KR20210090607A - Compositions of clofazimine, combinations comprising them, methods for their preparation, uses and methods comprising them - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhalation comprising clofazimin in a therapeutically effective dose, wherein the clofazimin is provided in the form of a suspension, and a method for preparing the same. The invention also provides a pharmaceutical combination comprising clofazimine in the form of an aerosol for pulmonary inhalation. The combinations and compositions provided by the present invention can be used in the treatment and/or prevention of pulmonary infections caused by mycobacteria and other gram-positive bacteria, and pulmonary fungal infections.

Description

Compositions of clofazimine, combinations comprising them, methods for their preparation, uses and methods comprising them

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

technical field

The present invention relates to a pharmaceutical composition for inhalation comprising clofazimin in a therapeutically effective dose, wherein the clofazimin is provided in the form of a suspension; their manufacturing method; and to uses and methods of treatment comprising them. The invention also provides a pharmaceutical combination comprising clofazimine in the form of an aerosol for pulmonary inhalation.

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

Clofazimin is a very hydrophobic riminophenazine antibiotic (Log P = 7.66) with anti-mycobacterial and anti-inflammatory activity, and was first described in 1957. Its structural formula is as follows:

The exact mechanism by which clofazimine exerts its antimicrobial effect is unknown. However, it is known to inhibit DNA replication and cell growth by preferentially binding to mycobacterial DNA. Other proposed mechanisms of action include membrane damage/destabilization, production of membrane-destabilized lysophospholipids, interference with potassium transport, and/or intracellular redox cycling. Although it has shown impressive activity against Mycobacterium tuberculosis (MTB) in vitro, including multi-drug resistant strains, until recently clofazimine was generally not shown to be effective in the treatment of pulmonary tuberculosis. (see, eg, Cholo M et al., J Antimicrob Chemother, 2012 Feb, 67(2):290-8).

Clofazimin is one of the three main drugs recommended by the World Health Organization (WHO) for the treatment of leprosy caused by Mycobacterium leprae, drug-resistant tuberculosis and non-tuberculous mycobacteria. (NTM) has been increasingly used in recent years for the treatment of other mycobacterial infections, such as those caused by .

Because clofazimine is substantially insoluble in water and exhibits high membrane permeability, it has been classified as a BCS (Biopharmaceutics Classification System) class II drug.

In order to solve the challenges associated with insufficient oral absorption and insufficient bioavailability of drugs, various strategies such as micronization, nanoscale, supercritical fluid recrystallization, spray freeze-drying into liquids, solid dispersions and solutions are used to optimize oral dosage forms. This was applied.

Clofazimine, classified as a BCS class II drug, is generally considered an ideal candidate for formulation into a solid dispersion for the improvement of oral bioavailability (e.g., Bhusnure et al. IJRPC 2014, 4(4), 906-918).

Accordingly, because of its lipophilicity, clofazimine is generally administered as a microcrystalline suspension in an oil-wax base to enhance oral absorption. Absorption in humans after oral administration varies considerably (45-62%). The adverse effects of clofazimine are dose-related and mainly affect the skin, eyes, gastrointestinal tract, and QT prolongation. Side effects include the development of reddish-brown discoloration of the skin and conjunctiva, which gradually reverts when stopped. These are the result of chronic systemic accumulation.

Mycobacterium is a genus of Actinobacteria along with its own genus, Mycobacteriaceae. Mycobacteria have a characteristic rod-shaped shape and a waxy outer coat.

Therefore, mycobacteria can be divided into three groups:

마이코박테리움 투버쿨로시스 (Mycobacterium tuberculosis) 복합체 - 결핵의 원인 병원체

Mycobacterium tuberculosis complex - the causative agent of tuberculosis

마이코박테리움 레프라에 (Mycobacterium leprae) - 한센병의 원인 병원체

Mycobacterium leprae - the causative agent of leprosy

비결핵성 마이코박테리아 (NTM) - 엠. 투버쿨로시스 (M. tuberculosis) 또는 엠. 레프라에 (M. leprae) 가 아닌 모든 다른 마이코박테리아, 예를 들어 마이코박테리움 압세수스 복합체 (Mycobacterium abscessus complex, MABSC), 마이코박테리움 아비움 복합체 (Mycobacterium avium complex, MAC) 를 포함함.

Non-tuberculous mycobacteria (NTM) - M. Tuberculosis (M. tuberculosis) or M. Including all other mycobacteria other than M. leprae, such as Mycobacterium abscessus complex (MABSC), Mycobacterium avium complex (MAC) box.

Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex bacteria. As one of the oldest documented infectious agents in humans, TB remains a significant cause of mortality and morbidity worldwide, with an estimated 10.4 million new TB infections in 2015 and 1.4 million deaths from active TB disease ( See, for example, the World Health Organization (WHO) Global Tuberculosis Report 2016). In addition to high morbidity and mortality, the incidence of multi-drug-resistant tuberculosis (MDR-TB) is a growing concern, with 580,000 patients developing drug-resistant TB infection in 2015. Co-morbidities such as human immunodeficiency virus (HIV) complicate treatment and accounted for 1.2 million TB cases in 2015.

To treat multi-drug resistance (MDR) infections, the WHO recommends implementing a 9 to 12 month treatment regimen of second-line anti-TB drugs. These regimens, such as the 9-12 month Bangladesh regimen, treated MDR-TB with a combination of gatifloxacin, ethambutol, pyrazinamide and clofazimine, leading to relapse-free treatment in 87.9% of patients (e.g., Sotgiu, G, et al., "Applicability of the shorter 'Bangladesh regimen' in high multidrug-resistant tuberculosis settings", see International Journal of Infectious Diseases (2017) 56 190-193).

Other studies investigating shortened TB treatment have demonstrated that clofazimine has no clinical benefit after 2 weeks of oral administration (e.g., Diacon, AH, et al., "Bactericidal Activity of Pyrazinamide and Clofazimine Alone and in Combinations with Pretomanid and Bedaquiline", American Journal of Respiratory and Critical Care Medicine (2015), 191 (8), 943-953). The lack of activity was due to the low bioavailability of the drug, as it was theorized to bind with high affinity to circulating serum proteins. Despite the fact that clofazimine has been empirically proven to be effective in the treatment of MDR-TB, and broad-drug resistant TB (XDR-TB), its insufficient bioavailability after systemic administration limits its biological activity during short-term treatment. (See, e.g., Swanson, RV, et al., "Pharmacokinetics and Pharmacodynamics of Clofazimine in a Mouse Model of Tuberculosis," Antimicrobial Agents and Chemotherapy (2015), 59 (6), 3042-3051).

Treatment of pulmonary infections with inhaled antibiotics, compared to systemic delivery, results in higher drug concentrations in the lung, reduces adverse effects (e.g., Touw, DJ, et al., "Inhalation of antibiotics in cystic fibrosis). ", European Respiratory Journal (1995), 8 , 1594-1604), which results in increased biological activity and efficacy (e.g., Hickey, AJ, "Inhaled drug treatment for tuberculosis: Past progress and future prospects", Journal of Controlled Release, (2016), 240 , 127-134). An in vivo mouse model demonstrated that aerosolized administration of clofazimin showed a significant improvement in bacilli clearance in a TB-infection model compared to oral administration of clofazimin at 28 days after initiation of treatment (e.g. , Verma, RK, et al., "Inhaled microparticles containing clofazimine are efficacious in treatment of experimental Tuberculosis in Mice", Antimicrobial Agents and Chemotherapy (2013), 57 (2), 1050-1052). This improved efficacy over the short term may be due to the direct delivery of clofazimin to the site of infection in the lung, resulting in higher clofazimin concentrations in lung macrophages in tuberculosis granulomas.

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

The group of non-tuberculous mycobacteria (NTM), previously termed atypical or ubiquitous mycobacteria, contains more than 150 species. NTMs are ubiquitous in nature and exhibit wide diversity. They can be detected in soil, land and drinking water, as well as in foods such as pasteurized milk or cheese. In general, NTM is considered less pathogenic. Nevertheless, they can cause serious illness in humans, especially those with weakened immune systems or who have previously suffered from lung disease. Currently, NTMs are classified according to their growth rate and divided into slow-growing (SGM) and fast-growing (RGM) mycobacteria.

The slow-growing Mycobacterium avium complex (MAC) is one of the most important and most frequent pathogenic NTMs, Mycobacterium avium, Mycobacterium chimaera and species of Mycobacterium intracellulare. Mycobacterium kansasii (Mycobacterium kansasii), Mycobacterium malmoens (Mycobaceterium malmoense), Mycobacterium xenopi (Mycobacterium xenopi), Mycobacterium simiae (Mycobacterium. simiae), Mycobacterium Like Mycobacterium abscessus, Mycobacterium gordonae, Mycobacterium fortuitum and Mycobacterium chelonae, they mainly cause lung infections do. Mycobacterium marinum is responsible for skin and soft tissue infections such as Aquarium granulomas.

In particular, RGM causes severe, life-threatening chronic lung disease and causes diffuse and often fatal infections. Infections are typically caused by invasive procedures including the injection and implantation of contaminated material and catheters, non-sterile surgical procedures, or foreign bodies. Exposure to shower heads and jacuzzis has also been reported as a risk for infection. NTMs typically cause opportunistic infections in patients suffering from chronic lung diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and other immunocompromised patients.

