH rheometer in stress control mode. A double gap geometry is used and the spindle is rotated continuously to ensure that the particles remain suspended during the temperature point. Viscosity measured at 20 ℃,25 ℃ and 30 ℃ respectively across 0.01, 0.05 and 0.1Pa stress. Two separate loads were performed to obtain the average viscosities shown in table 4 below.

TABLE 4

Animal models and efficacy testing

The compositions of the present invention have been tested for their ability to inhibit the growth of clinical NTM species in a mouse model of acute in vivo pulmonary infection to obtain preliminary data to establish clofazimine concentration levels in lung tissue following direct respiratory delivery as opposed to systemic administration. Two separate mouse models were used to study lung NTM infection depending on the bacterial species of interest. In the tests, strains of M.avium 2285 and M.abscessus 103 were used (see filing of strains details (9 months 2014) to "Phylogenetic analysis of Mycobacterium species using the world genes" Hazbon M.H., Riojas M.A., Damon A.M., Alalade R., Cantwell B.J., Monaco A., King S., Sohrabi A.) of the EMBL/GenBank/DDBJ database). These two were previously used in the literature as Models for NTM infection (Obregon-Henao et al 2015 antisense Agents Chemother; and Chan et al animal Models of Non-Tuberculus Mycobacterium Infections, Mycobacterium Dis 2016).

In vivo safety studies in Balb/C mice:

to ensure in vivo safety and tolerability, 6-8 week old Balb/C female mice were obtained from Charles River. Mice were allowed to rest for one week prior to dosing. For each clofazimine dose, three healthy mice were given a total of three doses every other day. At 10.0, 5.01 and 2.51mg/kg of clofazimineThe composition of example 1 was administered to mice. By passing

Aerosol intratracheal administration, 3 healthy mice were dosed with these compounds, 3 doses every other day.

Clofazimine was found to be safe at 20mg/kg doses (gavage, 200 μ l). The composition of example 1 showed no toxicity at the highest dose tested (10.0 mg/kg; 35 μ l intratracheal 0.2506 mg/dose). Thus, at a dose of 10.0mg/kg, the formula I composition is considered safe and well tolerated.

Determination of the minimum inhibitory concentration

By using

Hinton (mh) broth (cation adjustment) Minimal Inhibitory Concentration (MIC) testing was performed by microbhank (microboth) dilution with calcium and magnesium concentrations to the concentrations recommended in CLSI standard M7-a7(Becton Dickinson). MIC testing was also performed by microtissues dilution using 7H9 broth (Sigma-Aldrich). The reason for compound screening using MH and 7H9 broths was that anti-mycobacterial compounds have been shown to exhibit different MIC activities, depending on the broth used in the MIC assay. M. abscessus was grown in ambient air at 35-37 ℃ for 3 days (depending on the bacterial strain) on 7H11 agar plates (Sigma-Aldrich), and M.avium was grown in ambient air at 37 ℃ for 21-30 days on 7H11 agar plates (Sigma-Aldrich).

Colony Forming Units (CFU) were removed from agar plates and placed in MH or 7H9 broth containing 0.05% tween-80 and grown in ambient air at 35-37 ℃ until the Optical Density (OD) absorbance measured after 3 days of growth (M. abscessus) or 12 days (M. avium) was 0.08-0.1(0.5McFarland standard). Confirmation was then made by preparing a bacterial cell suspension in saline solution, matching (OD)0.08-0.1(0.5McFarland standard). Stock solutions of compounds were prepared by suspending the compounds in DMSO at a concentration of 1.28mg/ml and immediately used to test the range 64-0.062 pg/ml. Thereafter, 180. mu.l of the broth (MH or 7H9) was added to the first column of the 96-well plate, and 100. mu.l of the broth was added to the remaining columns of the 96-well plate. Mu.l of the compound stock was added to the wells of the first column and diluted continuously. Finally, 100 μ l NTM cell suspension was added to all wells except the medium only control well. QC reagents specific to each organism 1) bacteria only negative control 2) media only negative control 3) clarithromycin positive drug control.

