US20130164338A1 - Treatment of cystic fibrosis using calcium lactate, leucine and sodium chloride in a respiraple dry powder - Google Patents

Treatment of cystic fibrosis using calcium lactate, leucine and sodium chloride in a respiraple dry powder Download PDF

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US20130164338A1
US20130164338A1 US13/504,279 US201113504279A US2013164338A1 US 20130164338 A1 US20130164338 A1 US 20130164338A1 US 201113504279 A US201113504279 A US 201113504279A US 2013164338 A1 US2013164338 A1 US 2013164338A1
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formulation
dry powder
calcium
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Michael M. Lipp
Robert W. Clarke
David L. Hava
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Pulmatrix Inc
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Priority to PCT/US2011/049342 priority patent/WO2012030647A1/en
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/21Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/225Polycarboxylic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/13Amines
    • A61K31/131Amines acyclic
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/185Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic, hydroximic acids
    • A61K31/19Carboxylic acids, e.g. valproic acid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/185Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic, hydroximic acids
    • A61K31/19Carboxylic acids, e.g. valproic acid
    • A61K31/195Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/439Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/56Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives, e.g. steroids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
    • A61K33/06Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
    • A61K33/14Alkali metal chlorides; Alkaline earth metal chlorides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/007Pulmonary tract; Aromatherapy
    • A61K9/0073Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0075Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605Excipients; Inactive ingredients
    • A61K9/1611Inorganic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605Excipients; Inactive ingredients
    • A61K9/1617Organic compounds, e.g. phospholipids, fats
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/46Medical treatment of waterborne diseases characterized by the agent
    • Y02A50/468The waterborne disease being caused by a bacteria
    • Y02A50/473The waterborne disease being caused by a bacteria the bacteria being Escherichia coli, i.e. E. coli Infection

Abstract

The invention relates to a methods for treating cystic fibrosis, comprising administering an effective amount of a calcium salt formulation to the respiratory tract of an individual with cystic fibrosis. The calcium salt formulation is can be a dry powder formulation.

Description

    RELATED APPLICATION
  • This application claims the benefit of U.S. Patent Application No. 61/378,146 filed on Aug. 30, 2010, U.S. Patent Application No. 61/387,797 filed on Sep. 29, 2010, and U.S. Patent Application No. 61/431,205 filed on Jan. 10, 2011, the entire teachings of these applications are incorporated herein by reference.
  • BACKGROUND OF THE INVENTION
  • Cystic fibrosis (CF) is an inherited chronic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The mutations result in advanced or abolished CFTR activity and dysregulated ion transport across the epithelial membrane of airway epithelial cells. This defect causes the body to produce unusually thick, sticky mucus, that is not cleared well by mucociliary clearance and clogs the lungs, leading to lung infections, in particular recurrent pneumonia. The frequent lung infections and associated inflammation, over time, damage and produce structural changes in the lungs that diminish lung function. CF leads to early death in many affected individuals.
  • The poor clearance of mucus from the lungs leads to an airway environment that becomes more favorable to chronic infection as normal defense and clearance mechanisms are compromised. Bacteria have evolved mechanisms that allow colonization, persistent infection and the evasion of host defenses. While these mechanisms are diverse in nature, a common characteristic of several types of bacteria is the ability to form biofilms. Biofilms are complex, multicellular communities of bacteria that confer persistent survival in adverse conditions. Biofilm formation matures from the initial colonization of single bacteria in a freely motile or planktonic state to a multi-organism structure of bacteria. Once formed, biofilms are difficult to treat and become resistant to many antimicrobial treatments.
  • In the case of CF, Pseudomonas aeruginosa is a common bacterium that colonizes the airway and is associated with a decline in lung function and ultimately respiratory failure. Pseudomonas aeruginosa employs intricate regulatory signaling pathways to convert from the planktonic to biofilm state and to mature the structure of the biofilm. It is the formation and maintenance of the biofilm that is credited with the ability of Pseudomonas aeruginosa to persist in the airway following colonization.
  • Treatment of CF involves the use of antibiotics to treat and prevent lung infections, the use of recombinant human DNAse to reduce the viscosity of the airway lining fluid by breaking down DNA contained therein, and in some cases lung transplantation. Another therapy for CF involves the administration of aerosolized hypertonic saline solutions to the lungs. (See, Donaldson et al., N. Eng. J. Med. 254:241-250 (2006) and Elkins et al., N. Eng. J. Med. 254:229-240 (2006)). Hypertonic saline therapy is reported to improve mucociliary clearance and to reduce the number of exacerbations experienced by patients, resulting in improvements in lung function, less antibiotic being used to treat exacerbations, and a decrease in the number of days that patients are unable to work. Id. Hypertonic saline is thought to produce these benefits by improving the hydration of the airway lining fluid by the delivery of water to the lungs and by osmotic effects, which increases the volume of liquid at the airway surface and results in improved mucociliary clearance. Id.
  • Safe and effective therapies that are able to prevent bacterial colonization and biofilm formation may be a beneficial adjunct to traditional CF therapies. In this way, antibiotics may not be a favorable option because prophylactic treatments with antibiotics may lead to a greater incidence of antibiotic resistant organisms and thus further complicate treatment. A need exists for new methods for treating CF.
  • SUMMARY OF THE INVENTION
  • The invention relates to a method for treating cystic fibrosis, comprising administering to an individual with cystic fibrosis an effective amount of a calcium salt formulation. Preferably, the calcium salt formulation is a respirable dry powder containing respirable dry particles that comprise, on a dry basis, about 20% (w/w) leucine, about 75% (w/w) calcium lactate, and about 5% (w/w) sodium chloride; or about 37.5% (w/w) leucine, about 58.6% (w/w) calcium lactate, and about 3.9% (w/w) sodium chloride. Other preferred dry powder for use in treating CF include dry powders containing respirable dry particles that comprise, on dry basis:
  • about 60% to about 75% (w/w) calcium lactate, about 2% to about 5% (w/w) sodium chloride, about 15% to about 20% (w/w) leucine, and up to about 20% (w/w) of one or more additional therapeutic agents;
  • about 45% to about 58.6% (w/w) calcium lactate, about 1.9% to about 3.9% (w/w) sodium chloride, about 27.5% to about 37.5% (w/w) leucine, and up to about 20% (w/w) of one or more additional therapeutic agent;
  • about 75% (w/w) calcium lactate, about 5% (w/w) sodium chloride, about 0.01% to about 20% (w/w) of one or more additional therapeutic agents, and about 20% (w/w) or less leucine; or about 58.6% (w/w) calcium lactate, about 3.9% (w/w) sodium chloride, about 0.01% to about 37.5% (w/w) of one or more additional therapeutic agents, and about 37.5% (w/w) or less leucine.
  • The respirable dry particles have a volume median geometric diameter (VMGD) of 5 microns or less as measured at the one bar dispersion setting on ae HELOS/RODOS laser diffraction system. The respirable dry particles have a volume median geometric diameter (VMGD) of less than 5 microns, such as between 1 and 3 microns, as measured at the one bar dispersion setting on a HELOS/RODOS laser diffraction system.
  • The respirable dry powders have a Hausner Ratio of at least 1.5, preferably at least 2.0. In some embodiments, the dry powders have a Hausner Ratio of at least 1.4. The respirable dry powders have a dispersibility ratio at 1 bar/4 bar of less than 1.5, such as between 1.0 and 1.2, as measured at the 1 bar and 4 bar dispersion settings on the HELOS/RODOS laser diffraction system. The respirable dry powders have a dispersibility ratio at 0.5 bar/4 bar of less than 1.5, such as between 1.0 and 1.3, as measured by laser diffraction (HELOS/RODOS system).
  • The respirable dry powders have a Fine Particle Fraction (FPF) of less than 3.4 microns of at least 20% or at least 30%. The respirable dry powders have a Fine Particle Fraction (FPF) of less than 5.6 microns of at least 30%, or at least 40%, or at least 50%.
