Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
  • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• compositions of Clofazimine Compositions of Clofazimine, Combinations Comprising Them, Processes for Their Preparation, Uses and Methods Comprising Them
• the present invention relates to pharmaceutical compositions for inhalation comprising a therapeutically effective dose of clofazimine, wherein the clofazimine is provided in the form of a suspension; processes for their preparation; and uses and methods of treatment comprising them. Furthermore, the present invention provides pharmaceutical combinations comprising clofazimine in the form of an aerosol for pulmonary inhalation.
• 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, and of pulmonary fungal infections.
• clofazimine exerts its antimicrobial effect. 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 multidrug-resistant strains, clofazimine, until recently, was generally considered to be ineffective in the treatment of pulmonary tuberculosis (see, for example, Cholo M et al. , J Anti microb Chemother, 2012 Feb, 67(2):290-8).
• MTB Mycobacterium tuberculosis
• Clofazimine is one of the three principal drugs recommended by the World Health Organization 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.
• NTM nontuberculous mycobacteria
• 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.
• BCS Biopharmaceutics Classification System
• clofazimine is generally considered an ideal candidate for the formulation into solid dispersions for improvement of oral bioavailability (see, for example, Bhusnure et al. IJRPC 2014, 4(4), 906-918).
• 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, gastrointestinal tract, and QT elongation Side effects include the development of reddish-brown discoloration of the skin and conjunctiva and 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.
• Mycobacteria can be divided into three groups:
• NTM Nontuberculous mycobacteria which encompass all other mycobacteria that are not M. tuberculosis or M. leprae, including Mycobacterium abscessus complex (MABSC), Mycobacterium avium complex (MAC).
• MABSC Mycobacterium abscessus complex
• MAC Mycobacterium avium complex
• Tuberculosis is an infectious disease caused by 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 cases of TB infection, and 1.4 million people killed by active TB disease in 2015 (see, for example, World Health Organization (WHO) Global Tuberculosis Report 2016). In addition to the high prevalence and mortality rates, the incidence of multi-drug resistant tuberculosis (MDR-TB) is a growing concern, with 580,000 patients presenting with a drug-resistant TB infection in 2015. Co- morbidities, such as human immunodeficiency virus (HIV), complicate treatment, and were responsible for 1.2 million cases of TB in 2015.
• HAV human immunodeficiency virus
• MDR multi-drug resistant
• the WHO has recommended implementing a 9 to 12 month treatment regimen of second-line anti-TB drugs.
• These regimens such as the 9 to 12 month Bangladesh regimen, treat MDR-TB with a combination of gatifloxacin, ethambutol, pyrazinamide, and clofazimine, which led to a relapse-free cure in 87.9% of patients (see, for example, Sotgiu, G, et al. , “Applicability of the shorter ‘Bangladesh regimen’ in high multidrug-resistant tuberculosis settings”, International Journal of Infectious Diseases (2017) 56 190- 193).
• the use of an aerosolized administration of clofazimine in patients with MDR TB, or XDR-TB infections should further improve patient treatment outcomes, and may shorten the duration of current treatment regimens.
• NTM nontuberculous mycobacteria
• SGM slow-growing
• RGM rapid-growing
• the slow growing Mycobacterium avium complex 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.
• 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.
• COPD chronic obstructive pulmonary disease
• CF cystic fibrosis
• MABSC Mycobacterium abscessus complex
• 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%.
• Obregon-Henao A et al Antimicrobial Agents and Chemotherapy, November 2015, Vol 59, No 11 , p. 6904-6912; Qvist,T., Pressler,T., H0iby,N. and Katzenstein,TL, “Shifting paradigms of nontuberculous mycobacteria in cystic fibrosis”, Respiratory Research (2014), 15(1 ):pp.41 -47).
• NTM 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).
• 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 (see, for example, Sousa S. et al., International Journal of Mycobacterioiogy 4 (2015), 36-43).
• EPS extracellular polymeric substances
• mycobacterial EPS differ in nature from other biofilms, as mycobacteria do not produce exopolysaccharides (see, for example, Zambrano MM, Kolter R. Mycobacterial biofilms: a greasy way to hold it together. Cell. 2005 ⁇ .
• Mycobacterial biofilms vary between species, but can contain mycolic acids, glycopeptidolipids, mycolyl-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 (see, for example, Faria S. et al. , Journal of Pathogens, Vol 2015, Article ID 809014).
• Combinations of clofazimine and amikacin have been shown to act synergistically in vitro against both Mycobacterium abscessus and Mycobacterium avium (see, for example, van Ingen, J., et al.,“In Vitro Synergy between Clofazimine and Amikacin in Treatment of Nontuberculous Mycobacterial Disease”, Antimicrobial Agents and Chemotherapy 56 (12), 6324-6327 (2012)). Further, synergy has been shown with combinations of clofazimine and bedaquiline used against Mycobacterium tuberculosis (see, for example, Cokol, M. et al.
• Fungal pathogens have emerged as a leading cause of human mortality. Current estimates suggest death due to invasive fungal infections is on par with more well- known infectious diseases such as tuberculosis.
• Candida albicans, Cryptococus neoformans, and Aspergillis fumigatus represent the most prevalent fungal pathogens of humans. Each of these species is responsible for hundreds of thousands of infections annually with unacceptably high mortality rates due to poor diagnostics and limited treatment options.