M. abscessus was analyzed for OD on the third day, while M.avium was analyzed for OD on day 12. After these measurements, the plates were analyzed using the resazurin microtiter plate assay. Briefly, the method uses addition of resazurin (7-hydroxy-3H-phenothiazin-3-one 10-oxide) to 96-well plates. Resazurin is a blue dye that itself is weakly fluorescent until irreversibly reduced to pink and highly red fluorescent resorcinol. In MIC assays, it is used as a redox indicator to determine the viability of bacterial cells.

The analysis was performed in triplicate. The composition of example 1 was stored at 4 ℃ for 2 months and then analyzed for #1, #2 at 4 months and #3 at 5 months.

Minimum inhibitory concentration in the Presence and absence of CF sputum

The minimum inhibitory concentration analysis was performed as described above.

To investigate the effect of Cystic Fibrosis (CF) patient sputum on the antimicrobial activity of Clofazimine (CFZ) and the composition of example 1, sputum was collected from patients who had not received antibiotic treatment within the previous 48 hours and disinfected by exposure to uv light to clear endogenous bacteria. After sterilization, mycobacterium abscessus, mycobacterium avium, mycobacterium intracellulare and chimeric mycobacterium were cultured in 10% CF sputum prior to MIC testing. The MIC of the composition of example 1 was measured according to the same CLSI protocol as described above in the presence and absence of cystic fibrosis patient sputum. All studies were performed in duplicate.

Table 5 shows MIC values for clofazimine and the composition of example 1 in the presence and absence of sputum.

TABLE 5

The results in Table 5 show that the MICs of clofazimine and the composition of example 1 are consistent for a range of non-tuberculous mycobacterial species.

These data indicate that the composition of example 1 shows effective in vitro activity against mycobacterium abscessus and mycobacterium avium and is stable for at least this period of time.

Mouse model of mycobacterium abscessus in SCID mice

SCID female mice 6-8 weeks old were ordered from Charles River. Mice were rested one week prior to infection.

Prior to use, stock solutions of M.abscessus strain 103 were frozen in 1ml aliquots and stored at-80 ℃. For infection, aliquots were thawed, broken 20 times with a 1ml luer-lok syringe fitted with a 26g needle, and diluted in sterile 1 × PBS.

The acute SCID mouse model received 1X 10⁶Non-invasive intratracheal instillation of lung infections in CFU/mouse (M.abscessus strain 103).

3 mice were sacrificed 1 day post infection and bacterial uptake was determined. Whole lung, spleen and liver were extracted and homogenized in 4.5ml of 1 x PBS. Homogenization serial dilutions were made at a dilution of 1:10 and dilutions (0-1-2-3-4-5-6-7) were placed on 7H11 agar plates. The plates were placed in a 32 ℃ dry air incubator (depending on the strain) for 7 days.

The composition of example 1, 10.0mg/kg, was administered by the pulmonary route

(35 μ l) and clofazimine (gavage), amikacin (subcutaneous) in a volume of 200 μ l per mouse, starting on day 2 post-infection and continuing for 8 days every other day.

Mice were sacrificed 2 days after the last dose of compound. All groups of 6 mice (untreated control, clofazimine (gavage), the composition of example 1, and amikacin treated mice) were sacrificed and the bacterial load was determined. The lung homogenate was placed at 0-1-2-3-4-5-6-7, the spleen homogenate at 0-1-2-3-4-5-6-7, and the liver homogenate at 0-1-2-3-4-5-6-7.

A Log10 protection value of at least 0.60 indicates that the activity is statistically significant. CFU was first converted to logarithms for statistical analysis, then evaluated by one-way analysis of variance, and then multiple comparisons of variance were performed by one-way Tukey test (GraphPad Prism analysis software). At the 95% confidence level, the difference was considered significant.

Table 6 shows the mean Log after Mycobacterium abscessus infection in SCID mice₁₀CFU data and mean Standard Error (SEM), where "n" is the total number of animals in the group at time of sacrifice.

