WO2019110099A1

WO2019110099A1 - Inhalable clofazimine formulation

Info

Publication number: WO2019110099A1
Application number: PCT/EP2017/081721
Authority: WO
Inventors: Stefan Ufer; Thomas Hofmann
Original assignee: Qrumpharma Inc.
Priority date: 2017-12-06
Filing date: 2017-12-06
Publication date: 2019-06-13

Abstract

The present invention relates to pharmaceutical compositions comprising a therapeutically effective dose of clofazimine, wherein the clofazimine is solubilized in the form of an emulsion. Furthermore, the present invention provides pharmaceutical formulations comprising clofazimine in the form of an aerosol for inhalation. The formulations and compositions provided by the present invention may be used in the treatment and/or prophylaxis of pulmonary infection caused by mycobacteria and other gram-positive bacteria.

Description

Inhalable clofazimine formulation

Field of the invention

The present invention relates to liquid pharmaceutical compositions for inhalation comprising a therapeutically effective dose of clofazimine wherein the clofazimine is solubilized in the form of an emulsion. Furthermore, the present invention provides pharmaceutical formulations comprising clofazimine in the form of an aerosol for pulmonary inhalation.

The formulations and compositions provided by the present invention may be used in the treatment and/or prophylaxis of pulmonary infections caused by mycobacteria and other gram-positive bacteria.

Background of the invention

Clofazimine is an extremely hydrophobic riminophenazine antibiotic (Log P = 7.66) with anti-mycobacterial and anti-inflammatory activities and was originally described in 1957. Its structural formula is as follows:

Cl

Cl The exact mechanism through which clofazimine exerts its antimicrobial effect is unknown. However, it is known to bind preferentially to mycobacterial DNA, thereby inhibiting DNA replication and cell growth. Other suggested mechanisms of action include membrane damage/destabilization, generation of membrane-destabilizing lysophospholipids, interference of potassium transport, and/or intracellular redox cycling. While impressively active against Mycobacterium tuberculosis (MTB) in vitro, including MDR strains, clofazimine, until recently, was generally considered to be ineffective in the treatment of pulmonary tuberculosis (Cholo M et al.. J Antimicrob Chemother. 2012 Feb: 67(2):290-8).

Clofazimine is one of the three principal drugs recommended by the WHO for the treatment of leprosy which is caused by Mycobacterium leprae and has been increasingly used for the treatment of other mycobacterial infections such as drug resistant tuberculosis and infections caused by nontuberculous mycobacteria (NTM) in recent years.

Clofazimine has been classified as a Biopharmaceutics Classification System (BCS) class II drug as it is practically insoluble in water and shows high membrane permeability.

To overcome the problems associated with poor (oral) absorption and poor bioavailability of drugs, various strategies have been applied such as micronization, nanonization, supercritical fluid re-crystallization, spray freeze drying into liquid, solid dispersions and solutions in optimizing oral dosage forms.

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

In line with this, because of its lipophilicity, clofazimine is generally administered as a microcrystalline suspension in an oil-wax base to improve oral absorption. The absorption in humans after oral administration varies considerably (45-62%). Adverse effects of clofazimine are dose related and primarily affect the skin, eyes and gastrointestinal tract. Side effects include the development of reddish-brown discoloration of the skin and conjunctiva are gradually reversible on cessation. They are the result of chronic systemic accumulation. Mycobacterium is a genus Actinobacteria, with its own genus, Mycobacteriaceae. Mycobacteria have characteristic rod-like shapes and waxy outer coats.

As such Mycobacteria can be divided into three groups:

• Mycobacterium tuberculosis complex - causative pathogen of tuberculosis

• Mycobacterium leprae - causative pathogen of leprosy

• Nontuberculous mycobacteria (NTM) which encompass all other mycobacteria that are not M. tuberculosis or M. leprae, including Mycobacterium abscessus complex (MABSC), Mycobacterium avium complex (MAC).

The group of nontuberculous mycobacteria (NTM), formerly called atypical or ubiquitous mycobacteria, contains over 150 species. NTM can be found ubiquitously in nature and show a broad diversity. They can be detected in soil, ground and drinking water as well as in food like pasteurized milk or cheese. In general, NTM are considered to be less pathogenic. Nevertheless, they can cause severe illness in humans, especially in immune compromised persons or those who suffer from previous pulmonary diseases. Currently NTM are classified according to their growth rate and are divided into slow-growing (SGM) and rapid-growing (RGM) mycobacteria.

The slow growing Mycobacterium avium complex (MAC) comprises the species Mycobacterium avium, Mycobacterium chimaera and Mycobacterium intracellulare that are among the most important and most frequent pathogenic NTM. Just like Mycobacterium kansasii, Mycobaceterium malmoense, Mycobacterium xenopi, Mycobacterium simiae, Mycobacterium abscessus, Mycobacterium gordonae, Mycobacterium fortuitum, and Mycobacterium chelonae, they mostly cause pulmonary infections. Mycobacterium marinum is responsible for skin and soft tissue infections like aquarium granuloma.

In particular, RGM cause serious, life-threatening chronic lung diseases and are responsible for disseminated and often fatal infections. Infections are typically caused by contaminated materials and invasive procedures involving catheters, non-sterile surgical procedures or injections and implantations of foreign bodies. Exposure to shower heads and jacuzzis has also been reported as risks for infections. NTM typically cause opportunistic infections in patients with (chronic) pulmonary diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and (other) immune compromised patients.

In recent years, the rapidly growing (RGM) Mycobacterium abscessus group strains ( Mycobacterium abscessus complex, MABSC) comprising the subspecies Mycobacterium abscessus subsp. abscessus (M. a. abscessus), Mycobacterium abcessus bolletii. and Mycobacterium abscessus massiliense have emerged as important human pathogens and are associated with significantly higher fatality rates than any other RGM.

Mycobacterium abscessus infection in CF patients are particularly problematic, as it results in enhanced pulmonary destruction and is often impossible to treat with failure rates as high as 60-66%. (Obreqon-Flenao A et al. Antimicrobial Agents and Chemotherapy, November 2015, Vol 59, No 1 1 , p. 6904-6912; Qvist.T., Pressler.T., Floibv.N· and Katzenstein.TL.,“Shifting paradigms of nontuberculous mycobacteria in cystic fibrosis”. Respiratory Research (201 4). 15(1 ) :PP.41 -47).

Human infection with NTM became of greater relevance with the emergence of the human acquired immune deficiency syndrome pandemic. Mycobacteria from Mycobacterium avium complex (MAC) were identified as the major cause of opportunistic infections in patients infected with the human immunodeficiency virus (HIV).

Several species of NTM are known to form biofilms. Biofilms are microcolonies of bacteria embedded in the extracellular matrix that provide stability and resistance to human immune mechanisms. In recent years, some species of NTM have been shown to form biofilms that enhance resistance to disinfectants and antimicrobial agents. Biofilm assembly proceeds through several phases, including reversible attachment, irreversible attachment, biofilm formation via bacterial aggregation, organization, and signaling, and finally dispersion. During this process, bacteria develop a matrix containing extracellular polymeric substances (EPS), such as polysaccharides, lipids and nucleic acids, to form a complex three-dimensional structure (Sousa S. et al.. International Journal of Mvcobacterioloqy 4 (2015). 36-43). Specifically, mycobacterial EPS differ in nature from other biofilms, as mycobacteria do not produce exopolysaccharides (Zambrano MM, Kolter R. Mycobacterial biofilms: a greasy wav to hold it together. Cell. 2005). Mycobacterial biofilms vary between species, but can contain mycolic acids, glycopeptidolipids, mycolyl-diacylglycerols, lipooligosaccharides, lipopeptides, 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). The assembly in biofilms is known to enhance resistance to antimicrobial agents (Faria S. et al.. Journal of Pathogens. Vol 2015. Article ID 809014).

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

Multiple combination regimens with inhaled amikacin following initial treatment with parenteral aminoglycosides, tigecycline and other promising oral antibiotics such as linezolid, delamanid, and bedaquiline, and surgical intervention in selected cases have shown promising results in the treatment of NTM lung disease (Lu Ryu et al., Tuberc Respir Pis 2016;79:74-84). However, the increasing incidence and prevalence of NTM infections, in particular NTM lung disease and the limited treatment options necessitate the development of novel dosage forms/pharmaceutical formulation enhancing the bioavailability of the currently used antibiotics such as clofazimine. Inhalation may enhance efficacy and reduce adverse effects compared to oral and parenteral therapies.