• Clofazimine has been shown to exhibit efficacy as a combination agent against multiple fungi (see, for example, Robbins, N., et al., “An Antifungal Combination Matrix Identifies a Rich Pool of Adjuvant Molecules that Enhance Drug Activity against Diverse Fungal Pathogens”, Cell Reports 13, 1481 -1492, November 17, 2015). Fungi also play a role as commensals, colonizers and/or pathogens in cystic fibrosis (see, for example, Chotirmall, S.H. and McElvaney, N.G.,“Fungi in the cystic fibrosis lung: Bystanders or pathogens?”, The International 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 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.
• a pharmaceutical composition comprising:
• an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solutions
• clofazimine or the pharmaceutically acceptable derivative or salt thereof, is provided in the form of particles in a suspension
• particles of clofazimine, or the pharmaceutically acceptable derivative or salt thereof have a median size of less than 5 pm and a D90 of less than 6 pm.
• the particles of clofazimine, or the pharmaceutically acceptable derivative or salt thereof have a mean size of less than 2 pm and a D90 of less than 3 pm.
• an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solutions
• clofazimine is provided in the form of particles in a suspension
• particles of clofazimine have a median size of less than 5 pm and a D90 of less than 6 pm.
• the particles of clofazimine have a median size of less than 2 pm and a D90 of less than 3 pm.
• the aerosolization of the compositions 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.
• 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 pm, and preferably in the range from about 1 to about 3 pm.
• the particle size distribution is narrow and has a geometric standard deviation (GSD) of less than about 2.5.
• the present invention is based on the unexpected discovery 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 extremely hydrophobic BCS class II agent, which results in significantly increased therapeutic efficacy coupled with reduced systemic side effects.
• 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 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.
• 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.
• 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.
• 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.
• a prodrug is a derivative of a compound which, upon administration, is capable of providing the active form of the compound.
• Such derivatives may be an ester or amide of a carboxyl group, a carboxyl ester of a hydroxyl group, or a phosphate ester of a hydroxyl group.
• a therapeutically effective amount means an amount of clofazimine, or a pharmaceutically acceptable salt or derivative thereof, as disclosed for this invention, which has a therapeutic effect.
• the doses of clofazimine which are useful in treatment are therapeutically effective amounts.
• 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.
• 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/combination for prophylactic and/or therapeutic purposes.
• 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.
• therapeutic treatment refers to administering treatment to a patient already suffering from an infection.
• treating is the administration to a mammal (either for therapeutic or prophylactic purposes) of therapeutically effective amounts of clofazimine.
• inhalation is meant to refer to pulmonary inhalation.
• infection as used herein is meant to refer to pulmonary infections.
• the term“substantially” when used to refer to the purity of a compound indicates a purity of compound of 95% or greater purity.
• the term“appropriate particle size” refers to a particle size of clofazimine in a composition, or a composition that provides the desired therapeutic effect when administered to a patient.
• the term “appropriate concentration” refers to a concentration of a component in a composition or combination which provides a pharmaceutically acceptable composition or combination.
• 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. European Pharmacopoeia or National Formulary.
• Aqueous electrolyte solutions as used in accordance with the present invention as the aqueous liquid carrier may further comprise sodium chloride, potassium chloride, lithium chloride, magnesium chloride, calcium chloride or mixtures thereof.
• the aqueous liquid carrier is preferably isotonic saline solution (0.9% NaCI corresponding to about/approximately 150 mM NaCI, preferably 154 mM NaCI).
• Clofazimine has been shown to exist in at least four polymorphic forms (see, for example, 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 a triclinic form FI, a monoclinic form Fll, and an orthorhombic form Fill.
• a further form FIV has also been seen only at high temperatures.
• composition comprising:
• an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solutions
• clofazimine is provided in the form of particles in a suspension
• the particles of clofazimine have a median size of less than 5 pm and a D90 of less than 6 pm, preferably a median size of less than 2 pm and a D90 of less than 3 pm, and wherein the clofazimine is provided in a polymorphic form or forms selected from triclinic form FI, monoclinic form Fll and orthorhombic form Fill and mixtures of such forms.
• the clofazimine is provided substantially in orthorhombic form Fill.
• a pharmaceutical composition according to any of the composition embodiments herein described is provided wherein the nonionic surfactant is selected from polysorbate 20 (for example Tween ® 20, polysorbate 60 (for example Tween ® 60) , polysorbate 80 (for example Tween ® 80), stearyl alcohol, a polyethylene glycol derivative of hydrogenated castor oil with an Hydrophilic-Lipophilic Balance value of 14 to 16 (for example Cremophor ® RFI 40) , a polyethylene glycol derivative of hydrogenated castor oil with an Hydrophilic- Lipophilic Balance value of 15 to 17 (for example Cremophor ® RFI 60), sorbitan monolaurate (for example Span ® 20), sorbitan monopalmitate (for example Span ® 40), sorbitan monostearate (for example Span ® 60), polyoxyethylene (20) oleyl ether (for example Brij ® 020), polyoxyethylene (20) cetyl ether (
• a pharmaceutical composition according to any of the composition embodiments described herein is provided, wherein the non- ionic surfactant is polysorbate 80, and wherein the aqueous liquid carrier is distilled water, hypertonic saline or isotonic saline.
• a pharmaceutical composition is provided wherein the hypertonic saline is from 1 % to 7% (w/v) sodium chloride.