TABLE 6

The data in table 6 show that treatment with the composition of example 1 resulted in the greatest reduction in bacterial recovery in the lungs and spleen of animals infected with mycobacterium abscessus. This reduction in bacteria was statistically improved compared to amikacin or oral clofazimine treatment.

Mouse model of M.avium infection in Beige mice

6-8 week old Beige female mice were ordered from Charles River. Mice were rested one week prior to infection.

The acute Beige mouse model received 1X 10⁸Colony Forming Units (CFU)/ml (mycobacterium avium strain 2285rough) of non-invasive aerosol exposure to lung infection. Before use, the working stock of M.avium strain 2285 was frozen in 1ml aliquots and stored at-80 ℃. For theInfection, aliquots were thawed, broken 20 times with a 1ml luer-lok syringe fitted with a 26g needle, and diluted in sterile 1 x Phosphate Buffered Saline (PBS).

3 mice were sacrificed on day 1 and day 7 post-infection and bacterial uptake was determined. Whole lung, spleen and liver were extracted, homogenized in 4.5ml of 1 x PBS and diluted to 1: 10. Dilutions (0-1-2-3-4-5-6-7) were placed on 7H11/OADC, TSA and charcoal agar plates and incubated in a dry air incubator (depending on the strain) at 32 ℃ for 30 days.

(35. mu.l) and clofazimine (gavage) was administered in a volume of 200. mu.l per mouse, starting on day 7 post-infection and continuing for 10 days every other day.

Mice were sacrificed 5 days after the last dose of compound. All groups of 6 mice (untreated control, clofazimine (gavage), and the composition of example 1 mice) were sacrificed and bacterial load was determined. The lung homogenate was placed at 0-1-2-3-4-5-6-7, the spleen homogenate at 0-1-2-3-4-5-6-7, and the liver homogenate at 0-1-2-3-4-5-6-7.

Log10 protection values of at least 0.60 indicate that the activity is statistically significant. The CFU is first converted to logarithms for statistical analysis, then evaluated by one-way analysis of variance, and then subjected to multiple comparative analysis of variance by one-way Tukey test (SigmaStat software program). At the 95% confidence level, the difference was considered significant.

Table 7 shows the mean Log after Mycobacterium avium infection in Beige mice₁₀CFU data.

TABLE 7

The data in table 7 show that treatment with the composition of example 1 results in a greater reduction of bacterial recovery in the lungs and spleen of animals infected with mycobacterium avium.

Chronic Beige mouse model

Beige mice 6 to 8 weeks old were rested one week prior to infection. On day 0, mice received 1X 10⁸CFU of Mycobacterium avium 2285rough lung infection. 3 mice were sacrificed on day 1 and 6 mice were sacrificed on day 27 and bacterial uptake and pre-treatment bacterial load were determined. Whole lung, spleen and liver were extracted, homogenized in 4.5ml of 1 × PBS, and placed on 7H11 and charcoal agar plates at a dilution of (0-1-2-3-4-5-6-7). The plates were placed in a 37 ℃ dry air incubator for 25 to 30 days.

The remaining infected Beige mice were treated every other day for 14 treatments starting on day 28. The animals received one of the following treatments: physiological saline solution (

35. mu.l); clofazimine (oral gavage, 20mg/kg, 200 μ l); the composition of example 1 (IT,

10mg/kg，35μl)。

mice were sacrificed on day 57 (two days after final treatment). The plates were placed in a 37 ℃ dry air incubator for 30 days.

CFU's were first converted to logarithms for statistical analysis, then evaluated by one-way analysis of variance, and then multiple comparative analysis of variance was performed by one-way Tukey test. At the 95% confidence level, the difference was considered significant.

Table 8 shows the mean Log after chronic infection with Mycobacterium avium of Beige mice₁₀CFU data.

TABLE 8

These data indicate that clofazimine hardly penetrates granuloma-like structures formed by established "chronic" animal NTM infection models. It appears that the compositions of the invention do not present the same problems and are able to maintain anti-mycobacterial activity even after the infection has been well established.