Recently, the subspecies Mycobacterium absesus subsp. Abscessus (Mycobacterium abscessus subsp. abscessus) (M. A. abscessus (M. a. abscessus)), Mycobacterium abscessus boletii. (Mycobacterium abcessus bolletii.) and Mycobacterium abscessus massiliense, including strains of the rapidly growing (RGM) Mycobacterium abscessus group (Mycobacterium abscessus) Mycobacterium abscessus complex (MABSC) has emerged as an important human pathogen and is associated with significantly higher mortality than any other RGM.

Mycobacterium abscessus infection in CF patients is particularly problematic because it results in increased lung destruction and is often impossible to treat with a high failure rate of 60-66% (e.g., Obregon-Henao A et al, Antimicrobial Agents and Chemotherapy, November 2015, Vol 59, No 11, p. 6904-6912; Qvist, T., Pressler, T., Hoiby, N. and Katzenstein, TL., “Shifting paradigms. of nontuberculous mycobacteria in cystic fibrosis", Respiratory Research (2014), 15 (1): pp.41-47).

Human infection by NTM has more to do with the emergence of the human AIDS pandemic. Mycobacteria from Mycobacterium avium complex (MAC) have been identified as a major cause of opportunistic infections in patients infected with human immunodeficiency virus (HIV).

Several species of NTM are known to form biofilms. Biofilms are microcolonies of bacteria embedded in an extracellular matrix that provides stability and resistance to human immune mechanisms. Recently, some species of NTMs have been shown to form biofilms that enhance resistance to disinfectants and antimicrobials. Biofilm assembly proceeds through several steps including reversible attachment, irreversible attachment, bacterial aggregation, biofilm formation through tissue and signal transduction, and eventual dispersion. During this process, bacteria express a matrix containing extracellular polymeric substances (EPS) such as polysaccharides, lipids and nucleic acids to form complex three-dimensional structures (eg, Sousa S. et al., International Journal of Mycobacteriology). 4 (2015), see 36-43). In particular, mycobacterial EPS differs from other biofilms in properties because mycobacteria do not produce exopolysaccharides (see, e.g., Zambrano MM, Kolter R. Mycobacterial biofilms: a greasy way to hold it together. Cell. 2005). ). Mycobacterial biofilms differ from species to species, but may contain mycholic acid, glycopeptidolipid, mycholyl-diacylglycerol, lipooligosaccharide, lipopeptide, and extracellular DNA (Overview and original research from: Rose SJ, Babrak). LM, Bermudez LE (2015) Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics. PLoS ONE). Assembly of biofilms is known to improve resistance to antimicrobial agents (see, eg, Faria S. et al., Journal of Pathogens, Vol 2015, Article ID 809014).

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

Multiple combination therapy with inhaled amikacin after initial treatment with parenteral aminoglycosides, tigecycline and other promising oral antibiotics such as linezolid, delamanide and bedaquiline, and surgical intervention in selected cases It has shown promising results in the treatment of NTM lung disease (Lu Ryu et al., Tuberc Respir Dis 2016;79:74-84). However, the increasing incidence and prevalence of NTM infections, particularly NTM lung disease, and limited treatment options require the development of novel dosage forms/pharmaceuticals that improve the bioavailability of currently used antibiotics such as clofazimine. Inhalation can improve efficacy and reduce adverse effects compared to oral and parenteral therapy.

The combination of clofazimin and amikacin has been shown to act synergistically in vitro against both Mycobacterium abscessus and Mycobacterium avium (e.g., van See Ingen, J., et al., "In Vitro Synergy between Clofazimine and Amikacin in Treatment of Nontuberculous Mycobacterial Disease", Antimicrobial Agents and Chemotherapy 56 (12), 6324-6327 (2012)). In addition, the use of a combination of clofazimine and bedaquiline has been shown to have synergistic action against Mycobacterium tuberculosis (see, e.g., Cokol, M. et al., "Efficient Measurement and factorization of high -order drug interactions in Mycobacterium tuberculosis", Sciences Advances 2017: 3 :e170881, see 11 October 2017). The clofazimine/bedaquiline combination also showed synergy against the non-tuberculous bacterium Mycobacterium abscessus (Ruth, MM et al., "A Bedaquiline/Clofazimine Combination Regimen Might Add Activity to the Treatment of Clinically Relevant Non-Tuberculous Mycobacteria", Journal of Antimicrobial Chemotherapy (2019), doi.org/10.1093/jac/dky526).

Fungal pathogens have emerged as a leading cause of human death. Current estimates suggest that deaths from invasive fungal infections are equivalent to more fully known infectious diseases such as tuberculosis. Candida albicans, Cryptococus neoformans and Aspergilus fumigatus represent the most prevalent human fungal pathogens. Each of these species causes hundreds of thousands of infections each year with unacceptably high mortality rates due to insufficient diagnosis and limited treatment options. Clofazimine has been shown to be efficacious as a combination agent against several fungi (see, e.g., Robbins, N., et al., "An Antifungal Combination Matrix Identifies a Rich Pool of Adjuvant Molecules that Enhance Drug Activity against Diverse Fungal Pathogens", Cell Reports 13, 1481-1492, November 17, 2015). Fungi also serve as commensal, colony-forming factors and/or pathogens in cystic fibrosis (see, e.g., Chotirmall, SH and McElvaney, NG, "Fungi in the cystic fibrosis lung: Bystanders or pathogens?", The International See Journal of Biochemistry & Cell Biology 52 (2014), 161-173).

The low solubility of clofazimine in water results in low oral bioavailability and high microbial resistance, and also drugs for formulation in liquid aqueous carriers, such as aerosolization by nebulizers to obtain lower lung deposition of aerosol particles. It requires specific techniques to dissolve and stabilize it.

In an 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 greater than 10; and

(c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solution;

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

The particles of clofazimine or a pharmaceutically acceptable derivative or salt thereof have a median size of less than 5 μm and a D90 of less than 6 μm.

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

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

(a) a therapeutically effective dose of clofazimine;

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

(c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solution;

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

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 clofazimin have a median size of less than 2 μm and a D90 of less than 3 μm.

Aerosolization of the compositions of the present invention by an appropriate nebulizer provides significantly increased delivery of aerosolized clofazimine to the lower lungs (i.e., bronchi, bronchioles, and alveoli of the central and lower peripheral lungs), thereby increasing therapeutic efficacy. improve substantially.

In addition, the inhalation device should preferably be further adapted for localized pulmonary delivery of an aerosol with an optimal particle size distribution for homogeneous deposition in the lower lung.

Therefore, the present invention provides an aerosol having aerosol particles of a size that facilitates delivery to the alveoli and bronchioles. A suitable aerodynamic particle size for targeting the alveoli and bronchioles is 1-5 μm. Larger particles are selectively deposited in the upper lungs, ie, the bronchi and trachea, and in the mouth and throat, ie, oropharynx region. Accordingly, the inhalation device is adapted to produce an aerosol having a mass median aerodynamic diameter (MMAD) in the range of about 1 to about 5 μm, and preferably in the range of about 1 to about 3 μm. In another embodiment, the particle size distribution is narrow and has a geometric standard deviation (GSD) of less than about 2.5.

The present invention provides that by pulmonary aerosol administration of clofazimine in the form of a suspension, lower (i.e. deeper) lung deposition of the active agent can be achieved, thereby significantly increasing the bioavailability of the microhydrophobic BCS class II agent and , which is based on the unexpected finding that it results in significantly increased therapeutic efficacy with reduced systemic side effects.

In another aspect, this finding is an improved antibiotic for infections caused by mycobacteria and gram-positive bacteria, in particular for lung infections caused by NTM, such as opportunistic infections in CF, COPD and immunocompromised patients, such as HIV patients. induce the provision of therapy.

In addition, the present invention provides a method of reducing the dosage and duration of treatment with clofazimine, as well as overcoming the systemic side effects of established oral treatment regimens for lung infections caused by Gram-positive bacteria, particularly TB and NTM infections of the lungs. aim to

It is understood by those skilled in the art that this application also discloses each and any combination of the individual features disclosed herein.

Justice

The term “pharmaceutically acceptable salts” refers to salts which retain the biological effects and properties of the compounds of the present invention and which are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or similar groups. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids 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 are, for example, acetic acid, propionic acid, naphthoic acid, oleic acid, palmitic acid, pamoic acid (embolic acid), stearic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid , fumaric acid, tartaric acid, citric acid, ascorbic acid, glucoheptonic acid, glucuronic acid, lactic acid, lactobionic acid, tartaric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. .

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

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

As used herein, the term "pharmaceutically acceptable derivative" means a compound disclosed, for example, in US 9,540,336, the disclosure of which is incorporated herein in its entirety. In addition, the 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. and Ma, Z., "Clofazimine Analogs with Efficacy against experimental Tuberculosis and reduced Potential for Accumulation" Antimicrobial Agents and Chemotherapy (2011), 55(11):pp.5185-5193]. Also, the term "pharmaceutically acceptable derivative" of a compound is, for example, a prodrug of said compound. In general, a prodrug is a derivative of a compound that, upon administration, is capable of providing the compound in an active form. Such derivatives may be, for example, esters or amides of carboxyl groups, carboxyl esters of hydroxyl groups, or phosphate esters of hydroxyl groups.

A “therapeutically effective amount,” “therapeutically effective dosage,” or “pharmaceutically effective amount” is an amount of clofazimine, or a pharmaceutically acceptable salt or derivative thereof, as disclosed for the present invention, that has a therapeutic effect. means A therapeutically useful dosage of clofazimine is a therapeutically effective amount. Thus, as used herein, a therapeutically effective amount refers to an amount of clofazimine that produces the desired therapeutic effect, as judged by clinical trial results and/or model animal infection studies.