Recently, it has been demonstrated that inhaled microparticles containing clofazimine are effective in the treatment of experimental tuberculosis in mice (Verma R et al.. Antimicrobial Agents and Chemotherapy, February 2013, Volume 57. No 2, 1050-

1052).

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

Summary of the invention

According to one aspect of the invention, there a pharmaceutical composition is provided comprising:

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

(b) an oil phase selected from a fatty acid, vegetable oil, vegetable oil derivative, MCT (medium chain triglycerides), LCT (long chain triglycerides), mono- di-, tri-olein and mixtures thereof,

(c) a nonionic surfactant with an HLB (Hydrophilic-Lipophilic Balance) > 10,

(d) a co-solvent/co-surfactant selected from propylene glycol, polyethylene glycol (including Lutrol^® E 300, Lutrol^® E 400, Kollisolv^® PEG 300 and Kollisolv^® PEG 400), isopropylalcohol, diethylene glycol monoethyl ether (including Transcutol™), propylene glycol monolaurate Type I (including Lauroglycol FFC™), ethanol and mixtures thereof, and

(e) an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solutions,

wherein the clofazimine is solubilized in the form of an emulsion.

Furthermore, the present invention provides a process for the preparation of the pharmaceutical composition according the present invention comprising the following steps:

(1 ) dissolving a therapeutic effective dose of clofazimine (a) in the oil phase (b),

(2) adding the resulting solution to the non-ionic surfactant (c), followed by homogenization to obtain a homogenized oily solution,

(3) preparation of the aqueous phase by dissolving the co-solvent (d) in the aqueous liquid carrier (e) and

(4) adding the aqueous phase obtained in (3) to the homogenized oily solution obtained in (2) followed by homogenization,

(5) adjusting the pH of the homogenate resulting from (4) to a pH between pH 5.5 and pH 7.0, preferably to pH 6.5 and

(6) adjusting the NaCI concentration to between 31 and 300 mM NaCI, preferably to 154 mM NaCI,

as well as the composition obtained by said process.

Step (5) is preferably carried out with NaOH, more preferably with 1 M NaOH, Step (6) is preferably carried out by addition of NaCI.

Also provided is a pharmaceutical formulation in the form of an aerosol for pulmonary inhalation prepared/to be administered by aerosolization/nebulizing of the composition according to the present invention by a nebulising device, preferably selected from an ultrasound nebulizer, a compression nebulizer, an electron spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer or a mechanical soft mist nebulizer.

A further aspect of the invention is the use of the pharmaceutical composition and the pharmaceutical formulation in the treatment and/or prophylaxis of pulmonary infections caused by mycobacteria or other gram-positive bacteria is provided as well as corresponding methods of treatment and/or prevention.

Thus the present invention also provides i.a. the following methods of treatment:

A method of treating and/or preventing a pulmonary infection caused by mycobacteria or other gram-positive bacteria in a patient, comprising administering a therapeutically effective amount of an aerosol of the pharmaceutical composition according to any one of claims 1 to 12 or 17 or the aerosol according to any one of claims 13 to 17 to the lower lung of the patient.

The method according to the above, wherein the infection is caused by a species of the genus mycobacterium selected from nontuberculous mycobacteria (NTM) (such as Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium avium), Mycobacterium leprae and/or tuberculosis (TB) (Mycobacterium tuberculosis complex). The method according to any of the above, wherein the infection is an opportunistic infection in patients with CF, COPD, HIV or AIDS, such as MAC pulmonary disease or opportunistic NTM infections associated with CF or COPD, or other immune suppression, iatrogenic or otherwise, or in patients with latent or active tuberculosis.

The method according to any of the above, wherein the infection is an opportunistic NTM (pulmonary) infection in patients with CF, in particular with Mycobacterium abscessus.

The method of above, comprising producing the aerosol with an ultrasound nebulizer, a compression nebulizer, an electron spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer or a mechanical soft mist nebulizer.

The method of any of the above, wherein about 1 to 5 ml emulsion is administered as an aerosol.

The method of any of the above, wherein about 5 to 10 mg clofazimine is administered to the lower lung.

The method of any one of the above, wherein the aerosol particles have an MMAD of about 1 pm to about 5 pm, preferably below about 3 pm.

The method according to any of the above, wherein the aerosol is adapted for once or twice daily administration.

Also provided is the use of the pharmaceutical composition of the invention for the preparation of an aerosol formulation for pulmonary inhalation.

Further provided is a system for use in providing antibiotic activity when treating and/or providing prophylaxis against a pulmonary infection caused by mycobacteria or gram-positive bacteria, wherein the system comprises:

(1 ) a nebulizable pharmaceutical formulation comprising:

(a) a therapeutically effective dose of clofazimine or a pharmaceutically acceptable derivative or salt thereof, (b) an oil phase selected from a fatty acid, vegetable oil, vegetable oil derivative, MCT, LCT, mono- di-, tri-olein and mixtures thereof,

(c) a nonionic surfactant with an HLB > 10,

(d) a co-solvent/co-surfactant selected from propylene glycol, polyethylene glycol (including Lutrol^® E 300, Lutrol^® E 400, Kollisolv^® PEG 300 and Kollisolv^® PEG 400), isopropylalcohol, diethylene glycol monoethyl ether (including Transcutol™), propylene glycol monolaurate Type I (including Lauroglycol FFC™), ethanol and mixtures thereof,

(e) an aqueous liquid carrier selected from water, isotonic saline buffered saline and aqueous electrolyte solutions,

wherein the clofazimine is solubilized in the form of an emulsion; and

2) a nebulizer.

Also provided is an aerosolized pharmaceutical formulation for use in providing antibiotic activity when treating and/or providing prophylaxis against a pulmonary infection caused by mycobacteria or other gram-positive bacteria in a patient wherein the formulation comprises the components (a) to (e) as defined above,

wherein the clofazimine is solubilized in the form of an emulsion.

The aerosolization of the composition of the invention by an appropriate nebulizer provides significantly increased delivery of the aerosolized clofazimine into the lower lung (i.e. to the bronchi, bronchioli, and alveoli of the central and lower peripheral lungs), thereby substantially enhancing the therapeutic efficacy.

The inhalation device should, moreover, preferably be further adapted for localized pulmonary delivery of an aerosol having an optimal particle size distribution for homogenous deposition in the lower lung.

The invention therefore provides for an aerosol having aerosol particles of sizes that facilitate delivery to the alveoli and bronchiole. A suitable aerodynamic particle size for targeting the alveoli and bronchiole is between 1 and 5 pm. Particles larger than that are selectively deposited in the upper lungs, namely bronchi and trachea and in the mouth and throat, i.e. oropharyngeal area. Accordingly, the inhalation device is adapted to produce an aerosol having a mass median aerodynamic diameter (MMAD) in the range from about 1 to about 5 mhi, and preferably in the range from about 1 to about 3 pm. In a further embodiment, the particle size distribution is narrow and has a geometric standard deviation (GSD) of less than about 2.5.

Brief description of the Figures

Figures 1A to 1C depict the bacterial counts (bacterial burden, Log₁₀ CFU) in the lungs (A), spleen (B) and liver (C) of SCID mice with intratracheal pulmonary infection M. abscessus 103 after treatment with the pulmonary aerosolized formulation of the invention (6.3 mg/kg), compared with saline (control) and clofazimine by gavage (20 mg/kg)) on days 1 and 11 after infection.

Figure 1 : Acute SCID Treatment Mouse Model.

Bacterial counts in the lungs (Fig.1 A), spleen (Fig.1 B), and liver (Fig1C) of SCID mice with an intratracheal pulmonary infection with 1x10⁶ CFUs of M. abscessus 103. The SCID mice were treated starting on day 2 for a total of 8 days with saline (□), QRM-003 6.3 mg/kg (pulmonary aerosol) (_■), clofazimine 20 mg/kg (gavage) («). Experimental groups of mice were evaluated for bacterial burden on day 1 , 2 and 12 after infection by plating serial dilutions of organ homogenates on nutrient 7H11 agar and counting CFUs after 7 days incubation at 32°C. Results represent the average of one experiment (n=6 mice per experiment) bacterial load in each group is expressed as average Logio CFU (± SEM) cells (± SEM).