Effect of the composition of example 3 on Barrier integrity and inflammation following in vitro Exposure to Lung epithelial cells

Cell survival rate

Survival of lung epithelial cells was assessed under two in vitro conditions using three different cell types: calu-3; a549; and hAELVI cells. Under "submerged conditions" (i.e. in transwell)^TMIn cell culture medium on a plate) or "gas-liquid interface" simulated conditions (ALI), and cell culture medium is removed from the topside of the cells. Calu-3 cells were exposed to three doses of the example 3 composition (10%, 50% or 100%) for 4 hours under "immersion conditions". To assess cell viability, cells were stained using acridine orange/propidium iodide (AO/PI) staining to distinguish live/dead cells. Red fluorescence indicates cell death.

Macrophage uptake

THP-1 cells differentiated into macrophage-like cells after 3 days of incubation with 124ng/ml phorbol 12-myristic acid 13 acetate (M MA). Once the cells had matured, they were exposed to the composition of example 3 (diluted 1:200 in Hank buffered saline (HBSS)) for 4 hours. Cells were stained by AO/PI as described above to determine cell viability after exposure.

Transepithelial electrical resistance (TEER) measurement

In transwell^TM3460 at 1 × 10⁵Cells/well were seeded with Calu-3 cells and left for 12 days to grow to confluence. According to the manufacturer's instructionsTEER measurements were performed using EVOM2(World Precision Instruments, Friedberg, Germany). After inoculation, Calu-3 cells were exposed to saline solution (negative control) or the composition of example 3 (concentration: 20mg/ml, 10mg/ml or 20. + -.2 mg/ml). Cells were exposed for 2 to 4 hours before TEER was measured.

Production of inflammatory cytokines

Differentiated THP-1 cells (dTHP-1) were exposed to the composition of example 3 for 4 hours or 24 hours (1:200HBSS dilution). HBSS exposure alone was used as a negative control and Lipopolysaccharide (LPS) (100ng/ml) was administered as a positive control.

After incubation, supernatants were removed from cells after either t 4 hours or 24 hours and the supernatants were combined. The pooled supernatant samples were subjected to enzyme-linked immunosorbent assay (ELISA). According to the manufacturer's instructions, the use of separate TNF alpha IL-6, IL-8 ELISA kit.

Statistical analysis was performed by one-way analysis (ANOVA) of variables and by subsequent Tukey post hoc tests. It was statistically significant to determine a probability value < 0.05.

Results

Under "submerged" conditions, the composition of example 3 did not cause a visual decrease in cell viability in 4 hours of culture at any applied concentration.

Under "ALI" conditions, three different cell types (Calu-3, a549, and haeldi cells) were studied at three different time points (five hours, two days, and seven days). Little or no cytotoxicity was observed at 4 hours in any cells, and little or no cytotoxicity was observed at 2 days in Calu-3 cells. Some toxicity was observed on days 2 and 7 in a549 cells and on day 7 in Calu-3 cells. Technical limitations do not allow quantification of cell death.

For macrophage uptake, differentiated THP-1 cells were incubated for 4 hours at 1:200HBSS to determine macrophage survival after exposure. The composition of example 3 did not induce cell death, but showed uptake of clofazimine by 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 sampled at different time points throughout the exposure process. A > 50% reduction in TEER at any given time point compared to the control group is considered a significant loss of barrier integrity.

The composition of example 3 had no effect on barrier integrity after one hour exposure to Calu-3 cells. An exposure of 20mg/ml resulted in a significant (i.e.. gtoreq.50%) reduction after two hours. At a concentration of 10mg/ml, a slight decrease (i.e. 25-35%) was shown at all time points after two hours. Exposure of 2mg/ml did not show any reduction in barrier function throughout the study period.

Inflammatory cytokine production

The positive control LPS performed as expected in this model. The composition of example 3 showed no significant change in cytokines at any time point studied.

The results are shown in table 9.

Cytokine production following dTHP-1 cell exposure

Table 9 (. p < 0.05)

In vivo safety and tolerability

In 6-8 weeks, pld Balb/C female mice were given three doses every other day. The composition of example 1 was administered to mice at doses of clofazimine of 10.0, 5.01, and 2.51 mg/kg. The composition was passed through a volume of 35. mu.l/mouse

Aerosol Intratracheal (IT) administration. After instillation, mice were observed at 10 minutes, 1, 2, and 4 hours after dosing, and then daily.