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

“Therapeutic effect” includes alleviating to some extent one or more of the symptoms of an infection and curing the infection. "Cure" means eliminating the symptoms of an active infection, including, in whole or substantially, removal of excess members of the surviving microorganisms involved in the infection, to a point below the threshold of detection by conventional measures. However, even after healing has occurred, certain long-term or permanent effects of the infection (eg, excessive tissue damage) may exist. As used herein, "therapeutic effect" is defined as a statistically significant reduction in bacterial loading in a host, development of resistance, or amelioration of symptoms of infection, as measured by human clinical outcomes or animal studies.

As used herein, “treat”, “treatment”, or “treating” refers to administering a pharmaceutical composition/combination for prophylactic and/or therapeutic purposes.

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

Unless otherwise specified herein, the term “inhalation” is meant to refer to pulmonary inhalation.

Unless otherwise specified herein, as used herein, the term “infection” is meant to refer to a pulmonary infection.

Unless otherwise specified, when used to refer to the purity of a compound, the term "substantially" refers to a purity of the compound of at least 95% purity.

Unless otherwise specified, the term “suitable particle size” refers to the particle size of clofazimine in a composition, or composition, which, when administered to a patient, provides the desired therapeutic effect.

Unless otherwise specified, the term "suitable concentration" refers to the concentration of a component in the composition or combination that provides for a pharmaceutically acceptable composition or combination.

Pharmaceutical Compositions and Combinations

The following water classes are particularly applicable to the present invention: sterile purified water, sterile water for injection, sterile water for irrigation, sterile water for inhalation (USP) and the corresponding water class according to, for example, the European Pharmacopoeia or National Pharmacopoeia. .

The aqueous electrolyte solution as used according to the invention as aqueous liquid carrier 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 corresponding to about/approximately 150 mM NaCl, preferably 154 mM NaCl).

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

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

(a) a therapeutically effective dose of clofazimine;

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

(c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solution;

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

The particles of clofazimin 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, wherein the clofazimin is triclinic form FI, monoclinic form provided in polymorphic form selected from FII and orthorhombic form FIII, and mixtures of these forms.

In another embodiment, clofazimine is provided in the substantially orthorhombic form FIII.

In still further embodiments, a non-ionic surfactant is polysorbate 20 (e.g. Tween ^® 20), polysorbate 60 (e.g. Tween ^® 60), polysorbate 80 (e.g. Tween ^® 80), stearyl alcohol, of 14 to 16 hydrophilic-pro-polyethylene glycol derivative having an oil balance values (such as Cremophor ^® RH 40), 15 to 17 hydrophilic-pro-hydrogenated castor significantly polyethylene having an oil balance value glycol derivatives (eg Cremophor ^® RH 60), sorbitan monolaurate (eg Span ^® 20), sorbitan monopalmitate (eg Span ^® 40), sorbitan monostearate (eg Span) ^® 60), polyoxyethylene (20) oleyl ether (eg Brij ^® O20), polyoxyethylene (20) cetyl ether (eg Brij ^® 58), polyoxyethylene (10) cetyl ether (eg Brij ® 58) For Brij ^® C10), polyoxyethylene (10) oleyl ether (eg Brij ^® O10), polyoxyethylene (100) stearyl ether (eg Brij ^® S100), polyoxyethylene (10) stearyl Ethers (eg Brij ^® S10), polyoxyethylene (20) stearyl ether (eg Brij ^® S20), polyoxyethylene (4) lauryl ether (eg Brij ^® L4), polyoxyethylene ( 20) cetyl ether (eg Brij ^® 93), polyoxyethylene (2) cetyl ether (eg Brij ^® S2), caprylocaproyl polyoxyl-8 glycerides (eg Labrasol ^® ), polyethylene glycol ( 20) stearate (eg Myrj™ 49), polyethylene glycol (40) stearate (eg Myrj™ S40), polyethylene glycol (100) stearate (eg Myrj™ S100), polyethylene glycol (8) Stearates (eg Myrj™ S8), and polyoxyl 4 There is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the pharmaceutical composition is selected from 0 stearate (eg Myrj™ 52), and mixtures thereof.

In another embodiment of the present invention, the pharmaceutical composition according to any one of the composition embodiments described herein, wherein the nonionic surfactant is polysorbate 80 and the aqueous liquid carrier is distilled water, hypertonic saline or isotonic saline is provided In another embodiment of the present invention, there is provided a pharmaceutical composition, wherein the hypertonic saline is 1% to 7% (w/v) sodium chloride. In another embodiment of the present invention, there is provided a pharmaceutical composition, wherein the nonionic surfactant is ultrapure polysorbate 80 (eg NOF Corporation polysorbate 80 (Hx2)) and the aqueous liquid carrier is isotonic saline .

In another embodiment of the present invention, there is provided a pharmaceutical composition according to any one of the composition embodiments described herein, wherein the osmotic pressure of the composition is in the range of 200-700 mOsm/kg. In another embodiment, the osmotic pressure of the composition is in the range of 300-400 mOsm/kg.

In another embodiment of the present invention, the nonionic surfactant is in the range of 0.001% to 5% (v/v) of the total composition, and the amount of clofazimin is 0.1% to 20% (w/v) of the total composition A pharmaceutical composition according to any one of the composition embodiments described herein is provided.

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 method comprising the steps of:

(1) homogenizing a suspension of clofazimine, a nonionic surfactant and water to obtain a suspension comprising clofazimine of an appropriate particle size;

(2) adjusting the pH of the suspension produced from (1) above to a pH of pH 5.5 to pH 7.5, and

(3) adjusting the sodium chloride concentration to an appropriate concentration, and

(4) adjusting the osmotic pressure to an appropriate level.

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

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 method comprising the steps of:

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

(2) isolating clofazimine;

(3) adding clofazimine to a nonionic surfactant and water;

(4) adjusting the pH of the suspension produced from (3) above to a pH of pH 5.5 to pH 7.5, and

(5) adjusting the sodium chloride concentration to an appropriate concentration.

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

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

(1) micronizing clofazimin to obtain clofazimine of an appropriate particle size;

(2) adding clofazimine to a nonionic surfactant and water;

(3) adjusting the pH of the suspension produced from (2) above to a pH of pH 5.5 to pH 7.5, and

(4) adjusting the sodium chloride concentration to an appropriate concentration.

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

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

In another embodiment, the composition contains an appropriate concentration of sodium chloride and is adjusted to a pH of between pH 5.5 and pH 7.5 by homogenizing a suspension of clofazimine, a non-ionic surfactant and water to obtain an appropriate particle size of clofa. There is provided a pharmaceutical composition according to any one of the composition embodiments described herein, prepared by a method comprising the step of obtaining jimin. In another embodiment, the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154 mM sodium chloride. In another embodiment, the homogenization is performed by high pressure homogenization, high shear homogenization, wet milling, ultrasonic homogenization, or a combination of these treatments. In another embodiment, the homogenization of clofazimine is carried out in several stages of homogenization. In another embodiment, clofazimine of suitable particle size is a particle having an average size of less than 5 μm and a D90 of less than 6 μm. In another embodiment, clofazimine of suitable particle size is a particle having an average size of less than 2 μm and a D90 of less than 3 μm.

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

(1) homogenizing a suspension of clofazimine, a nonionic surfactant and water to obtain a suspension comprising clofazimine of an appropriate particle size;

(2) adjusting the pH of the suspension produced from (1) above to a pH of pH 5.5 to pH 7.5, and

(3) adjusting the sodium chloride concentration to an appropriate concentration, and

(4) adjusting the osmotic pressure to an appropriate level.

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

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

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

(2) isolating clofazimine;

(3) adding clofazimine to a nonionic surfactant and water;

(4) adjusting the pH of the suspension produced from (3) above to a pH of pH 5.5 to pH 7.5, and

(5) adjusting the sodium chloride concentration to an appropriate concentration.

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

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

(1) micronizing clofazimin to obtain clofazimine of an appropriate particle size;

(2) adding clofazimine to a nonionic surfactant and water;

(3) adjusting the pH of the suspension produced from (2) above to a pH of pH 5.5 to pH 7.5, and

(4) adjusting the sodium chloride concentration to an appropriate concentration.

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

In another embodiment, the composition contains an appropriate concentration of sodium chloride and is adjusted to a pH of between pH 5.5 and pH 7.5 by homogenizing a suspension of clofazimine, a non-ionic surfactant and water to obtain an appropriate particle size of clofa. There is provided a method for preparing a pharmaceutical composition according to any one of the pharmaceutical composition embodiments described herein, prepared by a method comprising the step of obtaining jimin. In another embodiment, the pH is 7.4 and an appropriate concentration of sodium chloride is 154 mM sodium chloride. In another embodiment, the homogenization is performed by high pressure homogenization, wet milling, ultrasonic homogenization, or a combination of these treatments. In another embodiment, the homogenization of clofazimine is carried out in several stages of homogenization. In another embodiment, clofazimine of suitable particle size is a particle having an average size of less than 5 μm and a D90 of less than 6 μm. In another embodiment, clofazimine of suitable particle size is a particle having an average size of 2 μm and a D90 of less than 3 μm.

In another embodiment, the steps of: (a) homogenizing a suspension of clofazimine, a nonionic surfactant and water to obtain a suspension comprising clofazimine of appropriate particle size; (b) adjusting the pH of the resulting suspension to a pH of pH 5.5 to pH 7.5; (c) adjusting the sodium chloride concentration to an appropriate concentration; and (d) adjusting the osmotic pressure to an appropriate level; Steps (b), (c) and (d) are (b), (c), (d); (b), (d), (c); (c), (b), (d); (c), (d), (b); (d), (b), (c); or (d), (c), (b), there is provided a method for preparing a pharmaceutical composition according to any one of the composition embodiments described herein.