  • The respirable dry powders are characterized by a high emitted dose. For example, a Capsule Emitted Powder Mass (CEPM) of at least about 80% of said respirable dry powder contained in a unit dose container that contains 50 mg of said dry powder, in a dry powder inhaler is achieved when a total inhalation energy of less than about 1 Joule is applied to said dry powder inhaler. Alternatively, a Capsule Emitted Powder Mass (CEPM) of at least about 80% of said respirable dry powder contained in a unit dose container that contains 40 mg of said dry powder, in a dry powder inhaler is achieved when a total inhalation energy of less than about 1 Joule is applied to said dry powder inhaler.
  • The respirable dry powders can contain amorphous and/or crystalline states. For example, calcium lactate can be amorphous and the sodium chloride and/or leucine can be crystalline, or calcium lactate and leucine can be amorphous. Alternatively, the calcium lactate and sodium chloride are substantially in the amorphous phase and the leucine is in either the crystalline and/or amorphous phase.
  • The respirable dry powders can further comprise an additional therapeutic agent.
  • The invention also relates to a method of reducing the formation of a biofilm in a cystic fibrosis patient comprising administering to an individual with cystic fibrosis an effective amount of a calcium salt formulation (e.g., a dry powder described herein).
  • The invention also relates to a method of disrupting or dispersing a biofilm in a cystic fibrosis patient comprising administering to an individual with cystic fibrosis an effective amount of a calcium salt formulation (e.g., a dry powder described herein).
  • The invention also relates to a method for treating or preventing an acute exacerbation of cystic fibrosis comprising administering to the respiratory tract of a patient in need thereof an effective amount of calcium salt formulation (e.g., a dry powder described herein).
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1A is a schematic showing an in vitro simulated cough system. Bottled compressed air, filtered to remove particles >0.01 micrometers in diameter is used to fill the Pressurized Chamber to a set pressure to mimic the flow of a cough maneuver. To initiate a cough maneuver, the solenoid valve is actuated, releasing the compressed air through a pneumotachometer, which records the air flow rate, and a low resistance HEPA filter. Air enters the trough with airflow passing over the mucus mimetic and generating aerosol particles. The drip trap prevents any bulk motion of the mucus mimetic from entering the holding chamber while the generated aerosol enters the expandable holding chamber. After completion of the cough, the optical particle counter sizes and counts the aerosol particles in the holding chamber as it draws the air out of the chamber.
  • FIG. 1B is a graph showing calcium chloride is more effective than 0.90% saline in the suppression of bioparticle formation in an in vitro model. Mean (±SEM) cumulative particle counts were measured following simulated cough over mucus mimetic (MM) in a tracheal trough model (n=4 per condition). The effect of each test formulation was tested by topically treating the mimetic with nebulized aerosol prior to simulated cough and enumeration of the particles (0.3 to 25 μm) with an optical particle counter.
  • FIG. 1C is a graph showing suppression of pathogen containing bioparticle formation by exposure to 1.29% calcium chloride (0.12M) in 0.90% sodium chloride solution. Mucus mimetics were mixed with K. pneumoniae and added to the cough system. Following simulated cough, bioparticles were collected in liquid broth and the number of CFU determined. Mimetic treated with calcium aerosols reduced the number of particles containing K. pneumoniae by 75% relative to the untreated control.
  • FIG. 2A is a graph showing that mice infected with S. pneumoniae and treated two hours after infection with CaCl2-saline aerosol (1.29% calcium chloride (0.12M) in 0.90% sodium chloride) for fifteen minutes, have less bacterial burden than untreated controls. Each data point represents the data obtained from a single animal. The bar for each group represents the geometric mean of the group.
  • FIG. 2B is a graph showing that mice treated with CaCl2-saline aerosol (1.29% calcium chloride (0.12M) in 0.90% sodium chloride) for fifteen minutes, two hours before infection with S. pneumoniae, have less bacterial burden than untreated controls. Each data point represents the data obtained from a single animal. The bar for each group represents the geometric mean of the group.
  • FIG. 3A is a graph showing that mice infected with S. pneumoniae and treated with MgCl2-saline aerosol (0.12 M magnesium chloride in 0.90% sodium chloride) for fifteen minutes two hours before infection have a similar bacterial burden as untreated controls. Pooled data from multiple experiments are shown. Each data point represents the data obtained from a single animal. The bar for each group represents the geometric mean of the group. The data were statistically analyzed using a Mann-Whitney U test (ns=not significant).
  • FIG. 3B is a graph showing that mice infected with S. pneumoniae and pretreated with saline aerosol (0.90% sodium chloride) for fifteen minutes two hours before infection have a higher bacterial burden than animals pretreated with CaCl2-saline aerosol (1.29% calcium chloride (0.12M) in 0.9% sodium chloride). Pooled data from multiple experiments are shown. Each data point represents the data obtained from a single animal. The bar for each group represents the geometric mean of the group. The data were statistically analyzed using a Mann-Whitney U test.
  • FIG. 4A shows that formulations comprising calcium chloride and sodium chloride (Ca2+:Na+ at 8:1 molar ratio) reduced lung bacterial burden. Mice were treated with the indicated formulations using a PariLC Sprint nebulizer and subsequently infected with S. pneumoniae. The lung bacterial burden in each animal is shown. Each circle represents data from a single animal and the bar depicts the geometric mean with a 95% confidence interval. Data for the NaCl, 0.5× and 1× groups are pooled from two or three independent experiments. Data from the 2× and 4× groups are from a single experiment.
  • FIG. 4B shows that increasing calcium dose with longer nebulization times did not significantly impact therapeutic efficacy. Mice were treated with saline (NaCl) or a calcium:sodium formulation (1× tonicity=isotonic; 8:1 Ca2+:Na+ at 8:1 molar ratio) using a Pari LC Sprint nebulizer and subsequently infected with S. pneumoniae. The lung bacterial burden in each animal is shown. Each circle represents data from a single animal and the bar depicts the geometric mean. Dosing times of 3 minutes or greater significantly reduced bacterial burdens relative to controls (one-way ANOVA; Tukey's multiple comparison post-test).
  • FIG. 4C is a graph showing the inhibition of bacterial infection by ampicillin, Formulation 10 (1×), saline, and Formulation 10 plus Ampicillin (Ampicillin+1×). The data were collected from three independent experiments (n=5-6 per group per experiment) and each experiment was normalized to the respective saline control. Each data point represents the percent of the untreated control for a single animal and the bar depicts the geometric mean plus or minus the 95% confidence interval. Groups of data were analyzed by a Mann-Whitney U test. *** indicates p<0.001 compared to the saline control.
  • FIG. 5A, FIG. 5B, and FIG. 5C are graphs showing that a significant reduction in macrophage, neutrophil, and lymphocyte inflammation, as represented by cell counts, was seen when Tobacco Smoked Mice were treated q.d. with either prophylactic dosing or therapeutic dosing with Formulation 29, and with a positive control.
  • FIG. 6A and FIG. 6B are graphs showing that a significant reduction in KC and MIP2, two key neutrophil chemokines, was seen when TS Mice were treated q.d. with Formulations 30-A and IV-A.
  • FIG. 7 is a graph showing that a significant increase in mucociliary clearance was seen when sheep were treated with Formulations 29 and 13-A.
  • FIG. 8 is a graph showing a decrease in airway resistance was observed when mice were treated with Formulation XI and 14-A and then challenged with methacholine chloride (MCh) as compared to when the sham (Placebo-B) treatment group was challenged with MCh.
  • FIG. 9 is a graph showing a decrease in airway resistance was observed when mice were treated with Formulation XIV and 14-B and then challenged with methacholine chloride (MCh) as compared to when the sham (Placebo-B) treatment group was challenged with MCh.