• a pharmaceutical composition is provided wherein the non-ionic surfactant is ultrapure polysorbate 80 (for example NOF Corporation Polysorbate 80 (Hx2)), and wherein the aqueous liquid carrier is isotonic saline.
• a pharmaceutical composition according to any one of the composition embodiments described herein is provided wherein the osmolality of the composition is in the range of 200-700 mOsm/kg. In a further embodiment, the osmolality of the composition is in the range of 300-400 mOsm/kg.
• a pharmaceutical composition according to any one of the composition embodiments described herein, is provided wherein the nonionic surfactant is in the range of 0.001 % to 5% (v/v) of the total composition and the amount of clofazimine is in the range of 0.1 % to 20% (w/v) of the total composition.
• compositions according to any one of the composition embodiments described herein is provided, wherein the pharmaceutical composition is prepared by a process comprising the following steps:
• the pH is adjusted to 7.4, and the sodium chloride concentration is adjusted to 154 mM sodium chloride.
• the homogenization in step (1 ) is carried out by high pressure homogenization, high shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes.
• the homogenization of clofazimine is carried out in multiple steps of homogenization.
• the appropriate particle size of the clofazimine are particles having a mean size of less than 5 pm and D90 of less than 6 mGh.
• the appropriate particle size of clofazimine are particles having a mean size of less than 2 mhp and D90 of less than 3 pm.
• compositions according to any one of the composition embodiments described herein is provided, wherein the pharmaceutical composition is prepared by a process comprising the following steps:
• the pH is adjusted to 7.4, and the sodium chloride concentration is adjusted to 154 mM sodium chloride.
• the homogenization in step (1 ) is carried out by high pressure homogenization, high shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes.
• the homogenization of clofazimine is carried out in multiple steps of homogenization.
• the appropriate particle size of the clofazimine are particles having a mean size of less than 5 pm and D90 of less than 6 pm.
• the appropriate particle size of clofazimine are particles having a mean size of less than 2 pm and D90 of less than 3 pm.
• compositions according to any one of the composition embodiments described herein is provided, wherein the composition is prepared by a process comprising the following steps:
• the pH is adjusted to 7.4, and the sodium chloride concentration is adjusted to 154 mM sodium chloride.
• the micronization of the clofazimine is carried out by jet milling, spray drying, ball milling, or super critical fluids processing. In another embodiment, the micronization of clofazimine is carried out in multiple steps of micronization.
• the appropriate particle size of the clofazimine are particles having a mean size of less than 5 pm and D90 of less than 6 pm. In a further embodiment, the appropriate particle size of clofazimine are particles having a mean size of less than 2 mhp and D90 of less than 3 pm.
• a pharmaceutical composition according to any one of the composition embodiments described herein is provided, wherein the composition is prepared by a process comprising homogenization of a suspension of clofazimine in the nonionic surfactant, water containing an appropriate concentration of sodium chloride, and which has been adjusted to a pH of between pH 5.5 and pH 7.5, to obtain clofazimine of an appropriate particle size.
• the pH is adjusted to 7.4, and the sodium chloride concentration is adjusted to 154 mM sodium chloride.
• the homogenization is carried out by high pressure homogenization, high shear homogenization, wet milling, ultrasonic homogenization, or a combination of such processes.
• the homogenization of clofazimine is carried out in multiple steps of homogenization.
• the appropriate particle size of the clofazimine are particles having a mean size of less than 5 pm and D90 of less than 6 pm.
• the appropriate particle size of clofazimine are particles having a mean size of less than 2 pm and D90 of less than 3 pm.
• composition according to any of the composition embodiments described herein comprising the following steps:
• the pH is adjusted to 7.4, and the sodium chloride
• the homogenization is carried out by high pressure homogenization, wet milling, ultrasonic homogenization, or a combination of such processes.
• the homogenization of clofazimine is carried out in multiple steps of homogenization.
• the appropriate particle size of clofazimine are particles having a mean size of less than 5 mhp and a D90 of less than 6 mGh.
• the appropriate particle size of clofazimine are particles having a mean size of 2 mhp and a D90 of less than 3 mGh.
• the pH is adjusted to 7.4, and the sodium chloride
• the homogenization is carried out by high pressure homogenization, wet milling, ultrasonic homogenization, or a combination of such processes.
• the homogenization of clofazimine is carried out in multiple steps of homogenization.
• the appropriate particle size of clofazimine are particles having a mean size of less than 5 mhp and a D90 of less than 6 mGh.
• the appropriate particle size of clofazimine are particles having a mean size of 2 mhp and a D90 of less than 3 mGh.
• a process for the preparation of a pharmaceutical composition according to any one of the pharmaceutical composition embodiments as described herein comprising the following steps:
• the pH is adjusted to 7.4, and the sodium chloride
• the micronization of the clofazimine is carried out by jet milling, spray drying, ball milling, or super critical fluids processing. In a further embodiment, the micronization of clofazimine is carried out in multiple steps of micronization.
• the appropriate particle size of clofazimine are particles having a mean size of less than 5 mhp and a D90 of less than 6 mGh. In another embodiment, the appropriate particle size of clofazimine are particles having a mean size of 2 mhp and a D90 of less than 3 mGh.
• composition according to any one of pharmaceutical composition embodiment described herein is provided, comprising homogenization of a suspension of clofazimine in the nonionic surfactant, water containing an appropriate concentration of sodium chloride, and which has been adjusted to a pH of between pH 5.5 and pH 7.5, to obtain clofazimine of an appropriate particle size.