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

In another embodiment, the steps of: (a) micronizing clofazimine to obtain clofazimine of an appropriate particle size; and (b) adding clofazimine to water and a non-ionic surfactant containing an appropriate concentration of sodium chloride and adjusted to a pH of between pH 5.5 and pH 7.5. A method for preparing a pharmaceutical composition according to one is provided.

In another embodiment of the present invention, any one of the composition embodiments described herein with a nebulizing device selected from an ultrasonic nebulizer, an electronic spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer and a mechanical soft mist inhaler. A pharmaceutical combination is provided in the form of an aerosol for inhalation, wherein the aerosol particles produced by a nebulizing device have a mass median aerodynamic diameter of 1 to 5 μm, prepared by aerosolization of a composition according to In another embodiment, the aerosol for inhalation is for lower lung deposition. In another embodiment, the nebulizing device exhibits a yield rate of 0.1 to 1.0 ml/min. In another embodiment, the total inhalation volume is between 1 ml and 5 ml.

In another embodiment, nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth thiol, ethylene diamine tetraacetic acid, gentamicin Phosphatidylcholine-decorated gold nanoparticles loaded, chelator, cis-2-decenoic acid, D-amino acid, D-enantiomer peptide, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicylic acid, baicalin , naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antibacterial peptides, biofilm dispersing and / or disrupting agents, mucolytics and / or mucoactive agents, and / or reduction of biofilm formation There is provided a pharmaceutical composition according to any one of the composition embodiments described herein for use in combination with an agent. In another embodiment, the composition comprises bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and para-amino salicyl. rate, and mixtures thereof prior to, concurrently with, or subsequent to administration of an agent selected from the group consisting of:

In another embodiment, nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth thiol, ethylene diamine tetraacetic acid, gentamicin Phosphatidylcholine-decorated gold nanoparticles loaded, chelator, cis-2-decenoic acid, D-amino acid, D-enantiomer peptide, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicylic acid, baicalin , naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antibacterial peptides, biofilm dispersing and / or disrupting agents, mucolytics and / or mucoactive agents, and / or reduction of biofilm formation There is provided a pharmaceutical combination according to any one of the combination embodiments described herein for use in combination with an agent. In another embodiment, the combination is bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and para-amino salicy. sylates, and mixtures thereof, prior to, concurrently with or subsequent to administration of an agent selected from the group consisting of sylates, and mixtures thereof. In another embodiment, the composition is administered prior to, concurrently with or after administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof. In another embodiment, the composition is administered before, concurrently 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 as described herein for use in the treatment and/or prophylaxis of a lung infection caused by mycobacteria or other gram positive bacteria. In another embodiment, the infection is caused by a species of the genus Mycobacterium selected from non-tuberculous Mycobacterium and Mycobacterium tuberculosis complexes, and combinations thereof. In another embodiment, the non-tuberculous mycobacterium is Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium. Mycobacterium leprae, and combinations thereof. In another embodiment, the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient suffering from cystic fibrosis, chronic obstructive pulmonary or acquired immunodeficiency syndrome. In another embodiment, the infection is an opportunistic non-tuberculous mycobacterial infection in a patient suffering from cystic fibrosis. In another embodiment, the composition comprises bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and para-amino salicyl. rate, and mixtures thereof prior to, concurrently with, or subsequent to administration of an agent selected from the group consisting of: In another embodiment, the composition is administered prior to, concurrently with or after administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof. In another embodiment, the composition is administered before, concurrently with or after the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.

In another embodiment, there is provided a pharmaceutical combination according to any one of the combination embodiments as described herein, for use in the treatment and/or prophylaxis of lung infections caused by mycobacteria or other gram positive bacteria. do. In another embodiment, the infection is caused by a species of the genus Mycobacterium selected from non-tuberculous Mycobacterium and Mycobacterium tuberculosis complexes, and combinations thereof. In another embodiment, the non-tuberculous mycobacterium is Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium. Mycobacterium leprae, and combinations thereof. In another embodiment, the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient suffering from cystic fibrosis, chronic obstructive pulmonary or acquired immunodeficiency syndrome. In another embodiment, the infection is an opportunistic non-tuberculous mycobacterial infection in a patient suffering from cystic fibrosis. In another embodiment, the combination is bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and para-amino salicy. sylates, and mixtures thereof, prior to, concurrently with or after administration of an agent selected from the group consisting of sylates, and mixtures thereof. In another embodiment, the combination is prior to, concurrent with or after administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof, in the composition of the invention used to administer In another embodiment, the combination is used to administer the composition of the present invention prior to, concurrently with or after the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.

In another embodiment, there is provided a system for use in providing antibiotic activity when treating or providing prophylaxis against a lung infection caused by mycobacteria or other gram positive bacteria, comprising:

1) a nebulized pharmaceutical combination comprising:

(a) a therapeutically effective dose of clofazimine;

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

(c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solution;

and

2) nebulizer,

The clofazimine is provided in the form of a suspension,

The aerosol particles produced by the system have a mass median aerodynamic diameter of 1 to 5 μm.

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

In another embodiment, there is provided a pharmaceutical combination according to any one of the combination embodiments described herein for use in the treatment and/or prophylaxis of a pulmonary fungal infection or Clostridium difficile, or a combination thereof. . In another embodiment, there is provided a pharmaceutical combination according to any one of the combination embodiments described herein for use in the treatment and/or prophylaxis of a pulmonary fungal infection. In another embodiment, the pulmonary fungal infection is Candida albicans or Aspergilus 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 one of the composition embodiments described herein. In another embodiment, the infection is caused by a species of the genus Mycobacterium selected from non-tuberculous Mycobacterium and Mycobacterium tuberculosis complexes, and combinations thereof. In another embodiment, non-tuberculous Mycobacterium is Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus and Mycobacterium abscessus. Mycobacterium leprae, and combinations thereof. In another embodiment, the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient suffering from cystic fibrosis, chronic obstructive pulmonary disease or acquired immunodeficiency syndrome. In another embodiment, the infection is an opportunistic non-tuberculous mycobacterial infection in a patient suffering from cystic fibrosis.

In another embodiment, bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and para-amino salicylate; and mycobacteria in a patient in need thereof, comprising administering by inhalation a composition according to any one of the composition embodiments described herein prior to, concurrently with or after administration of an agent selected from mixtures thereof; Methods for treating or preventing lung infections caused by other gram-positive bacteria are provided. In another embodiment, the agent is bedaquiline or amikacin. In another embodiment, the agent is bedaquiline.

Particle size and distribution

The therapeutic effect of an aerosolized therapy depends on the dose deposited and its distribution. Aerosol particle size is one of the important variables in defining the dose deposited and the distribution of the drug aerosol in the lungs.

In general, inhaled aerosol particles are deposited by one of two mechanisms: impact, which is usually predominant for larger aerosol particles, and sedimentation, which is usually dominant for smaller aerosol particles. An impact occurs when the momentum of an inhaled aerosol particle is sufficiently large that the particle does not follow the airflow and encounters a physiological surface. Conversely, sedimentation occurs predominantly in the lower lungs when very small aerosol particles traveling with the inhaled air stream encounter physiological surfaces as a result of gravitational sedimentation.

Pulmonary drug delivery can be achieved by inhalation of an aerosol through the mouth and throat. Aerosol particles having an aerodynamic diameter greater than about 5 μm generally do not reach the lungs; Instead, they tend to affect the back of the throat and are swallowed and possibly absorbed orally. Aerosol particles having a diameter of about 3 to about 5 μm are small enough to reach the middle lung region (conducting airway) at the top, but too large to reach the alveoli. Smaller aerosol particles, ie, about 0.5 to about 3 μm, can reach the alveolar region. Aerosol particles having a diameter of less than about 0.5 μm tend to exhale during normal breathing, but may also deposit in the alveolar region by holding the breath.

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

Nevertheless, for explanatory purposes, the aerosol particle size of aerosol particles is determined according to the US Pharmacopeial Convention. In Process Revision <601> Aerosols, Nasal Sprays, Metered-Dose Inhalers, and Dry Powder Inhalers, Pharmacopeial Forum (2003), Volume Number 29, pages 1176-1210 also disclosed in Jolyon Mitchell, Mark Nagel "Particle Size Analysis of Aerosols from Medicinal Inhalers", KONA Powder and Particle Journal (2004), Volume 22, pages 32-65] will be given as MMAD as determined by measurement at room temperature with a Next Generation Impactor (NGI).

According to the present invention, the particle size of the aerosol is optimized to maximize the deposition of clofazimin at the site of infection and to maximize resistance. Aerosol particle size can be expressed as the mass median aerodynamic diameter (MMAD). Large particles (eg, MMAD > 5 μm) tend to deposit in the extrathoracic and upper airways because they are too large to detect bends in the airways. Tolerability (eg, cough and bronchospasm) may result from upper airway deposition of large particles.

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 less than 3 μm (< 3 μm).

However, with guided breathing exercises, central and lower lung deposition of aerosols will increase as larger particles can pass extrathoracic and upper airways and deeper into the lungs compared to during normal breathing. Guided breathing exercises can be as slow as 100 ml/min. Therefore, when used with induced breathing exercise, the MMAD of the preferred aerosol should be less than about 10 μm.