  • DETAILED DESCRIPTION OF THE INVENTION
  • The invention relates to methods for the treatment of CF and provides several advantages over prior approaches.
  • As used herein, “1×” tonicity refers to a solution that is isotonic relative to normal human blood and cells. Solutions that are hypertonic in comparison to normal human blood and cells are described relative to a 1× solution using an appropriate multiplier. For example, a hypertonic solution may have 1.1×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11× or greater tonicity.
  • The term “aerosol” as used herein refers to any preparation of a fine mist of particles (including liquid and non-liquid particles, e.g., dry powders), typically with a volume median geometric diameter of about 0.1 to about 30 microns or a mass median aerodynamic diameter of between about 0.5 and about 10 microns. Preferably the volume median geometric diameter for the aerosol particles is less than about 10 microns. The preferred volume median geometric diameter for aerosol particles is about 5 microns. For example, the aerosol can contain particles that have a volume median geometric diameter between about 0.1 and about 30 microns, between about 0.5 and about 20 microns, between about 0.5 and about 10 microns, between about 1.0 and about 3.0 microns, between about 1.0 and 5.0 microns, between about 1.0 and 10.0 microns, between about 5.0 and 15.0 microns. Preferably the mass median aerodynamic diameter is between about 0.5 and about 10 microns, between about 1.0 and about 3.0 microns, or between about 1.0 and 5.0 microns.
  • The term “dry powder” as used herein refers to a composition that contains finely dispersed respirable dry particles that are capable of being dispersed in an inhalation device and subsequently inhaled by a subject. Such a dry powder or dry particle may contain up to about 25%, up to about 20%, or up to about 15% water or other solvent, or be substantially free of water or other solvent, or be anhydrous.
  • The invention provides methods for the treatment of CF. As used herein, treatment is not necessarily curative, but is aimed at maintenance therapy or alleviating symptoms and/or discomfort. The method can be used as stand alone or front line therapy or incorporated into a larger therapeutic regimen.
  • In one aspect of the invention, the method comprises administering to an individual with CF an effective amount of a hypertonic calcium salt formulation as described herein. The hypertonic calcium salt formulation is administered to the respiratory tract as an aerosol, preferably by inhalation.
  • If desired the hypertonic calcium salt formulation can be administered as a dry powder. As used herein, hypertonic calcium salt formulation includes hypertonic liquid formulations, and also includes dry powders that contain calcium in an amount that improves lung mucus hydration, which is preferably determined or assessed by an increased rate of mucociliary clearance (Groth et al, Thorax, 43(5):360-365 (1988)). The method provides several advantages. Without wishing to be bound by any particular theory, it is believed that the hypertonic calcium salt formulations will increase the volume of liquid on the airway surface through osmotic effects and that this hydrating effect will be more persistent than the hydration achieved using hypertonic saline. This is because sodium ions are more readily absorbed from the airway lining fluid than calcium ions. Thus, the osmotic hydrating effects of calcium salts are expected to be more persistent that those of sodium salts, and dosing may be less frequent.
  • In addition, calcium ions are inotropic and increase ciliary beat frequency, thus directly increase mucociliary clearance. Accordingly, the method of the invention will improve mucociliary clearance directly by increasing ciliary beat frequency, and indirectly by hydrating the airway lining fluid and thinning the mucus layer.
  • In addition, calcium inhibits the ability of certain pathogens to cross mucus layers and inhibits viral infectivity and replication. Hypertonic calcium and dry powder salt formulations can prevent and treat bacterial infection. Accordingly, these activities of calcium provide an added benefit of reducing exacerbations caused by lung infections. Additionally, these activities of calcium reduce the occurrence and severity of lung infections.
  • These advantages would have been unexpected to coexist by a person of ordinary skill in the art. A person of ordinary skill would be concerned that administering calcium to the respiratory tract of a CF patient would exacerbate the disease, because it is known that calcium can increase mucus viscosity and gelation. Thus, a person of skill in the art would have expected that administering calcium to the respiratory tract of a CF patient would make the CF airway mucus even thicker, and thereby exacerbate the disease. This is especially true for calcium dry powders, because no extra liquid is administered to the patient.
  • Calcium salt formulations, such as hypertonic calcium and dry powder salt formulations, can inhibit or prevent the formation of biofilms, and can disrupt and/or disperse pre-existing biofilms. Thus, in another aspect, the invention provides methods to delay, reduce or prevent infection and pathogen colonization of the lungs of an individual with CF. The method comprises administering to an individual with CF an effective amount of a calcium salt formulation, such as a hypertonic calcium salt formulation as described herein, The calcium salt formulation is administered to the respiratory tract as an aerosol, preferably by inhalation.
  • In one embodiment, the invention relates to a method of reducing or preventing the formation of a biofilm in a CF patient comprising administering to an individual with CF an effective amount of a calcium salt formulation, wherein the calcium salt formulation is administered as an aerosol to the respiratory tract of said individual.
  • The invention also relates to a method of disrupting or dispersing a biofilm in a CF patient comprising administering to an individual with CF an effective amount of a calcium salt formulation, wherein the calcium salt formulation is administered as an aerosol to the respiratory tract of said individual.
  • CF patients may experience exacerbation caused by infections by Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus spp., Streptococcus spp., Streptococcus agalactiae, Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, Enterobacter spp., Acinetobacter spp., Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, Stenotrophomonas maltophilia, Burkholderia cepatia, influenza virus, respiratory syncytial virus, adenovirus, metapneumovirus, cytomegalovirus, herpes simplex virus and combinations thereof. CF patient may also experience exacerbations caused by infections with other pathogens. The exacerbations may be acute exacerbations or mild exacerbations.
  • Mild exacerbations of CF can also be caused by all of the below, namely, by the causes of influenza, influenza-like illness, and community associate pneumonia. In addition, exacerbations can be caused by opportunitistic bacterial pathogens, such as Pseudomonas aeruginosa, Burkholderia cepacia, Burkholderia pseudomallei, and the like, that characterize CF airway colonization, and also by atypical mycobacteria and Stenotrophomonas.
  • In certain embodiment, influenza is caused by either the influenza A or the influenza B virus.
  • In certain embodiments, an influenza-like illness is caused by RSV, rhinovirus, adenovirus, parainfluenza, human coronaviruses (including the virus that causes severe acute respiratory syndrome) and metapneumovirus.
  • In certain embodiments, community associated pneumonia (CAP) is caused by at least one of the following bacteria: Moraxella catarralis, Mycoplasma pneumoniae, Chlamydophilia pneumonia, or Chlamydia pneumoniae, strep pneumonia, Haemophilus influenzae, chlamydophia, mycoplasma, and Legionella. Alternatively, or in addition to the previously mentioned bacteria, CAP may also be caused by at least one of the following fungi: Coccidiomycosis, histoplasmosis, and cryptococcocus. Alternatively, CAP can be caused by Gram-positive or Gram-negative bacteria associated with causing pneumonia.
  • In one embodiment, the invention relates to a method for treating or preventing an acute exacerbation of CF comprising administering to the respiratory tract of a patient in need thereof an effective amount of calcium salt formulation.
  • Another advantage provided by the method of the invention is a reduction in contagion. Specific concerns over patient-to-patient spread of infectious agents including Burkholderia cepatia in the CF clinic have been a concern of care providers in recent years. Administration of calcium salts in accordance with the method of the invention decreases the amount of particles that are exhaled by an individual with CF. This reduces the spread of pathogens that are present in the exhaled particles. Accordingly, the spread of lung infection from an individual with CF to others (e.g. care providers, family members, other individuals with CF) is reduced.