• the pH is 7.4, and the appropriate concentration of sodium chloride is 154 mM sodium chloride.
• the homogenization is carried out by high pressure homogenization, wet milling, ultrasonic homogenization, or a combination of such processes.
• the homogenization of clofazimine is carried out in multiple steps of homogenization.
• the appropriate particle size of clofazimine are particles having a mean size of less than 5 mhp and a D90 of less than 6 mGh. In another embodiment, the appropriate particle size of clofazimine are particles having a mean size of 2 mhp and a D90 of less than 3 mGh.
• composition according to any one of composition embodiments described herein, comprising the following steps: (a) homogenization of a suspension of clofazimine, the non-ionic surfactant and water to obtain a suspension comprising clofazimine of an appropriate particle size; (b) adjusting the pH of the resulting suspension a pH of between pH 5.5 and pH 7.5; (c) adjusting the sodium chloride concentration to an appropriate concentration, and (d) adjusting the osmality to an appropriate level; and wherein steps (b), (c) and (d), may occur in the order of (b),
• composition according to any one of the composition embodiments described herein, comprising the following steps: (a) homogenization of a suspension of clofazimine and a non-aqueous liquid to obtain a suspension comprising clofazimine of the appropriate particle size; (b) isolation of the clofazimine; (c) addition of the clofazimine to the nonionic surfactant and water; (d) adjusting the pH of to 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; and wherein steps (d) and (e) may occur in the order of (d), (e); or (e), (d).
• a process for the preparation of a pharmaceutical composition comprising the following steps: (a) micronization of clofazimine to obtain clofazimine of an appropriate particle size, and (b) addition of the clofazimine to the nonionic surfactant, water containing an appropriate concentration of sodium chloride, and which has been adjusted to a pH of between between pH 5.5 and 7.5.
• a pharmaceutical combination in the form of an aerosol for inhalation is provided, prepared by aerosolization of the composition according to any one of the composition embodiments described herein, by a nebulizing device selected from an ultrasonic nebulizer, an electron spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer and a mechanical soft mist inhaler, and
• the aerosol particles produced by the nebulizing device have a mass median aerodynamic diameter of 1 to 5 pm.
• the aerosol for inhalation is for lower lung deposition.
• the nebulizing device exhibits an output rate of 0.1 to 1.0 ml/min.
• the total inhalation volume is between 1 ml and 5 ml.
• a pharmaceutical composition according to any one of the composition embodiments described herein is provided which is for use in combination with an agent for dispersing and/or destruction of biofilm, with mucolytic and/or mucoactive agents, and/or agents that reduce biofilm formation selected from nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nano particles, bismuth thiols, ethylene diamine tetraacetic acid, gentamicin loaded phosphatidylcholine-decorated gold nanoparticles, chelators, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic
• composition for the use is administered before, simultaneously, or subsequently to the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitine, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-amino salicylate, and mixtures thereof.
• an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitine, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-amino salicylate, and mixtures thereof.
• a pharmaceutical combination according to any of the combination embodiments described herein is provided which is for use in combination with an agent for dispersing and/or destruction of biofilm, with mucolytic and/or mucoactive agents, and/or agents that reduce biofilm formation selected from nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nano particles, bismuth thiols, ethylene diamine tetraacetic acid, gentamicin loaded phosphatidylcholine-decorated gold nanoparticles, chelators, cis-2-decenoic acid, D-amino acids, D-enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid
• the combination for the use is used to administer a composition of the present invention before, simultaneously, or subsequently to the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitine, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-amino salicylate, and mixtures thereof.
• the composition is administered before, simultaneously or subsequently to the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof.
• the composition is administered before, simultaneously or subsequently to the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.
• a pharmaceutical composition according to any one of the composition embodiments as described herein is provided for use in the treatment and/or prophylaxis of a pulmonary infection caused by mycobacteria or other gram positive bacteria.
• the infection is caused by a species of the genus mycobacterium selected from nontuberculous mycobacteria and Mycobacterium tuberculosis complex, and a combination thereof.
• the nontuberculous mycobacteria is selected from Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium leprae, and a combination thereof.
• the infection is an opportunistic infection, selected from MAC pulmonary disease and nontuberculous infection, in a patient with cystic fibrosis, chronic obstructive pulmonary or acquired immune deficiency syndrome.
• the infection is an opportunistic nontuberculous mycobacteria infection in patients with cystic fibrosis.
• the composition for the use is administered before, simultaneously, or subsequently to the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitine, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-amino salicylate, and mixtures thereof.
• the composition is administered before, simultaneously or subsequently to the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof.
• the composition is administered before, simultaneously or subsequently to the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.
• a pharmaceutical combination according to any of the combination embodiments as described herein is provided for use in the treatment and/or prophylaxis of a pulmonary infection caused by mycobacteria or other gram positive bacteria.
• the infection is caused by a species of the genus mycobacterium selected from nontuberculous mycobacteria and Mycobacterium tuberculosis complex, and a combination thereof.
• the nontuberculous mycobacteria is selected from Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium leprae, and a combination thereof.
• the infection is an opportunistic infection, selected from MAC pulmonary disease and nontuberculous infection, in a patient with cystic fibrosis, chronic obstructive pulmonary or acquired immune deficiency syndrome.
• the infection is an opportunistic nontuberculous mycobacteria infection in patients with cystic fibrosis.