Detailed description of the invention

The present invention is based on the unexpected discovery that by pulmonary aerosol administration of clofazimine in the form of an emulsion, lower lung deposition of the active agent can be achieved, thereby significantly increasing the bioavailability of the extremely hydrophobic BCS class II agent, which results in significantly increased therapeutic efficacy coupled with reduced systemic side effects.

In another aspect, this finding leads to the provision of an improved antibiotic therapy for infections caused by mycobacteria and gram-positive bacteria, in particular of pulmonary infections with NTM, such as opportunistic infections in CF, COPD and immune compromised patients such as HIV patients.

The present invention, moreover, aims at overcoming (systemic) side effects of established oral treatment regimens for (pulmonary) infections with gram positive bacteria, in particular TB and NTM infections of the lungs as well as at the reduction of dose and of duration of treatment with clofazimine.

It is understood by the person of skill in the art that the present application also discloses each and any combination of the individual features disclosed herein.

Definitions

The term“pharmaceutically acceptable salt” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which are not biologically or otherwise undesirable. In many cases, the compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. 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 include, for example, acetic acid, propionic acid, naphtoic acid, oleic acid, palmitic acid, pamoic (emboic) acid, stearic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, ascorbic acid, glucoheptonic acid, glucuronic acid, lactic acid, lactobionic acid, tartaric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, histidine, arginine, lysine, benethamine, N-methyl-glucamine, and ethanolamine. Other acids include dodecylsufuric acid, naphthalene-1 , 5-disulfonic acid, naphthalene-2-sulfonic acid, and saccharin.

In accordance with the present invention, apart from the free base, the use of the methanesulfonic acid, maleic acid, isonicotinic acid, nicotinic acid, malonic acid, and salicylic acid salts, and in particular of clofazimine mesylate is preferred.

By the term “pharmaceutically acceptable derivative” as used herein compounds disclosed in US 9,540,336 are meant, the disclosure of US 9,540,336 is incorporated herein in its entirety. In addition, derivates are meant as described in Lu.Y., Zhen.M., Wanq.B., Fu.L, Zhao.W., Li.P.. Xu,J., Zhu.H., Jin.H., Yin.D., Huang, H., Uoton.AM. and Ma.Z.,“Clofazimine Analogs with Efficacy against experimental Tuberculosis and reduced Potential for Accumulation” Antimicrobial Agents and Chemotherapy (201 1 ). 55(1 1 ) :DD.51 85-51 93.

By“therapeutically effective amount” or“pharmaceutically effective amount” is meant an amount of clofazimine, as disclosed for this invention, which has a therapeutic effect. The doses of clofazimine which are useful in treatment are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount means those amounts of clofazimine which produce the desired therapeutic effect as judged by clinical trial results and/or model animal infection studies.

The amount of the clofazimine and daily dose can be routinely determined by one of skill in the art, and will vary, depending on several factors, such as the particular microbial strain involved. This amount can further depend upon the patient’s height, weight, sex, age and medical history. For prophylactic treatments, a therapeutically effective amount is that amount which would be effective to prevent a microbial infection.

A“therapeutic effect” relieves, to some extent, one or more of the symptoms of the infection, and includes curing an infection. “Curing” means that the symptoms of active infection are eliminated, including the total or substantial elimination of excessive members of viable microbe of those involved in the infection to a point at or below the threshold of detection by traditional measurements. However, certain long-term or permanent effects of the infection may exist even after a cure is obtained (such as extensive tissue damage). As used herein, a“therapeutic effect” is defined as a statistically significant reduction in bacterial load in a host, emergence of resistance, or improvement in infection symptoms as measured by human clinical results or animal studies.

“Treat”, “treatment”, or “treating” as used herein refers to administering a pharmaceutical composition/formulation for prophylactic and/or therapeutic purposes. The term“prophylactic treatment” refers to treating a patient who is not yet infected, but who 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 preferred embodiments, treating is the administration to a mammal (either for therapeutic or prophylactic purposes) of therapeutically effective amounts of clofazimine.

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

The term“emulsion” as used herein is meant to refer to an emulsion having emulsion particles in the size range in the order of approximately 10 nm to 100 pm. The term“micro-emulsion” as used herein is meant to refer to an emulsion having emulsion particles in the size range in the order of 1 -1000 nm (in accordance with IUPAC 1 to 100 nm, usually 10 to 50 nm).

The term“nano-emulsion” as used herein is meant to refer to an emulsion having emulsion particles in the size range in the order of 1 -1000 nm (preferably as in accordance with IUPAC 1 to 100nm, usually 10 to 50 nm) generated by mechanical shear forces.

Definitions and characterization of the terms micro-emulsions and nano-emulsions as used in accordance with the present invention and also methods for their preparation are described in Vatsrai et al. Journal of Nanoscience. 2014. Article ID 268293 and Kale and Deore, Svs Rev Pharm. 2017; 8(1 ); 29-47.

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

Pharmaceutical compositions and formulations

As stated above, the present invention provides a nebulizable or aerosolized pharmaceutical composition comprising the components (a) to (e) as defined above, wherein the clofazimine is solubilized in the form of an emulsion,

and

wherein the emulsion droplets have a diameter of less than 1 pm.

In accordance with the present invention, the fatty acid may be selected from oleic acid, myristic acid, caprylic acid, capric acid, or mixtures thereof.

For the aqueous liquid carrier, the use of isotonic saline (0.9% NaCI (w/v), about 150 mM NaCI) is particularly preferred.

The following water grades are particularly applicable to the present invention: sterile purified water, sterile water for injection, sterile water for irrigation, sterile water for inhalation (USP) and corresponding water grades in accordance with e.g. Ph. Eur. or NF.

Aqueous electrolyte solutions as used in accordance with the present invention as the aqueous liquid carrier may further comprise potassium chloride, lithium chloride, magnesium chloride, calcium chloride or mixtures thereof.

In accordance with the present invention, the vegetable oil may be selected from any pharmaceutically grade vegetable oils including coconut oil, olive oil, soya bean oil, safflower oil, peanut oil, grapeseed oil, sunflower oil, corn oil or mixtures thereof.

In accordance with the present invention, the vegetable oil derivative is selected from hydrogenated vegetable oils, mixed partial glycerides, polyoxylglycerides and mixtures thereof.

Oleic acid and particularly ultra-pure oleic acid (available as EXTRA OLEIN 99) is particularly preferred for the oil phase. EXTRA Olein 99™ is known for low levels of peroxide compounds, a characteristic which is of importance for pulmonary applications.

According to the present invention, polyethylene glycol 400 (PEG 400) (such as Kollisolv^® PEG 400 or Lutrol^® E 400) is particularly preferred as the co-solvent.

The aqueous liquid carrier is preferably isotonic saline solution (0.9% NaCI corresponding to about/approximately 150 mM NaCI, preferably 154 mM NaCI).

According to the present invention, the non-ionic surfactant is selected from polysorbates such as Tween^® 20, Tween^® 60, Tween^® 80 (preferably ultraoure oolvsorbate 80): stearyl alcohol, PEG 40 hydrogenated castor oil (including Cremophor^® RH 40) PEG 60 hydrogenated castor oil including Cremophor^® RH 60), sorbitan monostearates (including Span^® 20, Span^® 40 and Span^® 60), polyalkylene glycol ethers including Brij^® 020, Brij^® 58, Brij^® C10, Brij^® 010, Brij^® S100, Brij^® S10, Brij^® S20, Brij^® L4, Brij^® 93 and Brij^® 52), caprylocaproyl polyoxyl-8 glycerides (Labrasol^®), polyoxyethylene stearates (including Myrj™ 49, Myrj™ S40, Myrj™ S100, Myrj™ S8 and Myrj™ 52) and mixtures thereof. Tween^® 80 and particularly ultra pure Tween^® 80/polysorbate 80 (available as polysorbate 80(HX2)™ are particularly preferred. Polysorbate 80(HX2)™ is known for low allergic reaction, low toxicity and low level of peroxide compounds, characteristics which are of importance for pulmonary application.

Moreover, the present invention provides a pharmaceutical composition as defined above,

wherein the oil phase is oleic acid, preferably ultra-pure oleic acid,

wherein the non-ionic surfactant is polysorbate 80, preferably ultra pure polysorbate 80,

wherein the co-solvent is polyethylene glycol 400 (PEG 400) and

wherein the aqueous liquid carrier is water or isotonic saline, preferably isotonic saline.