Another equally important factor (besides the aerosol particle size) is the particle size and size distribution of the solid particles, in this case the clofazimine particle size and distribution. The size of the solid particle in a given aerosol particle must be smaller than the aerosol particle it contains. Larger aerosol particles may contain one or more solid particles. Also, when handling dilute suspensions, the majority of aerosol particles may not contain solid particles.

For this reason, it is desirable to have solid drug particles that are significantly smaller than the MMAD of the aerosol particles. For example, if the MMAD of the aerosol particle is 3 μm, the desired solid particle will be 1 μm or less.

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

The solid particle size in suspension can be provided by the average size of the particles, and also by the distribution of the particles. The D90 value indicates that 90% of the particles in the suspension are below average size.

Nebulizer

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

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

The device preferably controls the patient's inhalation flow rate by an electrical or mechanical process.

In another preferred embodiment, the aerosol generation by the device is triggered by inhalation of the patient, for example using an AKITA device.

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

Use in treatment and/or prophylaxis

The pharmaceutical compositions and pharmaceutical combinations (aerosols, aerosolized formulations) and systems according to the present invention can be used with mycobacteria or other clofazimin sensitive bacteria, such as Staphylococcus aureus (methicillin resistant and vancomycin intermediates). resistant strains), Streptococcus pneumoniae and Enterococcus spp. (Enterococcus spp.) for use in the treatment and/or prevention of lung infections caused by. The pharmaceutical compositions and pharmaceutical combinations of the present invention may also be used in the treatment and/or prophylaxis of pulmonary fungal infections.

Dosage of Clofazimine

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

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

The daily lung dose (ie the dose deposited in the lung) of clofazimine to be administered according to the present invention is about 5-10 mg, which is equivalent to M. Corresponds to a nominal dose of 15-30 mg (device dose) in case of M. abscessus infection.

One of ordinary skill in the art will know that, for the minimum inhibitory concentration (MIC) of clofazimin for each bacterial strain well established in the art, the pulmonary dose of clofazimin to be administered (and thus the fill/nominal dose/volume to be nebulized) is understood to be routinely adjusted.

Depending on the frequency of administration, the once or twice daily, daily lung dose will be divided accordingly.

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

It will be clear to those skilled in the art that the above amounts relate to clofazimine free base, and accordingly the dosages for derivatives and salts will have to be adjusted for the MIC of each compound and strain.

Mucolytics/biofilm modifiers

In order to reduce the sputum viscosity during aerosol treatment and also to destroy the biofilm present, the treatment and/or prophylaxis according to the present invention may comprise the further administration of a mucolytic agent and/or a biofilm disruptor.

These agents may be prepared as a fixed combination, or may be administered simultaneously with or after the pharmaceutical composition/aerosol combination comprising clofazimine according to the invention.

Biofilm dispersants/disruptors, mucolytics and/or mucoactive agents and/or reducing biofilm formation to be used according to the invention include nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, Sodium bicarbonate, tromethamine, silver nanoparticles, bismuth thiol, ethylene diamine tetraacetic acid, gentamicin loaded phosphatidylcholine-decorated gold nanoparticles, chelator, cis-2-decenoic acid, D-amino acid, D-enantiomer peptide , gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicylic acid, baicalin, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antibacterial peptides.

Moreover, other pharmaceutically active agents may also be used in combination with the pharmaceutical composition/aerosol combination according to the present invention. Such active agents include bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cepoxytin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and para-amino salicylate, and mixtures thereof. can be selected from

These agents may be prepared as a fixed combination or may be administered before, simultaneously with or after the pharmaceutical composition/aerosol combination comprising clofazimine according to the invention.

Example

The following examples serve not only to more fully explain how to use the invention described above, but also to suggest the best mode contemplated for carrying out various aspects of the invention. Embodiments according to the invention fall within the scope of the claims of the present application.

Experiment

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

Example 1

200 mg of clofazimine (triclinic form I), 90 mg of sodium chloride, and 9.5 ml of water were mixed twice in an Ultra-Turrax homogenizer at 10,000 rpm for 5 minutes. 0.5 ml of polysorbate 80 (NOF Hx2) were added. This mixture was treated with an ultrasonic probe (Branson Digital Sonifier ^TM 250D with Bandelin Sonoplus Probe MS73) 7 times, 3 minutes each, with an amplitude of 70%. The volume was adjusted to 10 ml with water. This suspension was filtered through a VWR folded qualitative filter paper (303, particle retention 5-13 μm, size: 150 mm) to obtain the composition of Example 1. The composition of Example 1 had a median particle size of clofazimine of 3.9 μm, and a D90 of 6.7 μm. The concentration of clofazimin was determined by UV/visible spectroscopy at 280 nm, calibrated with a stock solution of 1 mg/ml of clofazimin diluted in the mobile phase, and was determined to be 7.16 mg/ml.

The composition of Example 1 is shown in Table 1.

Table 1

Preparation of clofazimine of orthorhombic form III

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

Example 2

0.5% polysorbate 80 (NOF Hx2), and 0.6% of a suspension containing 100 ml of orthorhombic form of the water III claw wave Jimin 6 g containing sodium chloride by using the Ultra-Turrax ^® about 40 to 10,000 rpm pre-micronized for seconds. The preliminary formulation was prepared by adding 0.6% sodium chloride to water to create a volume of 300 ml. 300 ml of this suspension was added to the inlet of a homogenizer, M-110EH-30 Microfluidizer (Microfluidics, Westwood, MA, USA), and the suspension was circulated at 5,000 psi through a H30Z chamber to perform a pre-homogenization step for 15 minutes. did. A second H10Z chamber was then installed in series with the first chamber and the suspension was further homogenized at 25,000 psi for 23 minutes. Particle size analysis was performed with HORIBA LA 950, which showed a median particle size of 0.83 μm and a D90 value of 1.17 μm. The concentration of clofazimin at 16.05 mg/ml was determined by ultraviolet/visible spectroscopy at 280 nm, calibrated with a stock solution of 1 mg/ml clofazimin diluted in the mobile phase.

The composition of Example 2 is shown in Table 2.

Table 2

Example 3

A suspension of clofazimine (crystal-modified orthorhombic form III) in a solution of water, sodium chloride and polysorbate 80 was treated with an M-110EH-30 Microfluidizer, having the configuration H30Z-G10Z, operated at a pressure of 28,250 psi for 30 minutes. ^® Processor (chambers: H30Z and G10Z) to prepare the composition of Example 3, and the resulting particles of clofazimin had a median particle size of 1.28 μm and a D90 of less than 2 μm.

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 a STRESSTECH Rheometer in stress control mode. A double gap geometry was used and the spindle was rotated continuously to ensure that particulates remained in suspension during the temperature point. Viscosity was measured over 0.01, 0.05 and 0.1 Pa stresses at 20 °C, 25 °C and 30 °C, respectively. Two separate loadings were performed to obtain the average viscosities shown in Table 4 below.

Table 4

Animal Models and Efficacy Testing

In order to obtain preliminary data for establishing clofazimin concentration levels in lung tissue following direct respiratory delivery as opposed to systemic administration, the compositions of the present invention inhibit the growth of clinical NTM species in an acute in vivo lung infection mouse model. Their ability was tested. Depending on the bacterial species of interest, two distinct mouse models are used to investigate lung NTM infection. For the test, Mycobacterium avium 2285 and Mycobacterium abscessus 103 bacterial strains were used (see "Phylogenetic analysis of Mycobacterial species using whole genome sequences" for strain details). Hazbon MH, Riojas MA, Damon AM, Alalade R., Cantwell BJ, Monaco A., King S., Sohrabi A. Submitted (SEP-2014) to the EMBL/GenBank/DDBJ databases). These two species have previously been used in the literature as models of NTM infection (Obregon-Henao et al. 2015 Antimicrob Agents Chemother; and Chan et al. Animal Models of Non-Tuberculous Mycobacterial Infections, Mycobact Dis 2016).

In vivo safety study in Balb/C mice:

For in vivo safety and tolerability, 6-8 week old Balb/C female mice are obtained from Charles River. Mice are rested for 1 week prior to dosing. For each dose of clofazimine, 3 healthy mice are given a total of 3 doses every other day. Mice received 10.0, 5.01 and 2.51 mg/kg of clofazimine in the composition of Example 1. Compounds were ^{given by Microsprayer ®} aerosol intratracheal administration to 3 healthy mice for a total of 3 doses every other day.

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

Determination of the minimum inhibitory concentration

The minimum inhibitory concentration (MIC) test was performed by the micro-broth dilution method using Mueller Hinton (MH) broth (Cation Adjusted) with calcium and magnesium ion concentrations recommended by the CLSI standard M7-A7 (Becton Dickinson). The MIC test was also performed by the microbroth dilution method using 7H9 broth (Sigma-Aldrich). The reason for using both MH and 7H9 broth for compound screening is that anti-mycobacterial compounds have been found to exhibit different MIC activities depending on the broth used in the MIC assay.

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

Colony forming units (CFU) were obtained from agar plates, placed in MH or 7H9 broth with 0.05% tween-80, and after 3 days of growth (M. abscessus) or after 12 days (M. abscessus). avium (M. avium)) until the obtained optical density (OD) absorbance (OD) of 0.08 - 0.1 (0.5 McFarland Standard) is grown at 35-37°C in ambient air. Bacterial cell suspensions are then prepared and confirmed in saline consistent with (OD) 0.08 - 0.1 (0.5 McFarland Standard). Compound stock solutions were prepared by suspending the compound in DMSO at a concentration of 1.28 mg/ml and used immediately for the test range 64-0.062 μg/ml. Then, 180 μl of broth (MH or 7H9) was added to the first column in a 96 well plate, and 100 μl of broth was added to the remaining column in a 96 well plate. 20 μl of the compound stock solution was added to the first column of the well and serially diluted. Finally, 100 μl NTM cell suspension was added to all wells except the media-only control wells. QC agent specific to each organism 1) negative control for bacteria only 2) negative control for medium only 3) positive drug control for clarithromycin.