• the combination for the use is used to administer a composition of the present invention before, simultaneously, or subsequently to the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitine, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-amino salicylate, and mixtures thereof.
• the combination for the use is used to administer a composition of the present invention before, simultaneously or subsequently to the administration of an agent selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, and amikacin, and mixtures thereof.
• the combination for the use is used to administer a composition of the present invention before, simultaneously or subsequently to the administration of bedaquiline or a pharmaceutically acceptable salt or derivative thereof.
• 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 comprising:
• a nebulized pharmaceutical combination comprising:
• an aqueous liquid carrier selected from water, isotonic saline, buffered saline and aqueous electrolyte solutions
• clofazimine is present in the form of a suspension
• aerosol particles produced by the system have a mass median aerodynamic diameter of 1 to 5 pm.
• a pharmaceutical composition according to any one of composition embodiments described herein is provided, for use in the treatment and/or prophylaxis of pulmonary fungal infections or Clostridium difficile, or a combination thereof.
• a pharmaceutical composition according to any one of composition embodiments described herein is provided, for use in the treatment and/or prophylaxis of pulmonary fungal infections.
• the pulmonary fungal infection is Candida albicans or aspergilus fumigatus, or a combination thereof.
• a pharmaceutical combination according to any one of the combination embodiments described herein is provided, for use in the treatment and/or prophylaxis of pulmonary fungal infections or Clostridium difficile, or a combination thereof
• a pharmaceutical combinations according to any one of combinations embodiments described herein is provided, for use in the treatment and/or prophylaxis of pulmonary fungal infections.
• the pulmonary fungal infection is Candida albicans or aspergilus fumigatus, or a combination thereof.
• a method of treatment or prophylaxis of a pulmonary infection comprising administering by inhalation a composition according to any one the composition embodiments described herein.
• the infection is caused by a species of the genus mycobacterium selected from nontuberculous mycobacteria and Mycobacterium tuberculosis complex, and a combination thereof.
• the nontuberculous mycobacterium is selected from Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium leprae, and a combination thereof.
• the infection is an opportunistic infection, selected from MAC pulmonary disease and nontuberculous infection, in a patient with cystic fibrosis, chronic obstructive pulmonary disease or acquired immune deficiency syndrome.
• the infection is an opportunistic nontuberculous mycobacteria infection in a patient with cystic fibrosis.
• a method of treatment or prophylaxis of a pulmonary infection caused by mycobacteria or other gram positive bacteria, in a patient in need thereof, comprising administering by inhalation a composition according to any one of the composition embodiments described herein, before, simultaneously, or subsequently to the administration of an agent selected from bedaquiline, or a pharmaceutically acceptable salt of derivative thereof, cefoxitine, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-amino salicylate, and mixtures thereof.
• the agent is bedaquiline or amikacin.
• the agent is bedaquiline.
• Aerosol particle size is one of the important variables in defining the dose deposited and the distribution of drug aerosol in the lung.
• impaction which usually predominates for larger aerosol particles
• sedimentation which is prevalent for smaller aerosol particles. Impaction occurs when the momentum of an inhaled aerosol particle is large enough that the particle does not follow the air stream and encounters a physiological surface.
• sedimentation occurs primarily in the lower lung when very small aerosol 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. Aerosol 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. Aerosol 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 aerosol particles, i.e. about 0.5 to about 3 pm, are capable of reaching the alveolar region. Aerosol 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 aerosol 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 aerosol particles larger than the MMD, and half the mass is contained in aerosol particles smaller than the MMD.
• MMD mass median diameter
• VMD volume median diameter
• 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 an aerosol 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.
• VMD, MMD and MMAD should be the same.
• MMAD determinations will be smaller than MMD and VMD due to dehydration.
• VMD, MMD and MMAD measurements are considered to be under controlled conditions such that descriptions of VMD, MMD and MMAD will be comparable.
• the aerosol 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.
• NGI Next Generation Impactor
• 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.
• MMAD mass median aerodynamic diameter
• 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).
• 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.
• the preferred MMAD of the aerosol should be less than about 10 pm.
• clofazimine particle size and distribution Another equally important factor (in addition to aerosol particle size) is the particle size and size distribution of the solid particles, in this case clofazimine particle size and distribution.
• the size of a solid particle in a given aerosol particle must be smaller than the aerosol particle in which it is contained.
• a larger aerosol particle may contain one or more solid particles. Further, when dealing with dilute suspensions, a majority of aerosol particles may contain no solid particle.
• solid drug particles that are significantly smaller than the MMAD of the aerosol particles. For example, if MMAD of the aerosol particles is 3 pm, than a desired solid particle would be 1 pm, or smaller.
• the formulation is pumped through orifices in a plate, which breaks up the suspension into droplets. It follows, then, that the solid particles must also be smaller than these orifices, in order to pass through.
• Solid particle size in the suspension may be given by the mean size of the particles, and also by the distribution of the particles. D90 values indicate that 90% of the particles within the suspension are of the mean size or smaller.
• nebulizers 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.
• 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 EssenceTM produced by Aradigm; Porta-Neb ® , Freeway FreedomTM, Sidestream, Ventstream and l-neb produced by Respironics, Inc.; and PARI LCPIus ® , PARI LC-Star ® , and e-Flow7m produced by PARI, GmbFI. Further non-limiting examples are disclosed in US 6,196,219.
• the pharmaceutical composition may be preferably aerosolized using a nebulising device selected from an ultrasonic nebulizer, an electron spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer or a mechanical soft mist inhaler.