Table 10 shows the general observations after administration. "BAR" means that the animal is cheerful, active and responsive.

Watch 10

Table 11 shows the body weights of the animals over the three day period tested.

TABLE 11

These data indicate that there was no significant change in body weight during the three day treatment period.

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

Claims

1. A pharmaceutical composition comprising:

2. The pharmaceutical composition of claim 1, wherein the particles of clofazimine or a pharmaceutically acceptable derivative or salt thereof have an average size of less than 2 μ ι η and D90 is less than 3 μ ι η.

3. A pharmaceutical composition comprising:

(a) a therapeutically effective dose of clofazimine;

wherein the clofazimine is provided in the form of particles in suspension,

and is

4. The pharmaceutical composition of claim 3, wherein the particles have an average size of less than 2 μm and a D90 of less than 3 μm.

5. Pharmaceutical composition according to any of claims 3 or 4, wherein the clofazimine is provided in one or more polymorphic forms selected from the triclinic form FI, the monoclinic form FII and the orthorhombic form FIII and mixtures of these forms.

6. The pharmaceutical composition of claim 5, wherein said clofazimine is provided substantially in an orthorhombic form FIII.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the non-ionic surfactant is selected from polysorbate 20, polysorbate 60, polysorbate 80, stearyl alcohol, polyethylene glycol derivatives of hydrogenated castor oil having a hydrophilic-lipophilic balance of 14 to 16, polyethylene glycol derivatives of hydrogenated castor oil having a hydrophilic-lipophilic balance of 15 to 17, sorbitol monolaurate, sorbitol monopalmitate, sorbitol monostearate, polyoxyethylene (20) oleyl ether, polyoxyethylene (20) 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 polyoxy-8 glyceride, Polyethylene glycol (20) monostearate, polyethylene glycol (40) stearate, polyethylene glycol (100) stearate, polyethylene glycol (8) stearate, and polyethylene glycol 40 stearate, and mixtures thereof.

8. The pharmaceutical composition according to any one of claims 1 to 7,

wherein the nonionic surfactant is polysorbate 80, and

wherein the aqueous liquid carrier is distilled water, a hypertonic salt solution, or an isotonic salt solution.

9. The pharmaceutical composition of claim 8, wherein the hypertonic salt solution is 1% to 7% (w/v) sodium chloride.

10. The pharmaceutical composition of claim 8, wherein the non-ionic surfactant is ultra-pure polysorbate 80, and wherein the aqueous liquid carrier is an isotonic salt solution.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the osmolality of the composition is 200-700 mOsm/kg.

12. The pharmaceutical composition according to any one of claims 1 to 10, wherein the osmolality of the composition is 300-400 mOsm/kg.

13. The pharmaceutical composition according to any one of claims 1 to 10,

the nonionic surfactant comprises 0.001% to 5% (v/v) of the total composition, and

the clofazimine is present in an amount of 0.1% to 20% (w/v) of the total composition.

14. The pharmaceutical composition according to any one of claims 1 to 13, prepared by a process comprising the steps of:

(3) adjusting the sodium chloride concentration to a suitable concentration and

(4) adjusting the osmolality to a suitable level.

15. The pharmaceutical composition according to any one of claims 1 to 13, prepared by a process comprising the steps of:

(1) homogenizing the suspension of clofazimine and non-aqueous liquid to obtain a suspension containing clofazimine of suitable particle size;

(2) separating the clofazimine, and then separating,

(3) adding the clofazimine into the nonionic surfactant and water,

(5) the sodium chloride concentration is adjusted to a suitable concentration.

16. The pharmaceutical composition according to any one of claims 1 to 13, prepared by a process comprising the steps of:

(2) adding the clofazimine into the nonionic surfactant and water,

(4) the sodium chloride concentration is adjusted to a suitable concentration.