In a further embodiment, the present invention provides a pharmaceutical composition as defined above, wherein

the oil phase is in the range of 1 %-30% (v/v) of parts (b) (c) and (d), (i.e. excluding (a) and (e)),

the surfactant (c):co-solvent (d) ratio is in the range of 1 :1 to 25:1 (v/v), the amount of the aqueous carrier (e) is in the range of 60-90% (v/v) of the total composition (a-e) and

the amount of clofazimine (a) is in the range of 0.1 % to 2% (w/w) of the total composition (a-e).

In a preferred embodiment, the present invention provides a pharmaceutical composition as defined above, wherein

the oil phase is about 15% (v/v) of parts (b) (c) and (d),

the surfactant (c):co-solvent (d) ratio is about 1 1.9:1 ,

the amount of the aqueous carrier (e) is about 84% (v/v) of the total composition (a-e) and the amount of clofazimine is about 0.9% (w/w).

Furthermore, the present invention provides a process for the preparation of the compositions of the invention comprising the following steps:

(1 ) dissolving a therapeutic effective dose of clofazimine (a) in the oil phase (b), (2) adding the resulting solution to the non-ionic surfactant (c),

followed by homogenization to obtain a homogenized oily solution,

(5) adjusting the pH of the homogenate resulting from (4) to a pH between pH 5.5 to pH 7.0, preferably to pH 6.5 and

(6) adjusting the NaCI concentration to between 31 and 300mM NaCI, preferably to 154 mM NaCI,

as well as the pharmaceutical composition according to the present invention prepared by said process.

Step (5) is preferably carried out with NaOH, more preferably with 1 M NaOH, Step (6) is preferably carried by addition of NaCI.

The homogenization in steps (2) and (4) may be carried out by means and devices such as ultrasonic bath, sonic probe, vortex mixer, (manual) shaking, high pressure homogenization (HPH), microfluidization by high pressure pump and spontaneous emulsification.

The homogenization/emulsification steps can be carried out for about 1 to 15 min at room temperature.

It is further preferred that the homogenization in step (2) is carried out in an ultrasonic bath for about 5 to 15 minutes, preferably for about 10 minutes at room temperature.

It is preferred that the homogenization in step (4) is carried out with an ultrasonic probe for about 0.5 to 5 minutes, preferably for about 1 minute with a 30% amplitude at room temperature.

In accordance with the present invention, nano- and micro-emulsions are preferred, nano-emulsions are particularly preferred. Size of emulsion droplets and size distribution

The size of the emulsion droplets was determined by DSL (Dynamic Light Scattering) using a ZetaSizer Nano ZS (Malvern Instruments, Malvern, United Kingdom).

In accordance with the present invention, the emulsion droplets have an MMD of < 1 pm as determined by dynamic light scattering in accordance with the method as described in Galliher-Becklev A et al.. Vaccine 2015, 33(25) 2903-8 and Pharmaceutical Forum Volume Number 31 (4). cage 1234. July 2005. <429>. Light Diffraction Measurement of Particle Size.

In one embodiment, the resulting liquid emulsion (i.e. the pharmaceutical composition) has a pH range from about 4.5 to about 7.5, preferably from about 5.5 to about 7.0, most preferred pH 6.5. This pH range improves tolerability. When the resulting aerosol is either acidic or basic, it can cause bronchospasm and cough. Although the safe range of pH is relative, some patients may tolerate a mildly acidic aerosol, while others will experience bronchospasm. Any aerosol with a pH of less than about 4.5 typically induces bronchospasm. Aerosols with a pH from about 4.5 to about 5.5 will cause bronchospasm occasionally. Any aerosol having pH greater than about 7.5 may have low tolerability because body tissues are generally unable to buffer alkaline aerosols. Aerosols with controlled pH below about 4.5 and over about 7.5 typically result in lung irritation accompanied by severe bronchospasm cough and inflammatory reactions. For these reasons as well as for the avoidance of bronchospasm, cough or inflammation in patients, the optimum pH for the aerosol formulation was determined to be between about pH 5.5 to about pH 7.0. Consequently, in one embodiment, aerosol formulations for use as described herein are adjusted to a pH between about 4.5 and about 7.5 with preferred pH range from about 5.5 to about 7.5. More preferred is a pH range from about 5.5 to about 7.0. , a resulting pH value of about 6.5 is particularly preferred

By non-limiting example, compositions may also include a buffer or a pH adjusting agent, typically a salt prepared from an organic acid or base, such as NaOH. Representative buffers include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine, hydrochloride, phosphate buffers or mixtures thereof. Many patients have increased sensitivity to various chemical tastes, including bitter, salt, sweet, metallic sensations.

To create well-tolerated drug products, by non-limiting example taste masking may be accomplished through the addition of taste-masking excipients, adjusted osmolality, and sweeteners.

In some embodiments, the osmolality of the pharmaceutical composition in accordance with the present invention is adjusted by providing excipients. In some cases, a certain amount of chloride or another anion is needed for successful and efficacious delivery of aerosolized clofazimine.

Bronchospasm or cough reflexes can be sufficiently controlled and/or suppressed when the osmolality of the diluent is in a certain range.

A preferred pharmaceutical composition according to the present invention of therapeutic compounds which is safe and tolerated has a total osmolality from about 200 to about 700 mOsmol/kg with a range of chloride concentration of from about 31 mM to about 300 mM.

Preferred is a total osmolarity of about 300-400 mOsmol/kg and a concentration of about 154mM of sodium chloride resulting in 0.9% NaCI, isotonic saline.

In a preferred embodiment, the pH of the pharmaceutical composition according the present invention is in the range of 5.5 and 7.0. A pH value of about 6.5-7.0 is particularly preferred.

Preferably, the viscosity of the liquid pharmaceutical composition of the invention is smaller than 1.5 cp. Viscosity is measured at room temperature with the Physica Modular Compact Rheometer MCR 501 (Anton Paar GmbH, Germany) in accordance with the method disclosed in Junii Matsuoka et al., J. Oleo Sci. 66, (1 1 ) 1285-1291 (2017), Structure of the Microemulsion of Polyqlvcerol Polyricinoleate Encapsulating Vitamin E. Measurement was performed at room temperature using the Physica MCR 501 with the following attachment: Rotation with a cone-plate (5 cm diameter), increasing shear rate of 0.1 -100/s.

The present invention provides a pharmaceutical formulation in the form of an aerosol for inhalation, preferably for lower lung deposition, prepared by aerosolization/nebulizing of the composition according to the present invention as defined above by a nebulizing device selected from an ultrasound nebulizer, a compression nebulizer, an electron spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer or a mechanical soft mist nebulizer,

and

wherein the emulsion droplets have a mass median diameter of less than 1 pm, wherein the aerosol particles have a mass median aerodynamic diameter of 1 to 5 pm, preferably below 3 pm.

The present invention also provides the use of the composition of the present invention as described above any for the preparation of an aerosol formulation for inhalation, preferably for lower lung deposition,

and

Furthermore, the present invention provides a system for use in providing antibiotic activity when treating or providing prophylaxis against a pulmonary infection caused by mycobacteria or other gram-positive bacteria, wherein the system comprises:

(1 ) a nebulized pharmaceutical formulation comprising the components (a) to (e) as defined above

and

2) a nebulizer,

wherein the clofazimine is present in an emulsion, preferably in a nano- or microemulsion,

and

wherein the emulsion droplets have a diameter (MMD) of less than 1 pm,

wherein the aerosol particles have a mean particle diameter (MMAD) of 1 to 5 pm, preferably below 3 pm.

Components (a) to (e) are as defined above. Particle Size and Distribution

The therapeutic effect of aerosolized therapies is dependent upon the dose deposited and its distribution. Aerosol particle size is one of the most important variables in defining the dose deposited and the distribution of drug aerosol in the lung.

Generally, inhaled particles are subject to deposition by one of two mechanisms: impaction, which usually predominates for larger particles, and sedimentation, which is prevalent for smaller particles. Impaction occurs when the momentum of an inhaled particle is large enough that the particle does not follow the air stream and encounters a physiological surface. In contrast, sedimentation occurs primarily in the lower lung when very small particles which have traveled with the inhaled air stream encounter physiological surfaces as a result of gravitational settling.