• a nebulising device selected from an ultrasonic nebulizer, an electron spray nebulizer, a vibrating membrane nebulizer, a jet nebulizer or a mechanical soft mist inhaler.
• the device controls the patient’s inhalation flow rate either by an electrical or mechanical process.
• 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.
• compositions and pharmaceutical combinations (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.
• the pharmaceutical compositions and pharmaceutical combinations of the present invention may also be used for the treatment and/or prophylaxis of pulmonary fungal infections.
• the pharmaceutical composition is delivered by nebulization in about 1 -5 ml, preferably 1 -2 ml of the pharmaceutical composition of the invention.
• the target fill dose is about 1 -5 ml corresponding to 20-100 mg clofazimine, based on a clofazimine concentration in the pharmaceutical composition of about 20 mg/ml.
• 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, which corresponds to a nominal dose of 15-30 mg (device dose) in the case of M. abscessus infections.
• the daily lung dose will be split accordingly.
• clofazimine is to be administered once or twice daily with a resulting total daily lung dose of about 5 to 10 mg.
• the treatment and/or prophylaxis according with the present invention can involve additional administration of mucolytic and/or biofilm destructing agents.
• agents can be prepared in fixed combination or be administered simultaneously or subsequently to the pharmaceutical composition/aerosol combination 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 nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate, sodium bicarbonate, tromethamine, silver nano particles, bismuth thiols, ethylene diamine tetraacetic acid, gentamicin loaded phosphatidylcholine- decorated gold nanoparticles, chelators, cis-2-decenoic acid, D-amino acids, D- enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic acid, fatty acids, host defense peptides, and antimicrobial peptide
• compositions/aerosol combinations in accordance with the present invention.
• active agents may be selected from bedaquiline or a pharmaceutically acceptable salt or derivative thereof, cefoxitine, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-amino salicylate, and mixtures thereof.
• agents can be prepared in fixed combination or be administered prior to, simultaneously or subsequently to the pharmaceutical composition/aerosol combination comprising clofazimine in accordance with the present invention.
• compositions and combinations below have been prepared in accordance with the processes described herein.
• Example 1 200 mg of clofazimine (as triclinic form I), 90 mg of sodium chloride, and 9.5 ml of water were mixed in an Ultra-Turrax homogenizer two times at 10,000 rpm for 5 minutes. 0.5 ml of polysorbate 80 (NOF Hx2) was added. This mixture was treated with an ultrasonic probe (Branson Digital SonifierTM 250D with Bandelin Sonoplus Probe MS73) seven times, 3 minutes each, with an amplitude of 70%. The volume was adjusted to 10 ml with water. This suspension was filtered through VWR folded qualitative filter paper (303, particle retention 5-13 pm, Size: 150 mm), to give the Composition of Example 1 .
• Example 1 had a median particle size of clofazimine of 3.9 pm, with a D90 of 6.7 pm.
• concentration of clofazimine was determined by ultraviolet/visible spectroscopy at 280 nm, calibrating with a stock solution of 1 mg/ml of clofazimine diluted in the mobile phase, and determined to be 7.16 mg/ml.
• Example 1 The composition of Example 1 is shown in Table 1
• a slurry of clofazimine (10 g) in toluene (20 ml) was stirred at 40°C in an oil bath for 72 hours using a magnetic stirrer at 800 rpm.
• the solid portion of the slurry was collected by filtration through a crucible and dried at a maximum temperature of 40°C under vacuum in an oven. This yielded 8.64 g of clofazimine as substantially pure (>98%) orthorhombic form III.
• a suspension containing 6g of clofazimine of orthorhombic form III in 100 ml of water containing 0.5% polysorbate 80 (NOF Hx2) and 0.6% sodium chloride was pre- micronized for approximately 40 seconds at 10,000 rpm using an Ultra-Turrax®.
• the pre-formulation was prepared by adding 0.6% sodium chloride in water to give a volume of 300 ml. 300 ml of this suspension was added into the inlet of the homogenizer, a M-110EH-30 microfluidizer (Microfluidics, Westwood, MA, USA) and a pre-homogenization step was performed for 15 minutes by circulation of the suspension through the H30Z chamber at 5,000 psi.
• the second H10Z chamber was installed in series with the first chamber and the suspension was further homogenized for 23 minutes at 25,000 psi.
• Particle size analysis was performed with a HORIBA LA 950 indicating a median particle size of 0.83 mhp with a D90 value of 1.17 mGh.
• a concentration of clofazimine of 16.05mg/ml was determined by ultraviolet/visible spectroscopy at 280 nm, calibrating with a stock solution of 1 mg/ml of clofazimine diluted in the mobile phase.
• Example 2 The composition of Example 2 is shown in Table 2
• Example 3 A suspension of clofazimine (crystal modification orthorhombic Form III) in a solution of water, sodium chloride and Polysorbate 80, was treated using a
• M-110EH-30 Microfluidizer Processor (chambers: H30Z and G10Z) operated for 30 minutes at a pressure of 28,250 psi, with the H30Z-G10Z configuration to produce the Compostion of Example 3, with the resulting particles of clofazimine having a median particle size of 1.28 mhp and a D90 below 2 mGh.
• Example 3 The composition of Example 3 is shown in Table 3.