17. The pharmaceutical composition according to any one of claims 1 to 13, which is prepared by a process comprising: a suspension of clofazimine in a nonionic surfactant, water containing a suitable concentration of sodium chloride, is homogenized to obtain clofazimine of a suitable particle size, and the pH of the suspension has been adjusted to a pH between pH 5.5 and pH 7.5.

18. The pharmaceutical composition of any one of claims 14, 15 or 16, wherein the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154mM sodium chloride.

19. The pharmaceutical composition of claim 17, wherein the pH is 7.4 and the suitable concentration of sodium chloride is 154mM sodium chloride.

20. The pharmaceutical composition of claim 16, wherein micronization of said clofazimine is by jet milling, spray drying, ball milling, or supercritical fluid processing.

21. The pharmaceutical composition according to any one of claims 14, 15, 17 or 19, wherein the homogenization in step (1) is performed by high pressure homogenization, high shear homogenization, wet milling, ultrasonic homogenization, or a combination of these methods.

22. The pharmaceutical composition of any one of claims 14, 15, 17, 18, 19 or 21, wherein homogenization of the clofazimine is performed in multiple homogenization steps.

23. The pharmaceutical composition according to any one of claims 16 or 20, wherein micronization of said clofazimine is carried out in multiple micronization steps.

24. The pharmaceutical composition of any one of claims 14 to 23, wherein the clofazimine of suitable particle size is a particle with an average size of less than 5 μ ι η and D90 of less than 6 μ ι η.

25. The pharmaceutical composition of any one of claims 14 to 23, wherein the clofazimine of suitable particle size is a particle having an average size of less than 2 μ ι η and D90 of less than 3 μ ι η.

26. A pharmaceutical combination in aerosol form for inhalation prepared by aerosolization of a composition according to any one of claims 1 to 25 by an aerosolization device selected from the group consisting of an ultrasonic nebulizer, an electrospray nebulizer, a vibrating membrane nebulizer, a jet nebulizer and a mechanical soft mist inhaler, and

wherein the mass median aerodynamic diameter of the aerosol particles produced by the nebulisation device is from 1 to 5 μm.

27. The pharmaceutical combination of claim 26, wherein the aerosol for inhalation is for lower lung deposition.

28. A pharmaceutical composition according to any one of claims 1 to 25 for use in combination with an agent that disperses and/or disrupts biofilm, a mucolytic and/or mucoactive agent, and/or an agent that reduces biofilm formation selected from the group consisting of: nebulized 4-7% hypertonic saline solution, metaperiodate, sodium lauryl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth mercaptane, ethylene diamine tetraacetic acid, gentamicin-loaded phosphatidylcholine-modified gold nanoparticles, chelating agents, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides.

29. A pharmaceutical combination according to any one of claims 26 or 27 for use in combination with an agent that disperses and/or disrupts biofilm, a mucolytic and/or mucoactive agent, and/or an agent that reduces biofilm formation selected from the group consisting of: nebulized 4-7% hypertonic saline solution, metaperiodate, sodium lauryl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth mercaptane, ethylene diamine tetraacetic acid, gentamicin-loaded phosphatidylcholine-modified gold nanoparticles, chelating agents, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides.

30. The pharmaceutical composition according to any one of claims 1 to 25 for use in the treatment and/or prevention of pulmonary infections caused by mycobacteria or other gram-positive bacteria.

31. The pharmaceutical combination according to any one of claims 26 or 27 for use in the treatment and/or prevention of pulmonary infections caused by mycobacteria or other gram-positive bacteria.

32. The pharmaceutical composition of claim 30, wherein the infection is caused by a species of mycobacterium selected from the group consisting of nontuberculous mycobacteria and a tuberculous mycobacteria complex and combinations thereof.

33. The pharmaceutical combination for use according to claim 31, wherein the infection is caused by a species of mycobacterium selected from the group consisting of nontuberculous mycobacteria and a complex of mycobacterium tuberculosis, and combinations thereof.

34. The pharmaceutical composition for the use according to claim 32, wherein the non-tuberculous mycobacterium is selected from the group consisting of mycobacterium avium, mycobacterium intracellulare, mycobacterium abscessus, and mycobacterium leprae, and combinations thereof.