  • The hypertonic calcium salt formulations or calcium salt formulations are administered to the respiratory tract, and can be administered in any suitable form, such as a solution, a suspension, an emulsion, a spray, a mist, a foam, a gel, a vapor, droplets, particles, or a dry powder. Preferably the hypertonic calcium formulation or calcium salt formulation is aerosolized for administration to the respiratory tract. Hypertonic calcium salt formulations or calcium salt formulations can be aerosolized for administration via the oral airways using any suitable method and/or device, and many suitable methods and devices are conventional and well-known in the art. For example, hypertonic calcium salt formulations or calcium salt formulations can be aerosolized using a nebulizer, an atomizer, a continuous sprayer, an oral spray, a metered dose inhaler (e.g., a pressurized metered dose inhaler (pMDI) including HFA propellant, or a non-HFA propellant) with or without a spacer or holding chamber, or a dry powder inhaler (DPI). Hypertonic calcium salt formulations or calcium salt formulations can be aerosolized for administration via the nasal airways using a nasal pump or sprayer, a metered dose inhaler (e.g., a pressurized metered dose inhaler (pMDI) including HFA propellant, or a non-HFA propellant) with or without a spacer or holding chamber, a nebulizer with or without a nasal adapter or prongs, an atomizer, a continuous sprayer, or a DPI. Hypertonic calcium salt formulations or calcium salt formulations can also be delivered to the nasal mucosal surface via, for example, nasal wash and to the oral mucosal surfaces via, for example, an oral wash. Hypertonic calcium salt formulations or calcium salt formulations can be delivered to the mucosal surfaces of the sinuses via, for example, nebulizers with nasal adapters and nasal nebulizers with oscillating or pulsatile airflows.
  • The geometry of the airways is an important consideration when selecting a suitable method for producing and delivering aerosols to the lungs. The lungs are designed to entrap particles of foreign matter that are breathed in, such as dust. There are three basic mechanisms of deposition: impaction, sedimentation, and Brownian motion (J. M. Padfield. 1987. In: D. Ganderton & T. Jones eds. Drug Delivery to the Respiratory Tract, Ellis Harwood, Chichester, U.K.). Impaction in the upper airways occurs when particles are unable to stay within the air stream, particularly at airway branches. Impacted particles are adsorbed onto the mucus layer covering bronchial walls and eventually cleared from the lungs by mucociliary action. Impaction mostly occurs with particles over 5 μm in aerodynamic diameter. Smaller particles (those less than about 3 μm in aerodynamic diameter) tend to stay within the air stream and to be advected deep into the lungs. Sedimentation often occurs in the lower respiratory system where airflow is slower. Very small particles (those less than about 0.6 μm) can deposit by Brownian motion. Deposition by Brownian motion is generally undesirable because deposition cannot be targeted to the alveoli (N. Worakul & J. R. Robinson. 2002. In: Polymeric Biomaterials, 2nd Ed. S. Dumitriu ed. Marcel Dekker. New York).
  • For administration, a suitable method (e.g., nebulization, dry powder inhaler) is selected to produce aerosols with the appropriate particle size for preferential delivery to the desired region of the respiratory tract, such as the deep lung (generally particles between about 0.6 microns and 3 microns in diameter), the upper airway (generally particles of about 3 microns or larger diameter), or the deep lung and the upper airway. It is well-known that particles with an aerodynamic diameter of about 1 micron to about 3 microns, can be delivered to the deep lung. Larger aerodynamic diameters, for example, from about 3 microns to about 5 microns can be delivered to the central and upper airways.
  • In certain embodiments, a dry powder formulation is administered to the small airways. In these embodiments, the dry powder preferably contains respirable particles that have a VMDG and/or MMAD that is suitable for delivery to the small airways, such as a VMGD and/or MMAD of about 0.5 μm to about 3 μm, about 0.75 μm to about 2 μm, or about 1 μm to about 1.5 μm.
  • An “effective amount” of hypertonic calcium salt formulation or calcium salt formulation is administered to an individual with CF. An effective amount is an amount that is sufficient to achieve the desired therapeutic or prophylactic effect under the conditions of administration, such as an amount sufficient to increase the rate of mucociliary clearance, to reduce pathogens in an individual, to inhibit pathogens passing through the lung mucus or airway lining fluid, to decrease the incidence or rate of infection with pathogens that cause pneumonia, and/or to decrease the shedding of exhaled particles containing pathogens that cause pneumonia. For example, an amount effective to reduce or prevent bacterial biofilm formation, or to disrupt and/or disperse pre-existing biofilms is administered. Because the hypertonic calcium salt formulations or calcium salt formulations are administered to the respiratory tract (e.g., lungs), generally by inhalation, the dose that is administered is related to the composition of the hypertonic calcium salt formulation or calcium salt formulation (e.g., calcium salt concentration), the rate and efficiency of aerosolization (e.g., nebulization rate and efficiency), and the time of exposure (e.g., nebulization time). For example, substantially equivalent doses can be administered using a concentrated liquid calcium salt formulation and a short (e.g., 5 minutes) nebulization time, or using a dilute liquid calcium salt formulation and a long (e.g., 30 minutes or more) nebulization time, or using a dry powder formulation and a dry powder inhaler. The clinician of ordinary skill can determine appropriate dosages based on these considerations and other factors, for example, the individual's age, sensitivity, tolerance and overall well-being. The hypertonic calcium salt formulations or calcium salt formulations can be administered in a single dose or multiple doses as indicated. In some aspects, the hypertonic calcium salt formulation or calcium salt formulations are administered two, three or four times per day.
  • Generally, an effective amount of a hypertonic calcium salt formulation or calcium salt formulation will deliver a dose of about 0.001 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.002 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.005 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose to about 60 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose to about 50 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose to about 40 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose to about 30 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose to about 20 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose to about 10 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose to about 5 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.02 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.03 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.04 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.05 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.1 mg Ca+2/kg body weight/dose to about 2 mg Ca+2/kg body weight/dose, about 0.1 mg Ca+2/kg body weight/dose to about 1 mg Ca+2/kg body weight/dose, about 0.1 mg Ca+2/kg body weight/dose to about 0.5 mg Ca+2/kg body weight/dose, about 0.2 mg Ca+2/kg body weight/dose to about 0.5 mg Ca+2/kg body weight/dose, about 0.18 mg Ca+2/kg body weight/dose, about 0.001 mg Ca+2/kg body weight/dose, about 0.005 mg Ca+2/kg body weight/dose, about 0.01 mg Ca+2/kg body weight/dose, about 0.02 mg Ca+2/kg body weight/dose, or about 0.5 mg Ca+2/kg body weight/dose. In some embodiments, a hypertonic calcium salt formulation or calcium salt formulation is administered in an amount sufficient to deliver a dose of about 0.1 mg Ca2+/kg body weight/dose to about 2 mg Ca2+/kg body weight/dose, or about 0.1 mg Ca2+/kg body weight/dose to about 1 mg Ca2+/kg body weight/dose, or about 0.1 mg Ca2+/kg body weight/dose to about 0.5 mg Ca2+/kg body weight/dose, or about 0.18 mg Ca2+/kg body weight/dose.
  • In some embodiments, the amount of calcium salt delivered to the respiratory tract (e.g., lungs, respiratory airway) is about 0.001 mg/kg body weight to about 60 mg/kg body weight/dose, or about 0.01 mg/kg body weight/dose to about 50 mg/kg body weight/dose, about 0.01 mg/kg body weight/dose to about 40 mg/kg body weight/dose, about 0.01 mg/kg body weight/dose to about 30 mg/kg body weight/dose, about 0.01 mg/kg body weight/dose to about 20 mg/kg body weight/dose, 0.01 mg/kg body weight/dose to about 10 mg/kg body weight/dose, about 0.1 mg/kg body weight/dose to about 10 mg/kg body weight/dose, or about 1 mg/kg body weight/dose to about 10 mg/kg body weight/dose, or about 0.01 mg/kg body weight/dose to about 1 mg/kg body weight/dose, or about 0.1 mg/kg body weight/dose to about 1 mg/kg body weight/dose.