• Example 3 The viscosity of the Composition of Example 3 was tested using a STRESSTECH Rheometer in stress control mode. A double gap geometry was utilized and the spindle was continuously rotated to ensure the particulates remained in suspension during temperature points. Viscosity was measured across 0.01 , 0.05, and 0.1 Pa stress each at 20°C, 25°C, and 30°C. Two separate loadings were performed to obtain the average viscosities shown in Table 4 below.
• compositions of the present invention have been tested for their ability to inhibit growth of clinical NTM species in an acute in vivo pulmonary infection mouse model to obtain preliminary data to establish clofazimine concentration levels in lung tissue after direct respiratory delivery as opposed to systemic administration.
• Two separate mouse models are used in order to investigate pulmonary NTM infection, dependent on the bacterial species of interest.
• 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 sequences”.
• mice For in vivo safety and tolerability, 6-8 week old Balb/C female mice are obtained from Charles River. The mice are rested for one week before dosing. For each dose of clofazimine, three healthy mice are given a total of three doses every other day. Mice were dosed at 10.0, 5.01 , and 2.51 mg/kg of clofazimine in the composition of Example 1. The compounds were given to 3 healthy mice for a total of three doses,
• Clofazimine was found to be safe at 20 mg/kg (gavage, 200 pi).
• the composition of Example 1 showed no toxicity at the highest dose tested (10.0 mg/kg; 0.2506 mg/dose in 35 mI intratracheally). Accordingly, the composition of Formula I was considered safe and well tolerated at 10.0 mg/kg.
• Minimum Inhibitory Concentration Minimum inhibitory concentration testing was performed by microbroth dilution method using MCieller Hinton (MH) broth (Cation Adjusted) to the calcium and magnesium ion concentration recommended in the CLSI standard M7-A7 (Becton Dickinson). MIC testing also was performed by microbroth dilution method using 7H9 broth (Sigma-Aldrich). The justification for use of both MH and 7H9 broth for compound screening is that antimycobacterial compounds have been shown to display different MIC activity depending on the broth that is used in the MIC assay.
• M. abscessus was grown on 7H11 agar plates (Sigma-Aldrich) for 3 days at 35-37°C in ambient air (depending on bacterial strain), and M. avium was grown on agar 7H11 plates (Sigma-Aldrich) for 21 -30 days at 37°C in ambient air.
• the colony forming units are taken from the agar plates and placed in either MH or 7H9 broth with 0.05% tween-80 and grown at 35-37°C in ambient air until the optical density (OD) absorbance taken after 3 days ⁇ M. abscessus) or 12 ⁇ M. avium) of growth is an (OD) 0.08 - 0.1 (0.5 McFarland Standard).
• the bacterial cell suspensions are then confirmed by preparing them in saline, matching the (OD) 0.08 - 0.1 (0.5 McFarland Standard).
• Compound stock solutions were made by
• Resazurin Microtiter Assay Plate method uses the addition of resazurin (7-Hydroxy-3/-/- phenoxazin-3-one 10-oxide) to the 96 well plate.
• Resazurin is a blue dye, itself weakly fluorescent until it is irreversibly reduced to the pink colored and highly red fluorescent resorufin. It is used as an oxidation-reduction indicator to determine bacterial cell viability in MIC assays. Assays were done in triplicate. Assay #1 was performed after storage of the
• Example 1 Composition of Example 1 at 4°C for 2 months, Assay #2 was performed at 4 months, and Assay #3 at five months.
• Minimum Inhibitory Concentrations in the Presence and Absence of CF Sputum Composition of Example 1 at 4°C for 2 months, Assay #2 was performed at 4 months, and Assay #3 at five months.
• sputum was collected from patients who had not received antibiotics for the previous 48 hours, and their sputum was sterilized by exposure to UV light to eliminate endogenous bacteria. Following sterilization, M. abscessus , M. avium, M. intracellulare, and M. Chimaera were incubated in 10% CF sputum before undergoing MIC testing. The MIC of the
• Example 1 Composition of Example 1 was measured following the same CLSI protocol as described above, in the presence and absence of cystic fibrosis patient sputum. All studies were performed in duplicate.
• Example 1 demonstrates potent in vitro activity against both M. abscessus and M. avium, and is stable at least over this time period.
• mice Three mice were sacrificed day 1 post-infection to determine bacterial uptake.
• Whole lungs, spleens, and livers are extracted, 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) plated on 7H11 agar plates. The plates are placed in 32°C dry-air incubator (strain dependent) for 7 days.
• Example 1 The Composition of Example 1 10.0 mg/kg was administered by a Microsprayer ® (35 pi) through the pulmonary route, and clofazimine (gavage), amikacin (subcutaneous) in a volume of 200 mI per mouse which begins 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 the compounds. Six mice of all groups (untreated control, clofazimine (gavage), composition of Example 1 , and amikacin treated mice) were sacrificed and bacterial loads were determined. Plating of lung homogenate at 0-1 -2-3-4-5-6-7, spleen at 0-1 -2-3-4-5-6-7 and liver at 0-1 -2-3-4-5-6-7.
• Log 10 protection values of at least 0.60 indicate activity is statistically significant.
• Statistical analysis was performed by first converting CFU to logarithms, which were then evaluated by a one-way ANOVA followed by a multiple comparison analysis of variance by a one-way Tukey test (GraphPad Prism analysis software). Differences are considered significant at the 95% level of confidence.