35. The pharmaceutical combination for use according to claim 33, wherein the non-tuberculous mycobacterium is selected from the group consisting of mycobacterium avium, mycobacterium intracellulare, mycobacterium abscessus and mycobacterium leprae, and combinations thereof.

36. The pharmaceutical composition for use according to claim 32, wherein the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient with cystic fibrosis, chronic obstructive pulmonary disease or acquired immunodeficiency syndrome.

37. The pharmaceutical combination for use according to claim 33, wherein the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient with cystic fibrosis, chronic obstructive pulmonary disease or acquired immunodeficiency syndrome.

38. The pharmaceutical composition for the use according to claim 36, wherein the infection is an opportunistic non-tuberculous mycobacterial infection in a patient with cystic fibrosis.

39. The pharmaceutical combination according to claim 37, wherein the infection is an opportunistic non-tuberculous mycobacterial infection in a patient with cystic fibrosis.

40. A system for providing antibiotic activity in the treatment or provision of prophylaxis of pulmonary infections caused by mycobacteria or other gram positive bacteria, wherein the system comprises:

1) an aerosolized pharmaceutical combination comprising:

(a) a therapeutically effective dose of clofazimine;

and

2) an atomizer is arranged on the bottom of the water tank,

wherein the clofazimine is provided in the form of a suspension,

and is

Wherein the mass median aerodynamic diameter of the aerosol particles produced by the system is from 1 to 5 μm.

41. Use according to any one of claims 28 to 39, wherein the atomising device exhibits an output rate of 0.1-1.0 ml/min.

42. The use of any one of claims 28 to 39, or 41, wherein the total inhaled volume is between 1 and 5 ml.

43. The use of any one of claims 28, 30, 32, 34, 36 or 38, wherein the composition is administered prior to, concurrently with, or subsequent to the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and p-aminosalicylate, and mixtures thereof.

44. The use of any one of claims 29, 31, 33, 35, 37 or 39, wherein the combination is administered prior to, simultaneously with or subsequent to the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and p-aminosalicylate and mixtures thereof.

45. The pharmaceutical composition according to any one of claims 1 to 25 for use in the treatment and/or prevention of pulmonary fungal infection or clostridium difficile, or a combination thereof.

46. The pharmaceutical combination according to any one of claims 26, 27, 29, 31, 33, 35, 37 or 39 for use in the treatment and/or prevention of pulmonary fungal infection or Clostridium difficile, or a combination thereof.

47. The pharmaceutical composition according to claim 45, for use in the treatment and/or prevention of a pulmonary fungal infection.

48. The pharmaceutical combination according to claim 46 for use in the treatment and/or prevention of pulmonary fungal infections.

49. The pharmaceutical composition of any one of claims 45 or 47, wherein the pulmonary fungal infection is Candida albicans or Aspergillus fumigatus, or a combination thereof.

50. The pharmaceutical combination according to any one of claims 46 or 48, wherein the pulmonary fungal infection is Candida albicans or Aspergillus fumigatus, or a combination thereof.

51. The use of claim 43, wherein the composition is administered prior to, concurrently with, or subsequent to the administration of an agent selected from the group consisting of bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and acamicin, and mixtures thereof.

52. The use of claim 43, wherein the composition is administered prior to, concurrently with, or subsequent to the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.

53. A method of treating or preventing a pulmonary infection in a patient in need thereof, comprising administering by inhalation a composition according to any one of claims 1 to 25.

54. The method of treatment or prevention according to claim 53, wherein the infection is caused by a species of Mycobacterium selected from the group consisting of non-Mycobacterium tuberculosis and Mycobacterium tuberculosis complex and combinations thereof.

55. A method of treatment or prevention according to claim 54, wherein the non-tuberculous mycobacteria are selected from M.

56. A method of treatment or prevention according to claim 53, wherein the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient with cystic fibrosis, chronic obstructive pulmonary disease or acquired immunodeficiency syndrome.

57. The method of treatment or prevention according to claim 56, wherein the infection is an opportunistic non-tuberculous mycobacterial infection in a patient with cystic fibrosis.