Pulmonary drug delivery may be accomplished by inhalation of an aerosol through the mouth and throat. Particles having an aerodynamic diameter of greater than about 5 pm generally do not reach the lung; instead, they tend to impact the back of the throat and are swallowed and possibly orally absorbed. Particles having diameters of about 3 to about 5 pm are small enough to reach the upper- to mid- pulmonary region (conducting airways), but are too large to reach the alveoli. Smaller particles, i.e. about 0.5 to about 3 pm, are capable of reaching the alveolar region. Particles having diameters smaller than about 0.5 pm tend to be exhaled during tidal breathing, but can also be deposited in the alveolar region by a breath hold.

Aerosols used in pulmonary drug delivery are made up of a wide range of particle sizes, so statistical descriptors are used. Aerosols used in pulmonary drug delivery are typically described by their mass median diameter (MMD), that is, half of the mass is contained in particles larger than the MMD, and half the mass is contained in particles smaller than the MMD. For particles with uniform density, the volume median diameter (VMD) can be used interchangeably with the MMD. Determinations of the VMD and MMD are made by laser diffraction. The width of the distribution is described by the geometric standard deviation (GSD). However, the deposition of a particle in the respiratory tract is more accurately described by the particle’s aerodynamic diameter, thus, the mass median aerodynamic diameter is typically used. MMAD determinations are made by inertial impaction or time of flight measurements. For aqueous particles, VMD, MMD and MMAD should be the same. However, if humidity is not controlled as the aerosol transits the impactor, MMAD determinations will be smaller than 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 descriptions of VMD, MMD and MMAD will be comparable.

Nonetheless, for the purpose of the description, the particle size of the aerosol particles will be given as MMAD as determined by measurement at room temperature with a Next Generation Impactor (NGI) in accordance with US Pharmacopeial Convention. In Process Revision <601 > Aerosols, Nasal Sprays, Metered-Dose Inhalers, and Dry Powder Inhalers, Pharmacopeial Forum (2003), Volume Number 29, pages 1 176-1210 also disclosed in Jolvon Mitchell. Mark Nagel“Particle Size Analysis of Aerosols from Medicinal Inhalers”. KONA Powder and Particle Journal (2004), Volume 22, pages 32-65.

In accordance with the present invention, the particle size of the aerosol is optimized to maximize the deposition of clofazimine at the site of infection and to maximize tolerability. Aerosol particle size may be expressed in terms of the mass median aerodynamic diameter (MMAD). Large particles (e.g., MMAD > 5 pm) tend to deposit in the extrathoracic and upper airways because they are too large to navigate bends in the airways. Intolerability (e.g., cough and bronchospasm) may occur from upper airway deposition of large particles.

Thus, in accordance with a preferred embodiment, the MMAD of the aerosol should be less than about 5 pm, preferably between about 1 and 5 pm, more preferably below 3 pm (< 3 pm).

However, a guided breathing maneuver can be used to allow larger particles to pass through the extrathoracic and upper airways and deeper into the lungs than during tidal breathing which will increase the central and lower lung deposition of the aerosol. A guided breathing maneuver may be as slow as 100 mL/min. Thus, when used with a guided breathing maneuver, the preferred MMAD of the aerosol should be less than about 10 pm.

Nebulizers may also only produce aerosol when the patient inhales. This does not impact the deposition of the aerosol, but increases the efficiency of delivery, i.e. it increases the percentage of the label claim dose that reaches the lung. Thus, a preferred embodiment of the invention includes a nebulizer that produces aerosol only while the patient inhales because it decreases the label claim dosage of drug required for efficacy.

Nebulizer

For aqueous and other non-pressurized liquid systems, a variety of nebulizers (including small volume nebulizers) are available to aerosolize the formulations. Compressor-driven nebulizers incorporate jet technology and use compressed air to generate the liquid aerosol. Such devices are commercially available from, for example, Healthdyne Technologies, Inc.; Invacare, Inc.; Mountain Medical Equipment, Inc.; Pari Respiratory, Inc.; Mada Medical, Inc.; Puritan-Bennet; Schuco, Inc., DeVilbiss Health Care, Inc.; and Hospitak, Inc. Ultrasonic nebulizers rely on mechanical energy in the form of vibration of a piezoelectric crystal to generate respirable liquid droplets and are commercially available from, for example, Omron Heathcare, Inc. and DeVilbiss Health Care, Inc. Vibrating mesh nebulizers rely upon either piezoelectric or mechanical pulses to respirable liquid droplets generate. Other examples of nebulizers for use with clofazimine described herein are described in U.S. Patent Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911 ; 4,510,929; 4,624,251 ; 5,164,740; 5,586,550; 5,758,637; 6,644,304; 6,338,443; 5,906,202;

5,934,272; 5,960,792; 5,971 ,951 ; 6,070,575; 6,192,876; 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 hereby incorporated by reference in their entirety. Commercial examples of nebulizers that can be used with the clofazimine compositions described herein include Respirgard II^®, Aeroneb^®, Aeroneb^® Pro, and Aeroneb^® Go produced by Aerogen; AERx^® and AERx Essence™ produced by Aradigm; Porta-Neb^®, Freeway Freedom™, Sidestream, Ventstream and l-neb produced by Respironics, Inc.; and PARI LCPIus^®, PARI LC-Star^®, and e-Flow7m produced by PARI, GmbH. Further non-limiting examples are disclosed in US 6,196,219.

In accordance with the present invention, the pharmaceutical composition may be preferably aerosolized using a nebulising device selected from an ultrasound nebulizer, an electron spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer or a mechanical soft mist nebulizer.

It is preferred that the device controls the patient’s inhalation flow rate either by an electrical or mechanical process.

In a further preferred embodiment, the aerosol production by the device is triggered by the patient’s inhalation, such as with an AKITA device.

Preferred (commercially available) examples of the above nebulizers/devices to be used in accordance with the present invention are Vectura fox, Pari eFIow, 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 formulations (aerosols, aerosolized formulations) and systems according to the present invention are intended for the use in the treatment and/or prophylaxis of pulmonary infections caused by mycobacteria or other clofazimine susceptible bacteria, such as Staphylococcus aureus (including methicillin-resistant and vancomycin intermediate- resistant strains), Streptococcus pneumoniae, and Enterococcus spp.

In one embodiment, the infection is caused by a species of the genus mycobacterium selected from nontuberculous mycobacteria (NTM) (RGM and SGM) and/or TB (mycobacterium tuberculosis complex).

In one embodiment of the invention, the infection is caused by a species of the genus mycobacterium, including, but not limited to Mcyobacterium leprae, Mycobacterium tuberculosis complex (causative agent of tuberculosis), or selected from nontuberculous mycobacteria (NTM) including, but not limited to Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus.

In a further embodiment, the NTM is selected from Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium abscessus.

In accordance with the present invention, it is particularly preferred that the infection is caused by Mycobacterium avium and/or Mycobacterium abscessus.

In a further embodiment of the present invention, the infection is an opportunistic infection in patients with CF, COPD, HIV or AIDS such as MAC pulmonary disease or opportunistic NTM infections in associated with CF or COPD, or other immune suppression, iatrogenic or otherwise, or in patients with latent or active tuberculosis.

In another embodiment, the infection is an opportunistic NTM (pulmonary) infection in patients with CF, in particular with Mycobacterium abscessus.

Dosing of clofazimine

In accordance with the present invention, the pharmaceutical formulation is delivered by nebulization in about 1 -5 ml, preferably 3 to 5 ml of the pharmaceutical composition of the invention (emulsion).

Thus, the target fill dose is about 1 -5 ml corresponding to 9-45 mg clofazimine, based on a clofazimine concentration in the pharmaceutical composition of about 9 mg/ml (8.9 mg/ml).

Based on the MIC of 0.5 mg/I for Mycobacterium abscessus, the daily lung dose (i.e. the dose deposited in the lung) of clofazimine to be administered in accordance with the present invention is about 5-10 mg (corresponding to 0.6 ml— 1 .1 ml of formulation example F6D according to the present invention), which corresponds to a nominal dose of 15-30 mg (device dose) (corresponding to 1 .7 ml-3.3 ml of formulation example F6D) in the case of M. abscessus infections.

It is understood that the person of skill in the art will routinely adjust the lung dose of clofazimine to be administered (and thus the fill/nominal dose/the volume to be nebulized) based on the MIC of clofazimine for the respective bacteria strain well established in the art. Depending on the dosing frequency, once or twice per day, the daily lung dose will be split accordingly.