• Table 6 shows the average Log-io CFU data and standard error of mean (SEM) following SCID mouse M. abscessus infection, where “n” is the total number of animals in group at time of sacrifice.
• Example 6 The data in Table 6 indicate that treatment with the composition of Example 1 led to the greatest reduction in bacterial recovery in the lungs and spleen of animals infected with M. abscessus. This bacterial reduction was statistically improved over treatment with amikacin, or oral clofazimine.
• the acute Beige mouse model received a non-invasive aerosol exposure pulmonary infection with 1x10 8 colony forming units (CFU)/ml ⁇ M. avium strain 2285 rough).
• Working stocks of M. avium strain 2285 rough were frozen in 1 ml aliquots and stored at -80°C before use. For infection an aliquot was thawed, disrupted 20 times with a 1 ml luer-lok syringe fitted with a 26 g needle, and diluted in sterile 1 x phosphate buffered saline (PBS).
• PBS sterile 1 x phosphate buffered saline
• mice Three mice were sacrificed on day 1 and day 7 post-infection to determine bacterial uptake.
• Whole lungs, spleens, and livers were extracted, homogenized in 4.5 ml of 1x PBS and diluted 1 :10. Dilutions (0-1 -2-3-4-5-6-7) are plated on 7H11/OADC, TSA and charcoal agar plates and incubated at 32°C in a dry-air incubator (strain dependent) for 30 days.
• the composition of Example 1 10.0 mg/kg was administered by a Microsprayer ® (35 pi) though the pulmonary route and clofazimine (gavage) in a volume of 200 mI per mouse which begins on day 7 post-infection and continued every other day for 10 consecutive days.
• mice were sacrificed 5 days after administration of the last dose of the compounds.
• Six mice of all groups (untreated control, clofazimine (gavage), and the composition of Example 1 ) were sacrificed and bacterial loads were determined.
• Log 10 protection values of at least 0.60 indicate activity is statistically significant.
• Statistical analysis was performed by first converting CFU to logarithms, which were then evaluated by a one-way ANOVA followed by a multiple comparison analysis of variance by a one-way Tukey test (SigmaStat software program). Differences are considered significant at the 95% level of confidence.
• Table 7 shows the average Log-io CFU data following Beige mouse M. avium infection.
• 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 mI); Clofazimine (oral gavage, 20 mg/kg, 200 mI); Composition of Example 1 (IT, Microsprayer®, 10 mg/kg, 35 mI).
• mice were sacrificed on Day 57, two days after the final treatment. Plates were placed in a 37°C dry-air incubator for 30 days.
• composition of the present invention does not have the same issues, and is able to maintain antimycobacterial activity even after the infection has become well-established.
• Calu-3 Three different cell types under two in vitro conditions were used to assess pulmonary epithelial cell viability: Calu-3; A549; and hAELVi cells.
• Cells were either treated under“submerged conditions” (i.e. in cell culture media on TranswellTM plates) or“air-liquid interface” mimicking conditions (ALI), which had cell culture media removed from the apical side of the cells.
• In“submerged conditions” Calu-3 cells were exposed to three doses of the Composition of Example 3 (10%, 50%, or 100%) for four hours.
• AO/PI acridine orange/propidium iodide
• THP-1 cells were differentiated to macrophage-like cells following incubation with 124 ng/ml phorbol 12-myristate 13 acetate (PMA) for 3 days. Once the cells were matured, they were exposed to the Composition of Example 3 (diluted 1 :200 in Hank’s Buffered Salt Solution (HBSS)) for four hours. Cells were stained via AO/PI, as described above, to determine cell viability following exposure.
• PMA phorbol 12-myristate 13 acetate
• TEER Transepithelial Electrical Resistance
• Calu-3 cells were seeded at 1x10 5 cells/well on a TranswellTM 3460, and left for 12 days to grow to confluence. TEER measurements were performed using an EVOM2 (World Precision Instruments, Friedberg, Germany) according to the manufacture’s instructions. Following seeding, Calu-3 cells were exposed to either saline (negative control) or the Composition of Example 3 (concentrations: 20 mg/ml, 10 mg/ml, or 2 mg/ml). The cells were exposed from 2 to 4 hours, before measuring TEER.
• THP-1 cells 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.
• LPS lipopolysaccharide
• Example 3 Under“submerged” conditions the Composition of Example 3 led to no visual reduction in cell viability over four-hour incubation at any of the concentrations administered.
• Example 3 With regard to macrophage uptake, differentiated THP-1 cells were incubated at 1 :200 HBSS for four hours to determine macrophage cell viability after exposure. The Composition of Example 3 did not induce cell death, but did show clofazimine uptake by the macrophages.
• Calu-3 cells were exposed to HBSS or three concentrations of the Composition of Example 3 for four hours, and TEER
• Example 3 The positive control LPS behaved as expected in this model.
• the Composition of Example 3 demonstrated no significant changes in cytokine at any timepoint investigated.
• mice 6-8-week-pld Balb/C female mice were given a total of three doses every other day. Mice were dosed at 10.0, 5.01 , and 2.51 mg/kg using the Composition of Example 1. The composition was given via Microsprayer® aerosol intratracheal (IT)
• mice were observed at 10 minutes, 1 , 2 and 4 hours after dosing, and then daily afterwards. Table 10 shows gross observations following administration. “BAR” indicates the animals were bright, active and responsive.
• Table 1 1 shows weights of the animals over the three days tested.
• compositions of the present invention are well tolerated at the doses tested.

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