M. M. abscessus OD was analyzed on day 3, and M. abscessus OD was analyzed on day 3. M. avium OD was analyzed on day 12. After these measurements, the plates were analyzed using the Resazurin Microtiter Assay Plate method. Briefly, the method uses the addition of resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) to a 96 well plate. Resazurin is a blue dye, which itself is weakly fluorescing until it is irreversibly reduced to pink and very red fluorescent resorufin. It is used as a redox indicator to determine bacterial cell viability in MIC assays.

Analysis was performed in triplicate. Analysis #1 was performed after storing the composition of Example 1 at 4° C. for 2 months, analysis #2 was performed at 4 months, and analysis #3 was performed at 5 months.

Minimum inhibitory concentration in the presence and absence of CF sputum

The minimum inhibitory concentration assay 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 antibiotics for the previous 48 hours, and their Endogenous bacteria were removed by sterilization of the sputum by exposure to UV light. After sterilization, M. Abscessus (M. abscessus), M. M. avium, M. Intracellulare (M. intracellulare) and M. Chimaera (M. Chimaera) was cultured in 10% CF sputum, followed by a MIC test. In the presence and absence of cystic fibrosis patient sputum, the same CLSI protocol as described above was performed to determine the MIC of the composition of Example 1. All studies were performed in duplicate.

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

Table 5

The results presented in Table 5 show consistent MICs of both clofazimin and the composition of Example 1 against various non-tuberculous mycobacterial species.

These data show that the composition of Example 1 was M. Abscessus (M. abscessus) and M. It shows potent in vitro activity against both M. avium, indicating that it is stable for at least this period.

M. in SCID mice. Mouse model of M. abscessus

6-8 week old SCID female mice were ordered from Charles River. Mice were rested for 1 week before infection.

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

The acute SCID mouse model received ^{non-invasive endotracheal infusion lung infection at 1×10 6} CFU/mouse (M. abscessus strain 103).

To determine bacterial uptake, three mice were sacrificed 1 day post-infection. Whole lung, spleen and liver are extracted and homogenized in 4.5 ml of 1x PBS. Homogenates were serially diluted in 1:10 dilutions and dilutions (0-1-2-3-4-5-6-7) were plated on 7H11 agar plates. Plates are placed in a 32° C. dry-air incubator (strain dependent) for 7 days.

10.0 mg/kg of the composition of Example 1 was administered ^{via the pulmonary route by Microsprayer ®} (35 μl), and clofazimin (gavage), amikacin (subcutaneous) was administered in a volume of 200 μl per mouse, which was It started on day 2 post infection and continued every other day for 8 consecutive days.

Mice were sacrificed 2 days after administration of the last dose of compound. Six mice of all groups (untreated control group, clofazimine (gavage), composition of Example 1, and amikacin treated mice) were sacrificed and the bacterial loading was determined. Lung homogenate from 0-1-2-3-4-5-6-7, spleen homogenate from 0-1-2-3-4-5-6-7, and 0-1-2-3-4 Plating of liver homogenates at -5-6-7.

A Log 10 protection value of 0.60 or greater indicates that the activity is statistically significant. Statistical analysis was performed first by converting the CFU to logarithm, and then using this to analyze multiple comparisons of change by one-way ANOVA followed by one-way Tukey test (GraphPad Prism analysis software). was evaluated by Differences are considered significant at the 95% confidence level.

Table 6 shows the SCID mouse M. _{Mean Log 10} CFU data after M. abscessus infection and standard error of the mean (SEM) are shown, where “n” is the total number of animals in the group at the time of sacrifice.

Table 6

The data in Table 6 shows that the treatment with the composition of Example 1 was M. It has been shown to induce a maximal reduction in bacterial recovery in the lungs and spleen of animals infected with M. abscessus. This bacterial reduction was statistically improved compared to treatment with amikacin or oral clofazimin.

M in beige mice. A mouse model of M. avium infection

6-8 week old beige female mice were ordered from Charles River. Mice were rested for 1 week before infection.

The acute beige mouse model received ^{non-invasive aerosol exposed lung infections at 1×10 8} colony forming units (CFU)/ml (M. avium strain 2285 rough). M. A working stock of M. avium strain 2285 rough was frozen in 1 ml aliquots and stored at −80° C. prior to use. For infection, aliquots were thawed, triturated 20 times with a 1 ml Luer-lock syringe fitted with a 26 g needle, and diluted in sterile 1×phosphate buffered saline (PBS).

To determine bacterial uptake, three mice were sacrificed 1 and 7 days post infection. Whole lung, spleen and liver were extracted, homogenized in 4.5 ml of 1x PBS and diluted 1:10. Dilutions (0-1-2-3-4-5-6-7) were plated on 7H11/OADC, TSA and charcoal agar plates and incubated at 32° C. for 30 days in a dry-air incubator (strain dependent). do.

10.0 mg/kg of the composition of Example 1 ^{was administered via the pulmonary route by Microsprayer ®} (35 μl) and clofazimine (gavage) in a volume of 200 μl per mouse, starting on day 7 after infection and continued every other day for 10 consecutive days.

Mice were sacrificed 5 days after administration of the last dose of compound. Six mice of all groups (untreated control, clofazimin (gavage), and the composition of Example 1) were sacrificed and the bacterial loading was determined. Lung homogenate from 0-1-2-3-4-5-6-7, spleen homogenate from 0-1-2-3-4-5-6-7, and 0-1-2-3-4 Plating of liver homogenates at -5-6-7.

A Log 10 protection value of 0.60 or greater indicates that the activity is statistically significant. Statistical analysis was performed first by converting the CFU to logarithm, and then applying this to multiple comparison analysis of change by one-way ANOVA followed by one-way Tukey test (SigmaStat software program). was evaluated by Differences are considered significant at the 95% confidence level.

Table 7 shows the beige mouse M. _{Average Log 10} CFU data after infection with M. avium are shown.

Table 7

The data in Table 7 shows that the treatment with the composition of Example 1 was M. It has been shown to induce a greater reduction in bacterial recovery in the lungs and spleen of animals infected with M. avium.

Chronic beige mouse model

Six to eight week old beige mice were rested for one week prior to infection. Mice on day 0 M. M. avium 2285 rough of 1x10 ⁸ CFU of lung infection was received. To determine bacterial uptake and pretreatment bacterial loading, 3 mice were sacrificed on day 1 and 6 mice were sacrificed on day 27. Whole lung, spleen and liver were extracted, homogenized in 4.5 ml of 1xPBS and plated on 7H11 and charcoal agar plates with (0-1-2-3-4-5-6-7) dilution. Plates were placed in a 37° C. dry-air incubator for 25-30 days.

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

Mice were sacrificed on day 57, 2 days after the final treatment. Plates were placed in a 37° C. dry-air incubator for 30 days.

Statistical analysis was performed by first converting the CFU to logarithm and then evaluating it by one-way ANOVA, followed by multiple comparative analysis of change by one-way Tukey test. . Differences are considered significant at the 95% confidence level.

beige mouse m. Table 8 shows the _{average Log 10} CFU data after chronic M. avium infection.

Table 8

These data suggest that clofazimin has a difficult time penetrating granuloma-like structures formed by the established "chronic" animal NTM infection model. The composition of the present invention does not have the same problem, and it appears that the anti-mycobacterial activity can be maintained in a sufficiently established state even after infection.

Effect of the composition of Example 3 on barrier integrity and inflammation after exposure to lung epithelial cells ex vivo

cell viability

To evaluate lung epithelial cell viability, three different cell types, Calu-3, under two ex vivo conditions; A549; and hAELVi cells were used. Cells were treated either under “immersion conditions” (ie, in cell culture medium on Transwell™ plates), or under “air-liquid interface” mimicking conditions (ALI) with cell culture medium removed from the apical side of the cells. In the “submerged state”, Calu-3 cells were exposed to three doses of the composition of Example 3 (10%, 50%, or 100%) for 4 hours. To estimate cell viability, viable/dead cells were differentiated by staining the cells using acridine orange/propidium iodide (AO/PI) staining. Red fluorescence signaled cell death.

Macrophage uptake

THP-1 cells were cultured with 124 ng/ml phorbol 12-myristate 13 acetate (PMA) for 3 days, and then differentiated into macrophage-like cells. Once the cells were mature, they were exposed to the composition of Example 3 (diluted 1:200 in Hank's Buffered Salt Solution (HBSS)) for 4 hours. Cells were stained via AO/PI as described above to determine cell viability after exposure.

Transepithelial electrical resistance (TEER) measurement

Calu-3 cells were seeded on Transwell™ 3460 at 1×10 ⁵ cells/well and allowed to stand for 12 days to grow to confluence. TEER measurements were performed using EVOM2 (World Precision Instruments, Friedberg, Germany) according to the manufacturer's instructions. After seeding, Calu-3 cells were exposed to saline (negative control) or the composition of Example 3 (concentration: 20 mg/ml, 10 mg/ml, or 2 mg/ml). After exposing the cells for 2 to 4 hours, TEER was measured.

Inflammatory cytokine production

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

After incubation, the supernatant was removed from the cells at t = 4 h or 24 h and pooled. On the pooled supernatant samples, ELISA (Enzyme Linked ImmunoSorbent Assay) was performed. Separate ELISA kits were used for TNF-α, IL-6, IL-8 and IL-10 according to the manufacturer's instructions.