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

It will be obvious to a person skilled in the art that the above amounts relate to clofazimine free base, the dosage amounts for derivatives, and salts will have to be adjusted accordingly based on the MIC of the respective compound and strain.

Mucolytic agents/biofilm modifying agents

In order to reduce sputum viscosity during aerosol treatment and to destroy existing biofilm, the treatment and/or prophylaxis according with the present invention can involve additional administration of mucolytic and/or biofilm destructing agents.

These agents can be prepared in fixed combination or be administered simultaneously or subsequently to the pharmaceutical composition/aerosol formulation comprising clofazimine in accordance with the present invention.

Agents for dispersing/destruction of the biofilm, mucolytic and/or mucoactive agents and/or agents that reduce biofilm formation to be used in accordance with the present invention are selected from hypertonic saline (4-7%, preferably 6%) (nebulized), metaperiodate, SDS, sodium bicarbonate, tromethamine (THAM), silver nanoparticles, bismuth thiols, EDTA, GPA NPs, chelators, C2DA, D-amino acids (D-AAs) (such as D-leucine, D-tryptophan, D-methionine, D-tyrosine), D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corsolilc acid, fatty acids, host defense peptides (HDPs), and synthetic antimicrobial peptides (including cathelicidins, defensins, bacteriocidins, and protegrins).

Furthermore, also other pharmaceutically active agents may be used in combination with the pharmaceutical compositions/aerosol formulations in accordance with the present invention. Such active agents may be selected from DNAse and antibiotics such as aztreonam, tobramycin, ciprofloxacin and amikacin.

EXAMPLES

The following examples serve to more fully describe the manner of using the above- described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. The Examples according to the invention are those falling within the scope of the claims herein.

Experimental

Formulation Examples

The exemplary compositions below have been prepared in accordance with the formulation process described above.

Formulation example F6D

Component Supplier Purity CAS

Clofazimine acros organics 98% 2030-63-9

Oleic acid VWR 65-88% 112-80-1

Chemicals

Polysorbate 80 NOF USP 9005-65-6

(Hx2) Corporation

Water R VWR Ph.Eur. 7732-18-5

Chemicals

Polyethylenglyc Roth Ph.Eur. 25322-68-3

ol 400

Sodium Roth Ph.Eur. 7647-14-5

chloride

Sodium VWR USP 1310-73-2

hydroxide 1

mol/ Composition

Clofazimine 90 mg

Oleic acid 1.5 ml

Polysorbate 80 (Hx2) 0.1 ml

Sodium chloride 87.66 mg

Water dest. with 0.1 % PEG 400 8.4 ml

(V/V)

Sodium hydroxide 1 mol/l q.s. until pH 6.5 is reached

(Approx. 100 pi)

Method for preparation

1. Dissolve 120 mg clofazimine in 2 ml oleic acid (targeted concentration 60 mg/ml).

2. Transfer 100 pi polysorbate 80 to an empty and clean glass vial and add 1500 pi of clofazimine solution 60 mg/ml.

3. Homogenize the solution for 10 minutes in ultrasonic bath and add 8400 pi PEG 400 (0.1 %) in water to the lipophilic phase.

4. Homogenize the emulsion for 1 minute with an ultrasound disperser (amplitude 30%, ~1 kJ).

5. Adjust pH and add sodium chloride.

The resulting composition shows a dark red color.

As the formulation separates in two phases, it must be shaken vigorously until a homogenous formulation is formed before using the composition for aerosolization. The shaking can be performed manually by shaking the composition for approximately 10 seconds. After approx. 15 min the composition again separates into a heterogeneous system.

The characterizing parameters of the composition are summarized in the table, below:

Parameter Target values Protocol (measured values)

pH 6.5 approx. 6.5

Osmolality 200-700 mOsm/kg (300 approx. 300 mOsm/kg targeted)

Chloride concentration 31 -300 mM approx. 150 mM

Viscosity <1.5 cp <1.5 cp

MMAD 3-5 pm 2.05 GSD 1.82

Diameter of oil droplets in Average 0.5 miti (PDI 0.43) formulation

Stability of Formulation >1 month (stored at room temperature, estimated by visual inspection)

Formulation example F9

Polysorbate 80 (Tween 80) 5 mI

Clofazimine in oleic acid (60mg/ml) 150 mI

Water dest with 0.1 % PEG 400 845 mI

Homogenization/emulsification by ultrasound bath 30 minutes, at 50^°C.

Method for preparation of F9

1. Dissolve 60 mg clofazimine in 1 ml oleic acid (targeted concentration 60 mg/ml) by sonication in Ultrasonic bath for 10 minutes at RT.

2. Transfer 5 pi polysorbate 80 to an empty and clean glass vial and add 150 mI of clofazimine solution 60 mg/ml. Homogenize the solution in Ultrasonic bath for 10 minutes at RT.

3. Add 845 mI PEG 400 (0.1 %) in water to the lipophilic phase and homogenize the solution for 30 minutes in ultrasonic bath at 50°C.

pH and osmolarity were not adjusted in Formulation F9.

Animal models and efficacy testing

QRM-003 (formulation example F6D) has been tested for its ability to inhibit growth of clinical NTM species in an acute in vivo pulmonary infection mouse model to obtain preliminary data to establish API concentration levels in lung tissue after direct respiratory delivery as opposed to systemic administration. Two separate mouse models will be used in order to investigate pulmonary NTM infection, dependent on the bacterial species of interest. For testing, Mycobacterium avium 2285, and Mycobacterium abscessus 103 bacterial strains have been used (Strain details can be found in “Phylogenetic analysis of Mycobacterial species using whole genome seguences”. Flazbon M.Fi.. Rioias M.A., Damon A.M., Alalade R., Cantwell B.J., Monaco A., King S.. Sohrabi A. Submitted (SEP-2014) to the EMBL/GenBank/DDBJ databases). These two species have been previously used in literature as models of NTM infection (Obregon-Flenao et al. 2015 Antimicrob Agents Chemother; and Chan et al. Animal Models of Non-Tuberculous Mycobacterial Infections. Mvcobact Pis 2016).

In vivo safety study in Balb/C mice:

Clofazimine was found to be safe at 20 mg/kg (gavage, 200 mI), Formulation F6D/QRM-003 showed toxicity at 12.6mg/kg (0.315 mg/dose in 35mI administered by inhalation (Inhalation Therapy, IT), but was safe at 6.3 mg/kg (0.1575 mg/dose in 17.5 mI IT).

1. Mouse model of M. avium infection in the Beige mouse

A mouse model for efficacy testing of QRM-003 (Formulation example F6D) against Mycobacterium avium is the Beige mouse model in accordance with the method described in detail in Andreiak et al., 2015 Antimicrob Agents Chemother.

This animal model has been repeatedly used, and is widely accepted as a model for the study of Mycobacterium avium complex infection. Mice are infected via high dose aerosol (HDA) of 1 x10⁸ CFU of Mycobacterium avium.

In the acute model seven days after infection, therapy is initiated and continued once daily for ten days (day 8-18). Therapy administered is either QRM-003 (Formulation F6D), (delivered by Microsprayer Device - Penn-Century, Inc.), or oral administration of clofazimine (administered dose in both cases 20 mg/kg). Throughout the course of the experiment, the mice are monitored for weight changes and survival. On day 1 , 7 and 19 the mice are euthanized, and organs are harvested. Lung, spleen and liver were aseptically dissected, weighed, gross histological evaluation completed.

In the chronic model after 28 days, therapy is initiated and continued for 1 or 2 months. Therapy administered is either QRM-003 (Formulation F6D), or oral administration of clofazimine (administered dose in both cases 20 mg/kg) as a control treatment. Throughout the course of the experiment, the mice are monitored for weight changes and survival. The mice are then harvested on day 1 , 27, 58 and/or 88, lung, spleen and liver are aseptically dissected, weighed, gross histological evaluation completed and homogenized.

In both models, saline serves as negative control.

2. Mouse model of M. abscessus infection in SCID and B6CFTR^{tm1 UNC}/CFTR^{tm1 UNC} mice

This mouse model tests the antimicrobial efficacy of QRM-003 (Formulation F6D) against Mycobacterium abscessus infection in accordance with the method described in detail in Obreqon-Flenao A. et al., Antimicrobial Agents and Chemotherapy, November 15, Volume 59, Number 1 1 , p, 6904-6912.