  • In some embodiments, a hypertonic calcium salt formulation, or calcium salt formulation, that comprises a sodium salt (e.g., sodium chloride) is administered in an amount sufficient to deliver a dose of about 0.001 mg Na+/kg body weight/dose to about 10 mg Na+/kg body weight/dose, or about 0.01 mg Na+/kg body weight/dose to about 10 mg Na+/kg body weight/dose, or about 0.1 mg Na+/kg body weight/dose to about 10 mg Na+/kg body weight/dose, or about 1.0 mg Na+/kg body weight/dose to about 10 mg Na+/kg body weight/dose, or about 0.001 mg Na+/kg body weight/dose to about 1 mg Na+/kg body weight/dose, or about 0.01 mg Na+/kg body weight/dose to about 1 mg Na+/kg body weight/dose, about 0.1 mg Na+/kg body weight/dose to about 1 mg Na+/kg body weight/dose, about 0.2 mg Na+/kg body weight/dose to about 0.8 mg Na+/kg body weight/dose, about 0.3 mg Na+/kg body weight/dose to about 0.7 mg Na+/kg body weight/dose, or about 0.4 mg Na+/kg body weight/dose to about 0.6 mg Na+/kg body weight/dose. In some embodiments the amount of sodium salt delivered to the respiratory tract (e.g., lungs, respiratory airway) is about 0.001 mg/kg body weight/dose to about 10 mg/kg body weight/dose, or about 0.01 mg/kg body weight/dose to about 10 mg/kg body weight/dose, or about 0.1 mg/kg body weight/dose to about 10 mg/kg body weight/dose, or about 1 mg/kg body weight/dose to about 10 mg/kg body weight/dose, or about 0.001 mg/kg body weight/dose to about 1 mg/kg body weight/dose, or about 0.01 mg/kg body weight/dose to about 1 mg/kg body weight/dose, or about 0.1 mg/kg body weight/dose to about 1 mg/kg body weight/dose.
  • Suitable intervals between doses that provide the desired therapeutic effect can be determined based on the severity of the condition (e.g., infection), the overall well being of the subject and the subject's tolerance to the salt formulations and other considerations. Based on these and other considerations, a clinician can determine appropriate intervals between doses. Generally, a salt formulation is administered once, twice, three or four times a day, as needed.
  • If desired or indicated, the hypertonic calcium salt formulation or calcium salt formulation can be administered with one or more other therapeutic agents, such as any one or more of the mucoactive agents, surfactants, cough suppressants, expectorants, steroids, bronchodilators, antihistamines, antibiotics, antiviral agents, or agents that promote airway secretion clearance described herein. The other therapeutic agents can be administered by any suitable route, such as orally, parenterally (e.g., intravenous, intra-arterial, intramuscular, or subcutaneous injection), topically, by inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), rectally, vaginally, and the like.
  • When an additional therapeutic agent is administered to a patient with hypertonic calcium salt formulation or calcium salt formulation, they can be administered to provide substantial overlap of pharmacological activity, and the additional therapeutic agent can be administered to the patient before, substantially at the same time, or after the hypertonic calcium salt formulation or calcium salt formulation (e.g., Formulation 29 or 30). For example a LABA such as formoterol, or a short-acting beta agonist such as albuterol can be administered to the patient before Formulation 29 or 30, or a dry powder based on Formulation 29 or 30, is administered. A dry powder for use in the invention (e.g., Formulation 29 or 30) and an additional therapeutic agent can be administered at substantially the same time as two or more separate formulations or as a single formulation (e.g., a blended dry powder, a dry powder formed by co-spray drying the components of Formulation 29 or 30 with an additional therapeutic agent). In one example, a dry powder of Formulation 29 or 30 can be administered immediately before or immediately after the dosing of Formulation 29 or 30.
  • In certain particular embodiments of the methods described herein, the hypertonic calcium salt formulation or calcium salt formulation (e.g., Formulation 29, Formulation 30, and/or dry powder based on Formulation 29 or 30 that contains an additional therapeutic agent) is administered to a patient who has been pretreated with a bronchodilator, or is administered concurrently with a bronchodilator. When the patient is pretreated with a bronchodilator it is preferred that the respirable dry powder is administered at a time after the bronchodilator when the onset of bronchodilatory effect is evident or, more preferably, maximal. For example, a short acting beta2 agonist such as albuterol, can be administered about 10 minutes to about 30 minutes, preferably, about 15 minutes, prior to administration of the respirable dry powder. Pretreatment with a short acting beta2 agonist such as albuterol is particularly preferred for CF patients. Some patients may already be taking bronchodilators, such as LABAs (e.g, fomoterol). Patients who are taking LABAs already receive some degree of bronchorelaxation due to the effects of the LABAs, and therefore further bronchodilation (e.g., using a short acting beta2 agonist) may not be required or desired. For these types of patients, hypertonic calcium salt formulation or calcium salt formulation (e.g., Formulation 29, Formulation 30, and/or dry powder based on Formulation 29 or 30 that contains an additional therapeutic agent) can be administered as substantially the same time or concurrently with the LABA, for example, in a single formulation (e.g., the respirable dry powder of Formulation XIX).
  • Hypertonic Calcium Salt Formulations and Calcium Salt Formulations
  • Hypertonic calcium salt formulations and calcium salt formulations for use in the methods described herein contain a calcium salt (e.g., calcium chloride, calcium lactate, calcium acetate) as an active ingredient, and can optionally contain additional salts or agents. Without wishing to be bound by a particular theory, it is believed that therapeutic benefits produced by the salt formulations and the methods described herein, result from an increase in the amount of calcium ion (Ca2+ provide upon dissolution of CaCl2) in the lung mucus or airway lining fluid after administration of the salt formulation. Calcium salts provide several advantages, including increasing ciliary beat frequency, drawing water into lungs, persisting longer than sodium, providing an antibacterial effect and modulating the surface viscoelasticity without significantly further increasing the bulk viscoelasticity of the CF mucus and without exacerbating disease.
  • In addition to calcium chloride, the hypertonic calcium salt formulation or calcium salt formulation can include any salt form of the elements lithium, sodium, potassium, magnesium, calcium, aluminum, silicon, scandium, titanium, vanadium, chromium, cobalt, nickel, copper, manganese, zinc, tin, silver and similar elements, that is non-toxic when administered to the respiratory tract.
  • Additional calcium salts that are suitable for use in the calcium salt formulation include, for example, calcium sulfate, calcium lactate, calcium citrate, calcium carbonate, calcium acetate, calcium phosphate, calcium alginate, calcium stearate, calcium sorbate, calcium gluconate and the like, and combinations thereof. Suitable sodium salts include, for example, sodium chloride, sodium acetate, sodium bicarbonate, sodium carbonate, sodium sulfate, sodium stearate, sodium ascorbate, sodium benzoate, sodium biphosphate, sodium phosphate, sodium bisulfite, sodium citrate, sodium lactate, sodium borate, sodium gluconate, sodium metasilicate, and the like, or a combination thereof. Suitable magnesium salts include, for example, magnesium carbonate, magnesium sulfate, magnesium stearate, magnesium trisilicate, magnesium chloride, and the like. Suitable potassium salts include, for example, potassium bicarbonate, potassium chloride, potassium citrate, potassium borate, potassium bisulfite, potassium biphosphate, potassium alginate, potassium benzoate, and the like. Additional suitable salts include cupric sulfate, chromium chloride, stannous chloride, and similar salts. Other suitable salts include zinc chloride, aluminum chloride and silver chloride.