Statistical analysis was performed by one-way analysis of variance (ANOVA) of change followed by the Tukey post-hoc test. Statistical significance was determined at probability values < 0.05.

result

Under "submerged" conditions, the composition of Example 3 did not result in a visual decrease in cell viability over 4 hours of incubation at any concentration administered.

Under the “ALI” condition, three different cell types (Calu-3, A549, and HAELVi cells) were irradiated over three different time points (5 hours, 2 days, and 7 days). Little or no cytotoxicity was observed at 4 hours in any cells, or at 2 days in Calu-3 cells. Some toxicity was seen on days 2 and 7 in A549 cells and at day 7 in Calu-3 cells. Technical limitations did not allow quantification of cell death.

Regarding macrophage uptake, differentiated THP-1 cells were cultured in 1:200 HBSS for 4 hours to determine macrophage cell viability after exposure. The composition of Example 3 did not induce apoptosis, but showed clofazimin uptake by macrophages.

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

Exposure of Calu-3 cells to the composition of Example 3 did not affect barrier integrity after 1 hour exposure. Exposure at 20 mg/ml resulted in a significant (ie ≧50%) decrease after 2 hours. At a concentration of 10 mg/ml, there was a slight decrease (ie 25-35%) at all time points after 2 h. Exposure at 2 mg/ml did not show any decrease in barrier function during the entire study period.

Inflammatory cytokine production

The positive control LPS worked as expected in this model. The composition of Example 3 demonstrated no significant changes in cytokines at any time point investigated.

A result is shown in Table 9.

Cytokine production after dTHP-1 cell exposure

Table 9 ( ^* P < 0.05)

In vivo safety and tolerability

6-8 week old pld Balb/C female mice received a total of 3 doses every other day. Mice were administered with the composition of Example 1 at 10.0, 5.01 and 2.51 mg/kg. The composition was ^{given via Microsprayer ®} aerosol intratracheal (IT) administration in a volume of 35 μl/mouse. After injection, the mice were observed at 10 minutes, 1, 2 and 4 hours after administration, and then daily thereafter.

Table 10 shows the overall observations after administration. "BAR" indicates that the animal was bright, active and responsive.

Table 10

Table 11 shows the body weights of the animals during the 3 days tested.

Table 11

These data indicate that there was no statistically significant change in body weight during 3 days of treatment.

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

Claims (75)