This model utilizes SCID mice in the acute infection model to examine QRM-003 (Formulation F6D) activity in the airways, and B6CFTR^{tm1 UNC}/CFTR^{tm1 UNC} mice, which have a non-functional CFTR to mimic the physiology observed by CF patients, to examine QRM-003 (Formulation F6D) activity in the airways for both acute and chronic infection models.

For the first acute model severe combined immunodeficient (SCID) mice were used, as this model has shown susceptibility to the development of rapid, acute M. abscessus infection. SCID mice were infected with a 1 x10⁶ CFU dose aerosol of Mycobacterium abscessus. In the acute model, therapy began two days after infection, and was continued for eight days. Therapy administered was either QRM-003 (Formulation F6D) (delivered via Microspray device every other day. 6.3 mg/kg), or oral administrations of clofazimine every other day (20 mg/kg). Throughout the course of the experiment, the mice were monitored for weight changes and survival. Mice were harvested on days 1 , and 1 1 , with lungs, spleen, and liver aseptically dissected, weighed, and homogenized. Tissue homogenate was serially diluted and plated onto Middlebrook 7H1 1 agar for quantification.

In the second acute model, B6CFTR^{tm1 UNC}/CFTR^{tm1 UNC} are infected with a 1 x10⁶ CFU dose aerosol of Mycobacterium abscessus to specifically mimic the physiology in CF patients. In the acute model, therapy begins seven days after infection, and is continued for ten days. Therapy administered is either QRM-003 (Formulation F6D) (delivered via Microspray device every other day, 6.3 mg/kg), or oral administrations of clofazimine every other day (20 mg/kg). Throughout the course of the experiment, the mice are monitored for weight changes and survival. Mice are harvested on days 1 , 7, and 19, with lungs, spleen, and liver aseptically dissected, weighed, and homogenized. Tissue homogenate is serially diluted and plated onto Middlebrook 7H1 1 agar for quantification.

In the corresponding chronic models, therapy is initiated seven days after infection and continued for four weeks. Therapy administered is either QRM-003 (Formulation F6D), or oral administrations of clofazimine (20 mg/kg). Throughout the course of the experiment, the mice are monitored for weight changes and survival. Mice are harvested on days 1 , 14, and 35. Lung, spleen, and liver tissues are aseptically removed, weighed, and undergo gross histological assessment.

Efficacy in the acute SCID mouse model

The results for the SCID mouse acute model are shown in Figure 1 depicting the results of recovered CFU of Mycobacterium abscessus from lung, spleen and liver in the acute model and confirm that the composition according to the invention can be successfully aerosolized in the animal experiment. In brief, bacterial recovery from the acute infection model of M. abscessus in SCID mice was as follows:

Table 1

Furthermore, - the 3-log decrease in Mycobacterium abscessus is substantial (a 99.9% pathogen reduction), and superior to the 2-log effect that is observed with oral clofazimine. Bacterial recovery from animals that received QRM-003 (Formulation F6D as pulmonary aerosol) was 1.33±0.02, 0.54±0.01 , and 0.61 ±0.10 Log CFU/ml lower than animals receiving oral clofazimine administration.

Results

The results of lung, spleen, and liver CFU counts recovered from SCID mice after M. abscessus acute infection are shown in Figure 1.

The mean lung, spleen and liver log- CFU at the start of drug treatment was 5.40±0.06, 5.56±0.44, and 5.06±0.02. Mice treated every other day with daily doses of QRM-003 6.3 mg/kg (pulmonary aerosol), clofazimine 20 mg/kg (gavage) and saline for a total of 8 days. Mice did not demonstrate any significant weight loss in any of the groups and all animals survived until the Day 1 1 endpoint of the assay.

The CFU lung, spleen and liver counts on day 12 of the mice treated with the different drugs were as follows: clofazimine (gavage) alone (3.58±0.10, 3.63±0.60, and 4.79±0.10) and QRM-003 at 6.3 mg/kg (pulmonary aerosol) (2.25±0.15, 3.09±1.4, and 4.18±1.5). The clofazimine (gavage) worked as expected in this model. The QRM-003 6.3 mg/kg (pulmonary aerosol) resulted in CFU lung, spleen and liver counts that were approximately 1.33±0.02, 0.54±0.01 , and 0.61 ±0.10 logic respectively, lower than those in mice receiving clofazimine 20 mg/kg (gavage) alone.

The results obtained from these studies show a significant reduction in bacterial recovery obtained from lung, liver, and spleen homogenate, as well as serum, after treatment with QRM-003 (formulation F6D) compared to control groups demonstrating the improved efficacy of the aerosolized composition according to the present invention against NTM in vivo, and provide a rationale for future experiments against CF-derived NTM strains.

Sacrifice observations: (based on lung lesions, color, etc)

Key: Lung: - uninfected

+ slightly infected

++ moderately infected

+++ heavily infected

Spleen: normal = uninfected

Table 2

Drug Distribution

The concentration of QRM-003 within the airways is quantified from the lungs of na^'ive Beige and B6CFTR^{tm1 UNC}/CFTR^{tm1 UNC} mice after treatment. In brief, the lung tissue from animals receiving QRM-003 are weighed and frozen, the concentration of the API (Clofazimine) in lung tissue homogenate is quantified via liquid chromatography coupled to tandem mass spectrometry (LC-MS) and compared to serum concentrations obtained. In addition, quantification of clofazimine within the NTM-induced granulomas in NTM-infected mouse models are obtained using lung tissue slices and are measured via laser capture microdissection (LCM) LC-MS analysis.

After instillation of therapeutic in Protocol #2, and after animal euthanasia, lung tissue samples are weighed, frozen, and API levels are quantified. For initial LC-MS quantification of API concentrations, tissue samples are weighed and homogenized in phosphate buffered saline using a FastPrep-24 instrument and 1 .4 mm zirconium oxide beads. Proteins are precipitated by addition of 1 :1 acetonitrile:methanol containing relevant standards to plasma or homogenized tissue. The mixtures are vortexed and then transferred for LC-MS analysis. Analysis are performed by Sciex Applied Biosystems Qtrap 4000 triple-quadrupole mass spectrometer coupled to an Agilent 1260 HPLC system. Clofazimine chromatography is performed on an Agilent Zorbax SB-C8 column (2.1 x 30mm; particle size 3.5 pm) using reverse phase gradient elution. The gradients use 0.1 % formic acid in Milli-Q deionized water for the aqueous mobile phase and 0.1 % formic acid in acetonitrile for the organic mobile phase. Multiple-reaction monitoring of parent/daughter transitions in electrospray positive-ionization mode are used to quantify the analysis, and data processing will be performed via Analyst software.

In the LCM-LCMS, whole lung tissues from NTM-infected animals are flash frozen in liquid nitrogen vapor tissue and undergo LCM to isolate granulomas. After isolation, drug concentrations of clofazimine in the granulomas are quantified, comparing accumulation of clofazimine in granulomas tissue after oral administration or QRM- 003 administration.

The present invention for the first time provides an inhalable liquid formulation of clofazimine, thus enabling deep and homogenous lung deposition (lower lung) of the BCS class II antibiotic clofazimine. The present invention provides an improved therapy for (pulmonary) gram positive, in particular TB and NTM pulmonary infections overcoming the side effects of established oral treatment regimens and enabling a reduction of dose and treatment duration.

Claims

1. Pharmaceutical composition comprising

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

(b) an oil phase selected from a fatty acid, a vegetable oil, vegetable oil derivative, MCT, LCT, mono- di-, tri-olein and mixtures thereof,

(c) a nonionic surfactant with an HLB value of >10,

(d) a co-solvent/co-surfactant selected from propylene glycol, polyethylene glycol (including Lutrol^® E 300, Lutrol^® E 400, Kollisolv^® PEG 300 and Kollisolv^® PEG 400), isopropylalcohol, diethylene glycol monoethyl ether (including Transcutol™), propylene glycol monolaurate Type I (including Lauroglycol FFC™), ethanol and mixtures thereof and

(e) an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solutions

wherein the clofazimine is solubilized in the form of an emulsion,

and

wherein the emulsion droplets have a diameter (MMD) of less than 1 pm.