  • The hypertonic calcium salt formulations or calcium salt formulations can contain about 1.3% to about 13% calcium chloride (w/v). For example, the hypertonic calcium salt formulation or calcium salt formulation can contain calcium chloride in an range of 1.3% to about 12.5%, 1.3% to about 12%, 1.3% to about 11.5%, 1.3% to about 11%, 1.3% to about 10.5%, 1.3% to about 10%, 1.3% to about 9.5%, 1.3% to about 9.0%, 1.3% to about 8.5%, 1.3% to about 8%, about 1.3% to about 7.5%, 1.3% to about 7%, 1.3% to about 6.5%, 1.3% to about 6%, 1.3% to about 5.5%, 1.3% to about 5%, about 1.3% to about 4.5%, about 1.3% to about 4%, about 1.3% to about 3.5%, about 1.3% to about 3%, about 1.3% to about 2.5%, about 1.3% to about 2%, about 2% to about 13%, about 2.5% to about 13%, about 3% to about 13%, about 3.5% to about 13%, about 4% to about 13%, about 4.5% to about 13%, about 5% to about 13%, about 5.5% to about 13%, about 6% to about 13%, about 6.5% to about 13%, about 7% to about 13%, about 7.5% to about 13%, about 8% to about 13%, about 8.5% to about 13%, about 9% to about 13%, about 9.5% to about 13%, about 10% to about 13%, about 10.5% to about 13%, about 11% to about 13%, about 11.5% to about 13%, about 12% to about 13%, about 1.3% to about 8%, about 2% to about 8%, about 2.5% to about 8%, about 3% to about 8%, about 3.5% to about 8%, about 4% to about 8%, about 4.5% to about 8%, about 5% to about 8%, about 5.5% to about 8%, about 6% to about 8%, about 6.5% to about 8%, about 7% to about 8%, about 3% to about 7%, or about 4% to about 6% (w:v).
  • In some embodiments, the hypertonic calcium salts further contain about 0.001% to about 0.9% sodium chloride (w:v). For example, the hypertonic calcium salt formulation or calcium salt formulation can contain sodium chloride in a range of about 0.1% to about 0.8%, 0.1% to about 0.7%, 0.1% to about 0.6%, 0.1% to about 0.5%, 0.1% to about 0.4%, 0.1% to about 0.3%, about 0.1% to about 0.2%, about 0.2% to about 0.9%, about 0.3% to about 0.9%, about 0.4% to about 0.9%, about 0.5% to about 0.9%, about 0.6% to about 0.9%, about 0.7% to about 0.9%, about 0.8% to about 0.9%, about 0.2% to about 0.8%, about 0.3% to about 0.7%, or about 0.4% to about 0.6% (w:v).
  • The hypertonic calcium salt formulation or calcium salt formulation can be a solution, emulsion, or suspension that can be aerosolized, for example using a nebulizer. Preferably, the hypertonic calcium salt formulation or calcium salt formulation is an aqueous solution. If desired, the hypertonic calcium salt formulation or calcium salt formulation can be dried to form a dry powder, for example by spray drying, cake drying and micronizing, for example, by milling, grinding, or using any other suitable method. The hypertonic salt formulations or calcium salt formulations can comprise multiple doses or be a unit dose composition as desired.
  • The hypertonic calcium salt formulation or calcium salt formulation is generally prepared in or comprises a physiologically acceptable carrier or excipient. For hypertonic calcium salt formulations or calcium salt formulations in the form of solutions, suspensions or emulsions, suitable carriers include, for example, aqueous, alcoholic/aqueous, and alcohol solutions, emulsions or suspensions, including water, saline, ethanol/water solution, ethanol solution, buffered media, propellants and the like. Any suitable excipient can be included, as desired.
  • For calcium salt formulations in the form of dry powders, suitable carriers or excipients include, for example, sugars (e.g., lactose, trehalose), sugar alcohols (e.g., mannitol, xylitol, sorbitol), amino acids (e.g., glycine, alanine, leucine, isoleucine, methionine, tyrosine, tryptophan), dipalmitoylphosphosphatidylcholine (DPPC), diphosphatidyl glycerol (DPPG), 1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohols, polyoxyethylene-9-lauryl ether, surface active fatty, acids, sorbitan trioleate (Span 85), glycocholate, surfactin, poloxomers, sorbitan fatty acid esters, tyloxapol, phospholipids, alkylated sugars, sodium phosphate, maltodextrin, human serum albumin (e.g., recombinant human serum albumin), biodegradable polymers (e.g., PLGA), dextran, dextrin, and the like. Amino acid excipients are preferably racemic, predominately L-isomer, or predominately D-isomer (e.g., the excipient can be L-leucine or D-leucine). If desired, the salt formulations can also contain additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., PA, 1985). The hypertonic calcium salt formulation or calcium salt formulation preferably contains a concentration of calcium salt that permits convenient administration of an effective amount of the formulation to the respiratory tract. For example, it is generally desirable that liquid formulations not be so dilute so as to require a large amount of the formulation to be nebulized in order to deliver an effective amount to the respiratory tract of a subject. In some embodiments however, it may be beneficial to dilute the liquid formulation so that a larger amount of solution is provided to thin the bulk mucus. Long administration periods are disfavored, and generally the formulation should be concentrated enough to permit an effective amount to be administered to the respiratory tract (e.g., by inhalation of aerosolized formulation, such as nebulized liquid or aerosolized dry powder) or nasal cavity in no more than about 120 minutes, no more than about 90 minutes, no more than about 60 minutes, no more than about 45 minutes, no more than about 30 minutes, no more than about 25 minutes, no more than about 20 minutes, no more than about 15 minutes, no more than about 10 minutes, no more than about 7.5 minutes, no more than about 5 minutes, no more than about 4 minutes, no more than about 3 minutes, no more than about 2 minutes, no more than about 1 minute, no more than about 45 seconds, or no more than about 30 seconds. For example, a liquid hypertonic calcium salt formulation can contain about 1.3% to about 13% calcium salt, such as calcium chloride (w/v). Liquid formulations can contain about 0.12M to about 1.2M calcium chloride.
  • Liquid calcium salt formulations of this type can vary in the degree of hypertonicity and in the concentrations of calcium salt and sodium salt that are present in the formulation. For example, the calcium salt formulation can contain 0.212 M CaCl2 and 0.027 M NaCl (2.35% CaCl2, 0.16% NaCl), or 0.424 M CaCl2 and 0.054 M NaCl (4.70% CaCl2, 0.31% NaCl).
  • In some embodiments, the calcium salt formulation is selected so that at least about 4 mL or at least about 5 mL of the formulation is aerosolized (e.g., nebulized) during administration. For example, at least 4 mL of the calcium salt formulation are aerosolized (e.g., nebulized) during a single administration, and the calcium salt formulation is administered, one, two, three or four times per day.
  • The hypertonic calcium salt formulation has at least about 1.1× tonicity, at least about 1.5× tonicity, at least about 2× tonicity, at least about 3× tonicity, at least about 4× tonicity, at least about 5× tonicity, at least about 6× tonicity, at least about 7× tonicity, at least about 8× tonicity, at least about 9× tonicity, at least about 10× tonicity or at least about 11× tonicity.
  • Dry powder formulations can contain at least about 10% calcium salt by weight, at least about 20% calcium salt by weight, at least about 30% calcium salt by weight, at least about 40% calcium salt by weight, at least about 50% calcium salt by weight, at least about 60% calcium salt by weight, at least about 70% calcium salt by weight, at least about 75% calcium salt by weight, at least about 80% calcium salt by weight, at least about 85% calcium salt by weight, at least about 90% calcium salt by weight, at least about 95% calcium salt by weight, at least about 96% calcium salt by weight, at least about 97% calcium salt by weight, at least about 98% calcium salt by weight, or at least about 99% calcium salt by weight. For example, some dry powder formulations contain about 20% to about 80% calcium salt by weight, about 20% to about 70% calcium salt by weight, about 20% to about 60% calcium salt by weight, or can consist substantially of calcium salt(s).