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 greater than 10; and
(c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solution;
The clofazimine or a pharmaceutically acceptable derivative or salt thereof is provided in the form of particles in suspension,
The particles of clofazimine or a pharmaceutically acceptable derivative or salt thereof have a median size of less than 5 μm and a D90 of less than 6 μm. The pharmaceutical composition according to claim 1, wherein the particles of clofazimine or a pharmaceutically acceptable derivative or salt thereof have an average size of less than 2 μm and a D90 of less than 3 μm. A pharmaceutical composition comprising:
(a) a therapeutically effective dose of clofazimine;
(b) a nonionic surfactant having a hydrophilic-lipophilic balance value greater than 10; and
(c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solution;
The clofazimine is provided in the form of particles in suspension,
The particles of clofazimin have a median size of less than 5 μm and a D90 of less than 6 μm. 4. The pharmaceutical composition of claim 3, wherein the particles have a median size of less than 2 μm and a D90 of less than 3 μm. 5. The method according to claim 3 or 4, wherein clofazimine is provided in a polymorphic form selected from triclinic form FI, monoclinic form FII and orthorhombic form FIII and mixtures of these forms. pharmaceutical composition. 6. The pharmaceutical composition of claim 5, wherein the clofazimine is provided in a substantially orthorhombic form FIII. 7. The nonionic surfactant according to any one of claims 1 to 6, wherein the nonionic surfactant has a hydrophilic-lipophilic balance value of 14 to 16, polysorbate 20, polysorbate 60, polysorbate 80, stearyl alcohol. Polyethylene glycol derivatives of hydrogenated castor oil, polyethylene glycol derivatives of hydrogenated castor oil having a hydrophilic-lipophilic balance value of 15 to 17, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, polyoxyethylene ( 20) oleyl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (10) cetyl ether, polyoxyethylene (10) oleyl ether, polyoxyethylene (100) stearyl ether, polyoxyethylene (10) Stearyl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (4) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (2) cetyl ether, caprylocaproyl polyoxyl-8 glycerides, polyethylene glycol (20) monostearate, polyethylene glycol (40) stearate, polyethylene glycol (100) stearate, polyethylene glycol (8) stearate, and polyoxyl 40 stearate, and mixtures thereof. composition. The pharmaceutical composition according to any one of claims 1 to 7, wherein the nonionic surfactant is polysorbate 80 and the aqueous liquid carrier is distilled water, hypertonic saline or isotonic saline. The pharmaceutical composition according to claim 8, wherein the hypertonic saline is 1% to 7% (w/v) sodium chloride. The pharmaceutical composition according to claim 8, wherein the nonionic surfactant is ultrapure polysorbate 80 and the aqueous liquid carrier is isotonic saline. 11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the osmotic pressure of the composition is in the range of 200-700 mOsm/kg. 11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the osmotic pressure of the composition is in the range of 300-400 mOsm/kg. 11. The method according to any one of claims 1 to 10, wherein the nonionic surfactant ranges from 0.001% to 5% (v/v) of the total composition, and the amount of clofazimine is from 0.1% to 20% of the total composition ( w/v). 14. The pharmaceutical composition according to any one of claims 1 to 13, which is prepared by a method comprising the steps of:
(1) homogenizing a suspension of clofazimine, a nonionic surfactant and water to obtain a suspension comprising clofazimine of an appropriate particle size;
(2) adjusting the pH of the suspension produced from (1) above to a pH of pH 5.5 to pH 7.5, and
(3) adjusting the sodium chloride concentration to an appropriate concentration, and
(4) adjusting the osmotic pressure to an appropriate level. 14. The pharmaceutical composition according to any one of claims 1 to 13, which is prepared by a method comprising the steps of:
(1) homogenizing the suspension of clofazimine and a non-aqueous liquid to obtain a suspension comprising clofazimine of appropriate particle size;
(2) isolating clofazimine;
(3) adding clofazimine to a nonionic surfactant and water;
(4) adjusting the pH of the suspension produced from (3) above to a pH of pH 5.5 to pH 7.5, and
(5) adjusting the sodium chloride concentration to an appropriate concentration. 14. The pharmaceutical composition according to any one of claims 1 to 13, which is prepared by a method comprising the steps of:
(1) micronizing clofazimin to obtain clofazimine of an appropriate particle size;
(2) adding clofazimine to a nonionic surfactant and water;
(3) adjusting the pH of the suspension produced from (2) above to a pH of pH 5.5 to pH 7.5, and
(4) adjusting the sodium chloride concentration to an appropriate concentration. 14. A suitable solution according to any one of claims 1 to 13, comprising homogenizing a suspension of clofazimine, a nonionic surfactant and water, containing an appropriate concentration of sodium chloride, adjusted to a pH of between pH 5.5 and pH 7.5. A pharmaceutical composition prepared by a method comprising the step of obtaining clofazimine in particle size. 17. The pharmaceutical composition according to any one of claims 14, 15 or 16, wherein the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154 mM sodium chloride. 18. The pharmaceutical composition according to claim 17, wherein the pH is 7.4 and the appropriate concentration of sodium chloride is 154 mM sodium chloride. 17. The pharmaceutical composition of claim 16, wherein the micronization of clofazimine is performed by jet milling, spray drying, ball milling, or supercritical fluid treatment. 20. The method according to any one of claims 14, 15, 17 or 19, wherein the homogenization in step (1) is carried out by high pressure homogenization, high shear homogenization, wet milling, ultrasonic homogenization, or a combination of these treatments. A pharmaceutical composition performed. 22. The pharmaceutical composition according to any one of claims 14, 15, 17, 18, 19 or 21, wherein the homogenization of clofazimine is carried out in several stages of homogenization. The pharmaceutical composition according to claim 16 or 20, wherein the micronization of clofazimine is carried out in several stages of micronization. 24. The pharmaceutical composition according to any one of claims 14 to 23, wherein the clofazimine of suitable particle size is a particle having an average size of less than 5 μm and a D90 of less than 6 μm. 24. The pharmaceutical composition according to 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 μm and a D90 of less than 3 μm. 26 by aerosolization of the composition according to any one of claims 1 to 25 by a nebulizing device selected from ultrasonic nebulizers, electronic spray nebulizers, vibrating membrane nebulizers, jet nebulizers and mechanical soft mist inhalers. manufactured,
A pharmaceutical combination in the form of an aerosol for inhalation, wherein said aerosol particles produced by a nebulizing device have a mass median aerodynamic diameter of 1 to 5 μm. 27. The pharmaceutical combination according to claim 26, wherein the aerosol for inhalation is for lower lung deposition. 26. The method of any one of claims 1 to 25, wherein nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth thiol, Ethylene diamine tetraacetic acid, gentamicin loaded phosphatidylcholine-decorated gold nanoparticles, chelator, cis-2-decenoic acid, D-amino acid, D-enantiomer peptide, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, par biofilm dispersing and/or disrupting agents, mucolytics and/or mucoactive agents, selected from tuline, penicylic acid, baicalin, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides and/or a pharmaceutical composition for use in combination with a reducing agent of biofilm formation. 28. The nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nanoparticles, bismuth thiol, ethylene diamine tetraacetic acid according to claim 26 or 27. , Gentamicin loaded phosphatidylcholine-decorated gold nanoparticles, chelator, cis-2-decenoic acid, D-amino acid, D-enantiomer peptide, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicylic acid , baicalin, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptides, dispersing and / or disrupting agents of biofilms, mucolytics and / or mucoactive agents, and / or biofilms A pharmaceutical combination for use in combination with a formation reducing agent. 26. A pharmaceutical composition according to any one of claims 1 to 25 for use in the treatment and/or prophylaxis of lung infections caused by mycobacteria or other gram positive bacteria. 28. A pharmaceutical combination according to claim 26 or 27 for use in the treatment and/or prophylaxis of lung infections caused by mycobacteria or other gram positive bacteria. 31. The pharmaceutical composition according to claim 30, wherein the infection is caused by a species of the genus Mycobacterium selected from non-tuberculous Mycobacterium and Mycobacterium tuberculosis complex, and combinations thereof. 32. The pharmaceutical combination according to claim 31, wherein the infection is caused by a species of the genus Mycobacterium selected from non-tuberculous Mycobacteria and Mycobacterium tuberculosis complexes, and combinations thereof. 33. The method of claim 32, wherein the non-tuberculous mycobacterium is Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus and Mycobacterium. A pharmaceutical composition selected from Mycobacterium leprae, and combinations thereof. 34. The method of claim 33, wherein the non-tuberculous mycobacterium is Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus and Mycobacterium. A pharmaceutical combination selected from Mycobacterium leprae, and combinations thereof. The pharmaceutical composition according to claim 32, wherein the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient suffering from cystic fibrosis, chronic obstructive pulmonary or acquired immunodeficiency syndrome. The pharmaceutical combination according to claim 33, wherein the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient suffering from cystic fibrosis, chronic obstructive pulmonary or acquired immunodeficiency syndrome. 37. The pharmaceutical composition of claim 36, wherein the infection is an opportunistic non-tuberculous mycobacterial infection in a patient suffering from cystic fibrosis. 38. The pharmaceutical combination according to claim 37, wherein the infection is an opportunistic non-tuberculous mycobacterial infection in a patient suffering from cystic fibrosis. A system for use in providing antibiotic activity when treating or providing prophylaxis against a lung infection caused by mycobacteria or other gram-positive bacteria, comprising:
1) a nebulized pharmaceutical combination comprising:
(a) a therapeutically effective dose of clofazimine;
(b) a nonionic surfactant having a hydrophilic-lipophilic balance value greater than 10; and
(c) an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solution;
and
2) nebulizer,
The clofazimine is provided in the form of a suspension,
The aerosol particles produced by the system have a mass median aerodynamic diameter of 1 to 5 μm. 40. Use according to any one of claims 28 to 39, wherein the nebulizing device exhibits a yield rate of 0.1-1.0 ml/min. 42. Use according to any one of claims 28 to 39 or 41, wherein the total inhalation volume is between 1 ml and 5 ml. 39. The composition of any one of claims 28, 30, 32, 34, 36 or 38, wherein the composition comprises bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, Use of an agent selected from micassin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and para-amino salicylate, and mixtures thereof prior to, concurrently with or after administration thereof 40. The method of any one of claims 29, 31, 33, 35, 37 or 39, wherein the combination comprises bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin; Use of an agent selected from amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and para-amino salicylate, and mixtures thereof prior to, concurrently with or after administration thereof. 26. A pharmaceutical composition according to any one of claims 1 to 25 for use in the treatment and/or prophylaxis of a pulmonary fungal infection or Clostridium difficile, or a combination thereof. The pulmonary fungal infection or Clostridium difficile according to any one of claims 26, 27, 29, 31, 33, 35, 37 or 39, or these A pharmaceutical combination for use in the treatment and/or prophylaxis of a combination of 46. A pharmaceutical composition according to claim 45 for use in the treatment and/or prophylaxis of pulmonary fungal infections. 47. A pharmaceutical combination according to claim 46 for use in the treatment and/or prophylaxis of pulmonary fungal infections. 48. The pharmaceutical composition according to claim 45 or 47, wherein the pulmonary fungal infection is Candida albicans or Aspergilus fumigatus, or a combination thereof. 49. The pharmaceutical combination according to claim 46 or 48, wherein the pulmonary fungal infection is Candida albicans or Aspergilus fumigatus, or a combination thereof. 44. The use according to claim 43, wherein the composition is administered prior to, concurrently with or after the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof. 44. The use according to claim 43, wherein the composition is administered before, concurrently with or after the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof. A method for treating or preventing a pulmonary infection in a patient in need thereof, comprising administering the composition according to any one of claims 1 to 25 by inhalation. 54. The method of claim 53, wherein the infection is caused by a species of the genus Mycobacterium selected from non-tuberculous Mycobacterium and Mycobacterium tuberculosis complex, and combinations thereof. 55. The method of claim 54, wherein the non-tuberculous Mycobacterium is Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus and Mycobacterium. A method of treatment or prophylaxis selected from Mycobacterium leprae, and combinations thereof. 54. The method of claim 53, wherein the infection is an opportunistic infection selected from MAC lung disease and non-tuberculous infection in a patient suffering from cystic fibrosis, chronic obstructive pulmonary disease or AIDS. 57. The method of claim 56, wherein the infection is an opportunistic non-tuberculous mycobacterial infection in a patient suffering from cystic fibrosis. bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitin, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin and para-amino salicylate, and mixtures thereof To 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 prior to, concurrently with or after administration of the agent. A method for treating or preventing a lung infection caused by 59. The method of claim 58, wherein the agent is bedaquiline or amikacin. 60. The method of claim 59, wherein the agent is bedaquiline. 14. A method 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 comprising clofazimine of an appropriate particle size;
(2) adjusting the pH of the suspension produced from (1) above to a pH of pH 5.5 to pH 7.5, and
(3) adjusting the sodium chloride concentration to an appropriate concentration, and
(4) adjusting the osmotic pressure to an appropriate level. 14. A method for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising the steps of:
(1) homogenizing the suspension of clofazimine and a non-aqueous liquid to obtain a suspension comprising clofazimine of appropriate particle size;
(2) isolating clofazimine;
(3) adding clofazimine to a nonionic surfactant and water;
(4) adjusting the pH of the suspension produced from (3) above to a pH of pH 5.5 to pH 7.5, and
(5) adjusting the sodium chloride concentration to an appropriate concentration. 14. A method for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising the steps of:
(1) micronizing clofazimin to obtain clofazimine of an appropriate particle size;
(2) adding clofazimine to a nonionic surfactant and water;
(3) adjusting the pH of the suspension produced from (2) above to a pH of pH 5.5 to pH 7.5, and
(4) adjusting the sodium chloride concentration to an appropriate concentration. Homogenizing a suspension of clofazimin, a nonionic surfactant and water, containing an appropriate concentration of sodium chloride, adjusted to a pH of between pH 5.5 and pH 7.5, to obtain clofazimine of an appropriate particle size, 14. A method for preparing a pharmaceutical composition according to any one of claims 1 to 13. 64. The process according to any one of claims 61, 62 or 63, wherein the pH is adjusted to 7.4 and the sodium chloride concentration is adjusted to 154 mM sodium chloride. 65. The process according to claim 64, wherein the pH is 7.4 and the appropriate concentration of sodium chloride is 154 mM sodium chloride. 64. The method of claim 63, wherein the micronization of clofazimine is performed by jet milling, spray drying, ball milling, or supercritical fluid treatment. 67. The method according to any one of claims 61, 62, 64 or 66, wherein the homogenization in step (1) is carried out by high pressure homogenization, wet milling, ultrasonic homogenization, or a combination of these treatments. . 69. A process according to any one of claims 61, 62, 64, 65, 66 or 68, wherein the homogenization of clofazimine is carried out in several stages of homogenization. The method according to claim 63 or 20, wherein the micronization of clofazimine is carried out in several stages of micronization. 71. The process according to any one of claims 61 to 70, wherein the clofazimine of suitable particle size is a particle having an average size of less than 5 μm and a D90 of less than 6 μm. 71. The process according to any one of claims 61 to 70, wherein the clofazimine of suitable particle size is a particle having an average size of less than 2 μm and a D90 of less than 3 μm. 14. A method 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 comprising clofazimine of an appropriate particle size;
(b) adjusting the pH of the resulting suspension to a pH of pH 5.5 to pH 7.5;
(c) adjusting the sodium chloride concentration to an appropriate concentration; and
(d) adjusting the osmotic pressure to an appropriate level;
Steps (b), (c) and (d) are (b), (c), (d); (b), (d), (c); (c), (b), (d); (c), (d), (b); (d), (b), (c); or (d), (c), (b) in the following order. 14. A method 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 a non-aqueous liquid to obtain a suspension comprising clofazimine of appropriate particle size;
(b) isolating clofazimine;
(c) adding clofazimine to the nonionic surfactant and water;
(d) adjusting the pH of the resulting suspension to a pH of between pH 5.5 and pH 7.5; and
(e) adjusting the sodium chloride concentration to an appropriate concentration;
Steps (d) and (e) are (d), (e); or (e), may occur in the order of (d). 14. A method for preparing a pharmaceutical composition according to any one of claims 1 to 13, comprising the steps of:
(a) micronizing clofazimin to obtain clofazimine of an appropriate particle size; and
(b) adding clofazimine to water and a non-ionic surfactant containing an appropriate concentration of sodium chloride, adjusted to a pH of between pH 5.5 and pH 7.5.