2. Pharmaceutical composition according to claim 1 , wherein the vegetable oil is selected from coconut oil, olive oil, soya bean oil, safflower oil, peanut oil, grapeseed oil, sunflower oil, corn oil or mixtures thereof.

3. Pharmaceutical composition according to claim 1 , wherein the fatty acid is selected from oleic acid, myristic acid, caprylic acid, capric acid, or mixtures thereof.

4. Pharmaceutical composition according to claim 1 , wherein the vegetable oil derivative is selected from hydrogenated vegetable oils, mixed partial glycerides, polyoxylglycerides and mixtures thereof.

5. Pharmaceutical composition according to any one of claims 1 to 4 wherein the non-ionic surfactant is selected from polysorbates such as Tween^® 20, Tween^® 60, Tween^® 80 (preferably ultrapure polysorbate 80); stearyl alcohol, PEG 40 hydrogenated castor oil (including Cremophor^® RH 40) PEG 60 hydrogenated castor oil (including Cremophor^® RH 60), sorbitan monostearates (including Span^® 20, Span^® 40 and Span^® 60), polyalkylene glycol ethers (including Brij^® 020, Brij^® 58, Brij^® C10, Brij^® 010, Brij^® S100, Brij^® S10, Brij^® S20, Brij^® L4, Brij^® 93 and Brij^® 52), caprylocaproyl polyoxyl-8 glycerides (Labrasol^®), polyoxyethylene stearates (including Myrj™ 49, Myrj™ S40, Myrj™ S100, Myrj™ S8 and Myrj™ 52) and mixtures thereof.

6. Pharmaceutical formulation according to any one of claims 1 to 5 wherein the clofazimine is solubilized in the form of a micro- or nanoemulsion.

7. Pharmaceutical composition according to any one of claims 1 , 3, or 5,

wherein the oil phase is oleic acid,

wherein the co-solvent is polyethylene glycol 400 (PEG 400) and

wherein the aqueous liquid carrier is distilled water or isotonic saline, preferably isotonic saline.

8. Pharmaceutical composition according to any one of claims 1 to 7, wherein the oil phase is in the range of 1 %-30% (v/v) of parts (b) (c) and (d), (i.e. excluding (a) and (e)),

the surfactant (c):co-solvent (d) ratio is in the range of 1 :1 to 25:1 (v/v), the amount of the aqueous carrier (e) is in the range of 60-90% (v/v) of the total composition (a-e) and the

amount of clofazimine (a) is in the range of 0.1 % to 2% (w/w) of the total composition (a-e).

9. Pharmaceutical composition according to any one of claims 1 to 8, wherein the the oil phase is about 15% (v/v) of parts (b) (c) and (d),

the surfactant (c):co-solvent (d) ratio is about 1 1.9:1 ,

10. Pharmaceutical composition according to any one of claims 1 to 9 prepared by a process comprising the following steps:

(2) adding the resulting solution to the non-ionic surfactant (c),

followed by ultrasonic homogenization to obtain a homogenized oily solution,

(3) preparation of the aqueous phase by dissolving the co-solvent (d) in the aqueous liquid carrier (e),

(6) adjusting the NaCI concentration to between 31 and 300mM NaCI, preferably to 154 mM NaCI.

11. Pharmaceutical composition according to claim 10, wherein the homogenization in step (2) is carried out in an ultrasonic bath for about 5 minutes to 15 minutes, preferably about 10 minutes at room temperature.

12. Pharmaceutical composition according to claim 10 or 11 , wherein the homogenization in step (4) is carried out with an ultrasonic probe for about 0.5 to 5 minutes, preferably for about 1 minute with a 30% amplitude at room temperature.

13. Pharmaceutical formulation in the form of an aerosol for inhalation, preferably for lower lung deposition, prepared by aerosolization of the composition according to anyone of claims 1 to 12 by a nebulizing device selected from an ultrasound nebulizer, an electron spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer or a mechanical soft mist inhaler,

and

wherein the emulsion droplets have a mass median diameter (MMD) of less than 1 pm,

wherein the aerosol particles have a mass median aerodynamic diameter (MMAD) of 1 to 5 mhi.

14. Pharmaceutical formulation according to claim 13, wherein the device is selected from any of Vectura fox, Pari eFIow, Pari Trek S, Philips Innospire mini, Philips InnoSpire Go, Medspray device, Aeroneb Go, Aerogen Ultra, Respironics Aeroneb, Akita, Medspray EcoMyst and Respimat.

15. Pharmaceutical formulation according to claim 13 or 14, wherein the device controls the patient’s inhalation flow rate either by an electrical or mechanical process, and

wherein the aerosol particles have a mass median aerodynamic diameter (MMAD) of 1 to 5 pm.

16. Pharmaceutical formulation according to any one of claims 13 to 15, wherein the aerosol production by the device is triggered by patient inhalation.

17. Pharmaceutical composition according to any one of claims 1 to 12 or pharmaceutical formulation according to any one of claims 13 to 16 which is to be used in combination with an agent for dispersing and/or destruction of the biofilm, with mucolytic and/or mucoactive agents, and/or agents that reduce biofilm formation selected from hypertonic saline (4-7%, preferably 6%) (nebulized), metaperiodate, SDS, sodium bicarbonate, tromethamine (THAM), silver nano particles, bismuth thiols, EDTA, GPA NPs, chelators, C2DA, D-amino acids (D-AAs) (such as D-leucine, D-tryptophan, D-methionine, D-tyrosine), D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides (HDPs), and antimicrobial peptides (including cathelicidins, defensins, bacteriocidins, lysozyme, lactoferrin, and protegrins).

18. Pharmaceutical composition according to any one of claims 1 to 12 or 17 or pharmaceutical formulation according to any one of claims 13, to 16 for use in the treatment and/or prophylaxis of pulmonary infections caused by mycobacteria or other gram positive bacteria.

19. Pharmaceutical composition or pharmaceutical formulation for use according to claim 18, wherein the infection is caused by a species of the genus mycobacterium selected from nontuberculous mycobacteria (NTM) (including Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus), Mycobacterium leprae, and/or tuberculosis (TB) ( Mycobacterium tuberculosis complex)

20. Pharmaceutical composition or formulation for use according to claim 19, wherein the infection is an opportunistic infection in patients with Cystic Fibrosis (CF), Chronic obstructive pulmonary disease (COPD), or AIDS such as MAC pulmonary disease or opportunistic NTM infections associated with CF or COPD.

21. Pharmaceutical composition or formulation for use according to claim 20, wherein the infection is an opportunistic NTM infection in patients with CF.

22. Use of composition of any one of claims 1 to 12 for the preparation of an aerosol formulation for inhalation, preferably for deep lung deposition, and

wherein the emulsion droplets have a mass median diameter (MMD) of less than 1 pm

23. Use according to claim 22, wherein the nebulizer is an ultrasonic nebulizer, an electron spray nebulizer a vibrating membrane nebulizer, jet nebulizer and mechanical soft mist nebulizers.

24. System for use in providing antibiotic activity when treating or providing prophylaxis against a pulmonary infection caused by mycobacteria or other gram-positive bacteria, wherein the system comprises: 1 ) a nebulized pharmaceutical formulation comprising:

(b) an oil phase selected from a fatty acid, a vegetable oil, vegetable oil derivatives, MCT, LCT, mono- di-, tri-olein and mixtures thereof,

(c) a nonionic surfactant with an HLB value of > 10,

and

2) a nebulizer,

and

wherein the emulsion droplets have a diameter (MMD) of less than 1 pm, wherein the aerosol particles have a mean particle diameter (MMAD) of 1 to 5 pm.

25. An aerosolized pharmaceutical formulation for use in providing antibiotic activity, when treating or providing prophylaxis against a pulmonary infection caused by mycobacteria or other gram-positive bacteria in a patient,

wherein the formulation comprises:

(a) a therapeutically effective amount of clofazimine or a pharmaceutically acceptable derivative, salt or ester thereof,

(c) a nonionic surfactant with an FILB value of > 10,

and

wherein the emulsion droplets have a diameter (MMD) of less than 1 pm,

wherein the aerosol particles have a mean particle diameter (MMAD) of 1 to 5 pm.

26. Use of any of the preceding claims, wherein the inhalation device exhibits an output rate of 0.1 -1.0 ml/min.

27. Use according to any of the preceding claims, wherein the total inhalation volume is between about 1 ml and 5 ml.