  • If desired, the hypertonic calcium salt formulation or calcium salt formulation can include one or more additional therapeutic agents, such as mucoactive or mucolytic agents, surfactants, antibiotics, antivirals, antihistamines, cough suppressants, bronchodilators, anti-inflammatory agents, steroids, vaccines, adjuvants, expectorants, macromolecules, therapeutics that are helpful for chronic maintenance of CF. The additional agent can be blended with a dry powder or co-spray dried as desired.
  • Examples of suitable mucoactive or mucolytic agents include MUC5AC and MUC5B mucins, DNA-ase, N-acetylcysteine (NAC), cysteine, nacystelyn, dornase alfa, gelsolin, heparin, heparin sulfate, P2Y2 agonists (e.g. UTP, INS365), nedocromil sodium, hypertonic saline, and mannitol.
  • Suitable surfactants include L-alpha-phosphatidylcholine dipalmitoyl (“DPPC”), diphosphatidyl glycerol (DPPG), 1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohols, polyoxyethylene-9-lauryl ether, surface active fatty, acids, sorbitan trioleate (Span 85), glycocholate, surfactin, poloxomers, sorbitan fatty acid esters, tyloxapol, phospholipids, and alkylated sugars.
  • If desired, the salt formulation can contain an antibiotic. The antibiotic can be suitable for treating any desired bacterial infection, and salt formulations that contain an antibiotic can be used to reduce the spread of infection, either within a patient or from patient to patient. For example, salt formulations for treating bacterial pneumonia or VAT, can further comprise an antibiotic, such as a macrolide (e.g., azithromycin, clarithromycin and erythromycin), a tetracycline (e.g., doxycycline, tigecycline), a fluoroquinolone (e.g., gemifloxacin, levofloxacin, ciprofloxacin and mocifloxacin), a cephalosporin (e.g., ceftriaxone, defotaxime, ceftazidime, cefepime), a penicillin (e.g., amoxicillin, amoxicillin with clavulanate, ampicillin, piperacillin, and ticarcillin) optionally with a β-lactamase inhibitor (e.g., sulbactam, tazobactam and clavulanic acid), such as ampicillin-sulbactam, piperacillin-tazobactam and ticarcillin with clavulanate, an aminoglycoside (e.g., amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, and apramycin), a penem or carbapenem (e.g. doripenem, ertapenem, imipenem and meropenem), a monobactam (e.g., aztreonam), an oxazolidinone (e.g., linezolid), vancomycin, glycopeptide antibiotics (e.g. telavancin), tuberculosis-mycobacterium antibiotics and the like.
  • If desired, the salt formulation can contain an agent for treating infections with mycobacteria, such as Mycobacterium tuberculosis. Suitable agents for treating infections with mycobacteria (e.g., M. tuberculosis) include an aminoglycoside (e.g. capreomycin, kanamycin, streptomycin), a fluoroquinolone (e.g. ciprofloxacin, levofloxacin, moxifloxacin), isozianid and isozianid analogs (e.g. ethionamide), aminosalicylate, cycloserine, diarylquinoline, ethambutol, pyrazinamide, protionamide, rifampin, and the like.
  • If desired, the salt formulation can contain a suitable antiviral agent, such as oseltamivir, zanamavir, amantidine, rimantadine, ribavirin, gancyclovir, valgancyclovir, foscavir, Cytogam® (Cytomegalovirus Immune Globulin), pleconaril, rupintrivir, palivizumab, motavizumab, cytarabine, docosanol, denotivir, cidofovir, and acyclovir. The salt formulation can contain a suitable anti-influenza agent, such as zanamivir, oseltamivir, amantadine, or rimantadine.
  • Suitable antihistamines include clemastine, asalastine, loratadine, fexofenadine and the like.
  • Suitable cough suppressants include benzonatate, benproperine, clobutinal, diphenhydramine, dextromethorphan, dibunate, fedrilate, glaucine, oxalamine, piperidione, opiods such as codeine and the like.
  • Suitable brochodilators include short-acting beta2 agonists, long-acting beta2 agonists (LABA), long-acting muscarinic anagonists (LAMA), combinations of LABAs and LAMAs, methylxanthines, short-acting anticholinergic agents (may also be referred to as short acting anti-muscarinic), and the like.
  • Suitable short-acting beta2 agonists include albuterol, epinephrine, pirbuterol, levalbuterol, metaproteronol, maxair, and the like.
  • Examples of albuterol sulfate formulations (also called salbutamol) include Inspiryl (AstraZeneca Plc), Salbutamol SANDOZ (Sanofi-Aventis), Asmasal clickhaler (Vectura Group Plc.), Ventolin® (GlaxoSmithKline Plc), Salbutamol GLAND (GlaxoSmithKline Plc), Airomir® (Teva Pharmaceutical Industries Ltd.), ProAir HFA (Teva Pharmaceutical Industries Ltd.), Salamol (Teva Pharmaceutical Industries Ltd.), Ipramol (Teva Pharmaceutical Industries Ltd), Albuterol sulfate TEVA (Teva Pharmaceutical Industries Ltd), and the like. Examples of epinephrine include Epinephine Mist KING (King Pharmaceuticals, Inc.), and the like. Examples of pirbuterol as pirbuterol acetate include Maxair® (Teva Pharmaceutical Industries Ltd.), and the like. Examples of levalbuterol include Xopenex® (Sepracor), and the like. Examples of metaproteronol formulations as metaproteronol sulfate include Alupent® (Boehringer Ingelheim GmbH), and the like.
  • Suitable LABAs include salmeterol, formoterol and isomers thereof (e.g. arformoterol), clenbuterol, tulobuterol, vilanterol (Revolair™), indacaterol, carmoterol, isoproterenol, procaterol, bambuterol, milveterol, olodaterol and the like.
  • Examples of salmeterol formulations include salmeterol xinafoate as Serevent® (GlaxoSmithKline Plc), salmeterol as Inaspir (Laboratorios Almirall, S.A.), Advair® HFA (GlaxoSmithKline PLC), Advair Diskus® (GlaxoSmithKline PLC, Theravance Inc), Plusvent (Laboratorios Almirall, S.A.), VR315 (Novartis, Vectura Group PLC) and the like. Examples of formoterol and isomers formulations (e.g., arformoterol) include Foster (Chiesi Farmaceutici S.p.A), Atimos (Chiesi Farmaceutici S.p.A, Nycomed Internaional Management), Flutiform® (Abbott Laboratories, SkyePharma PLC), MFF258 (Novartis AG), Formoterol clickhaler (Vectura Group PLC), Formoterol HFA (SkyePharma PLC), Oxis® (Astrazeneca PLC), Oxis pMDI (Astrazeneca), Foradil® Aerolizer (Novartis, Schering-Plough Corp, Merck), Foradil® Certihaler (Novartis, SkyePharma PLC), Symbicort® (AstraZeneca), VR632 (Novartis AG, Sandoz International GmbH), MFF258 (Merck & Co Inc, Novartis AG), Alvesco® Combo (Nycomed International Management GmbH, Sanofi-Aventis, Sepracor Inc), Mometasone furoate (Schering-Plough Corp), and the like. Examples of clenbuterol formulations include Ventipulmin® (Boehringer Ingelheim), and the like. Examples of tulobuterol formulations include Hokunalin Tape (Abbott Japan Co., Ltd., Maruho Co., Ltd.), and the like. Examples of vilanterol formulations include Revolair™ (GlaxoSmithKline PLC), GSK64244 (GlaxoSmithKline PLC), and the like. Examples of indacaterol formulations include QAB149 (Novartis AG, SkyePharma PLC), QMF149 (Merck & Co Inc) and the like. Examples of carmoterol formulations include CHF4226 (Chiese Farmaceutici S.p.A., Mitsubishi Tanabe Pharma Corporation), CHF5188 (Chiesi Farmaceutici S.p.A), and the like. Examples of isoproterenol sulfate formulations include Aludrin (Boehringer Ingelheim GmbH) and the like. Examples of procaterol formulations include Meptin clickhaler (Vectura Group PLC), and the like. Examples of bambuterol formulations include Bambec (AstraZeneca PLC), and the like. Examples of milveterol formulations include GSK159797C (GlaxoSmithKline PLC), TD3327 (Theravance Inc), and the like. Examples of olodaterol formulations include BI1744CL (Boehringer Ingelheim GmbH) and the like.