58. A method of treating or preventing a pulmonary infection caused by mycobacteria or other gram positive bacteria in a patient in need thereof comprising administering by inhalation a composition according to any one of claims 1 to 25 before, simultaneously with or after administering an agent selected from the group consisting of pharmaceutically acceptable salts of bedaquiline or its derivatives, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampicin, moxifloxacin, levofloxacin and p-aminosalicylate and mixtures thereof.

59. The method of treating or preventing according to claim 58, wherein the agent is bedaquiline or amikacin.

60. A method of treatment or prophylaxis according to claim 59, wherein the agent is bedaquiline.

61. A process for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising the steps of:

(1) homogenizing a suspension of clofazimine, a nonionic surfactant and water to obtain a suspension containing clofazimine of a suitable particle size;

(3) adjusting the sodium chloride concentration to a suitable concentration, an

(4) Adjusting the osmolality to a suitable level.

62. A process for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising the steps of:

(2) separating the clofazimine, and then separating,

(3) adding the clofazimine into a nonionic surfactant and water,

(5) the sodium chloride concentration is adjusted to a suitable concentration.

63. A process for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising the steps of:

(2) adding the clofazimine into a nonionic surfactant and water,

(4) the sodium chloride concentration is adjusted to a suitable concentration.

64. A process for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising homogenizing a suspension of clofazimine in a non-ionic surfactant, in water containing a suitable concentration of sodium chloride to obtain clofazimine of suitable particle size, and the pH of the suspension has been adjusted to a pH between pH 5.5 and pH 7.5.

65. The method of any one of claims 61, 62, or 63, wherein the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154mM sodium chloride.

66. The method of claim 64, wherein the pH is 7.4 and the suitable concentration of sodium chloride is 154mM sodium chloride.

67. The method of claim 63, wherein micronizing of said clofazimine is performed by jet milling, spray drying, ball milling, or supercritical fluid processing.

68. The process of any one of claims 61, 62, 64, or 66, wherein the homogenizing in step (1) is performed by high pressure homogenization, wet milling, ultrasonic homogenization, or a combination of these methods.

69. The method of any one of claims 61, 62, 64, 65, 66 or 68, wherein homogenization of said clofazimine is performed in multiple homogenization steps.

70. The process of any one of claims 63 or 20, wherein micronization of said clofazimine is carried out in a plurality of micronization steps.

71. The method of any one of claims 61-70, wherein the clofazimine of a suitable particle size is a particle having an average size of less than 5 μm and a D90 of less than 6 μm.

72. The method of any one of claims 61-70, wherein the clofazimine of a suitable particle size is a particle having an average size of less than 2 μm and a D90 of less than 3 μm.

73. A process for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising the steps of: (a) homogenizing a suspension of clofazimine, a nonionic surfactant and water to obtain a suspension containing clofazimine of a suitable particle size; (b) adjusting the pH of the resulting suspension to a pH between pH 5.5 and pH 7.5; (c) adjusting the sodium chloride concentration to a suitable concentration, and (d) adjusting the osmolality to a suitable level; and wherein steps (b), (b) and (d), may be (b), (c), (d); (b) (d), (c); (c) (b), (d); (c) (d), (b); (d) (b), (c); or (d), (c) and (b) occur sequentially.

74. A process for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising the steps of: (a) homogenizing the suspension of clofazimine and non-aqueous liquid to obtain a suspension containing clofazimine of suitable particle size; (b) separating the clofazimine; (c) adding the clofazimine to a nonionic surfactant and water; (d) adjusting the pH of the resulting suspension to a pH between pH 5.5 and pH 7.5; and (e) adjusting the sodium chloride concentration to a suitable concentration; and wherein steps (d) and (e) may be (d), (e); or (e) and (d) occur sequentially.

75. A process for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising the steps of: (a) micronizing clofazimine to obtain clofazimine of suitable particle size, and (b) adding said clofazimine to a non-ionic surfactant, water containing a suitable concentration of sodium chloride, and having adjusted its pH to a pH between pH 5.5 and pH 7.5.