  • Examples of LAMAs include tiotroprium, trospium chloride, glycopyrrolate, aclidinium, ipratropium and the like.
  • Examples of tiotroprium formulations include Spiriva (Boehringer-Ingleheim, Pfizer), and the like. Examples of glycopyrrolate formulations include Robinul® (Wyeth-Ayerst), Robinul® Forte (Wyeth-Ayerst), NVA237 (Novartis), and the like. Examples of aclidinium formulations include Eklira® (Forest Labaoratories, Almirall), and the like.
  • Examples of combinations of LABAs and LAMAs include indacaterol with glycopyrrolate, formoterol with glycopyrrolate, indacaterol with tiotropium, olodaterol and tiotropium, vilanterol with a LAMA, and the like.
  • Examples of combinations of indacaterol with glycopyrrolate include QVA149A (Novartis), and the like. Examples of combinations of formoterol with glycopyrrolate include PT003 (Pearl Therapeutics) and the like. Examples of combinations of olodaterol with tiotropium include BI1744 with Spiriva (Boehringer Ingelheim) and the like. Examples of combinations of vilanterol with a LAMA include GSK573719 with GSK642444 (GlaxoSmithKline PLC), and the like.
  • Examples of methylxanthines include aminophylline, ephedrine, theophylline, oxtriphylline, and the like.
  • Examples of aminophylline formulations include Aminophylline BOEHRINGER (Boehringer Ingelheim GmbH) and the like. Examples of ephedrine formulations include Bronkaid® (Bayer AG), Broncholate (Sanofi-Aventis), Primatene® (Wyeth), Tedral SA®, Marax (Pfizer Inc) and the like. Examples of theophylline formulations include Euphyllin (Nycomed International Management GmbH), Theo-dur (Pfizer Inc, Teva Pharmacetuical Industries Ltd) and the like. Examples of oxtriphylline formulations include Choledyl SA (Pfizer Inc) and the like.
  • Examples of short-acting anticholinergic agents include ipratropium bromide, oxitropium bromide, and tiotropium (Spiriva).
  • Examples of ipratropium bromide formulations include Atrovent®/Apovent/Inpratropio (Boehringer Ingelheim GmbH), Ipramol (Teva Pharmaceutical Industries Ltd) and the like. Examples of oxitropium bromide include Oxivent (Boehringer Ingelheim GmbH), and the like.
  • Suitable anti-inflammatory agents include leukotriene inhibitors, phosphodiesterase 4 (PDE4) inhibitors, other anti-inflammatory agents, and the like.
  • Suitable leukotriene inhibitors include montelukast (cystinyl leukotriene inhibitors), masilukast, zafirleukast (leukotriene D4 and E4 receptor inhibitors), pranlukast, zileuton (5-lipoxygenase inhibitors), and the like.
  • Examples of montelukast formulations (cystinyl leukotriene inhibitor) include Singulair® (Merck & Co Inc), Loratadine, montelukast sodium SCHERING (Schering-Plough Corp), MK0476C (Merck & Co Inc), and the like. Examples of masilukast formulations include MCC847 (AstraZeneca PLC), and the like. Examples of zafirlukast formulations (leukotriene D4 and E4 receptor inhibitor) include Accolate® (AstraZeneca PLC), and the like. Examples of pranlukast formulations include Azlaire (Schering-Plough Corp). Examples of zileuton (5-LO) formulations include Zyflo® (Abbott Laboratories), Zyflo CR® (Abbott Laboratories, SkyePharma PLC), Zileuton ABBOTT LABS (Abbott Laboratories), and the like. Suitable PDE4 inhibitors include cilomilast, roflumilast, oglemilast, tofimilast, and the like.
  • Examples of cilomilast formulations include Ariflo (GlaxoSmithKline PLC), and the like. Examples of roflumilast include Daxas® (Nycomed International Management GmbH, Pfizer Inc), APTA2217 (Mitsubishi Tanabe Pharma Corporation), and the like. Examples of oglemilast formulations include GRC3886 (Forest Laboratories Inc), and the like. Examples of tofimilast formulations include Tofimilast PFIZER INC (Pfizer Inc), and the like.
  • Other anti-inflammatory agents include omalizumab (anti-IgE immunoglobulin Daiichi Sankyo Company, Limited), Zolair (anti-IgE immunoglobulin, Genentech Inc, Novartis AG, Roche Holding Ltd), Solfa (LTD4 antagonist and phosphodiesterase inhibitor, Takeda Pharmaceutical Company Limited), IL-13 and IL-13 receptor inhibitors (such as AMG-317, MILR1444A, CAT-354, QAX576, IMA-638, Anrukinzumab, IMA-026, MK-6105, DOM-0910, and the like), IL-4 and IL-4 receptor inhibitors (such as Pitrakinra, AER-003, AIR-645, APG-201, DOM-0919, and the like), IL-1 inhibitors such as canakinumab, CRTh2 receptor antagonists such as AZD1981 (CRTh2 receptor antagonist, AstraZeneca), neutrophil elastase inhibitor formulations such as AZD9668 (neutrophil elastase inhibitor, from AstraZeneca), GW856553X Losmapimod (P38 kinase inhibitor, GlaxoSmithKline PLC), Arofylline LAB ALMIRALL (PDE-4 inhibitor, Laboratorios Almirall, S.A.), ABT761 (5-LO inhibitor, Abbott Laboratories), Zyflo® (5-LO inhibitor, Abbott Laboratories), BT061 (anti-CD4 mAb, Boehringer Ingelheim GmbH), Corns (inhaled lidocaine to decrease eosinophils, Gilead Sciences Inc), Prograf® (IL-2-mediated T-cell activation inhibitor, Astellas Pharma), Bimosiamose PFIZER INC (selectin inhibitor, Pfizer Inc), R411 (α4 (β1/α4 β7 integrin antagonist, Roche Holdings Ltd), Tilade® (inflammatory mediator inhibitor, Sanofi-Aventis), Orenica® (T-cell co-stimulation inhibitor, Bristol-Myers Squibb Company), Soliris® (anti-05, Alexion Pharmaceuticals Inc), Entorken® (Farmacija d.o.o.), Excellair® (Syk kinase siRNA, ZaBeCor Pharmaceuticals, Baxter International Inc), KB003 (anti-GMCSF mAb, KaloBios Pharmaceuticals), Cromolyn sodiums (inhibit release of mast cell mediators): Cromolyn sodium BOEHRINGER (Boehringer Ingelheim GmbH), Cromolyn sodium TEVA (Teva Pharmaceutical Industries Ltd), Intal (Sanofi-Aventis), BI1744CL (oldaterol (β2-adrenoceptor antagonist) and tiotropium, Boehringer Ingelheim GmbH), NFκ-B inhibitors, CXR2 antagaonists, HLE inhibitors, HMG-CoA reductase inhibitors and the like.
  • Anti-inflammatory agents also include compounds that inhibit/decrease cell signaling by inflammatory molecules like cytokines (e.g., IL-1, IL-4, IL-5, IL-6, IL-9, IL-13, IL-18 IL-25, IFN-α, IFN-β, and others), CC chemokines CCL-1-CCL28 (some of which are also known as, for example, MCP-1, CCL2, RANTES), CXC chemokines CXCL1-CXCL17 (some of which are also know as, for example, IL-8, MIP-2), growth factors (e.g., GM-CSF, NGF, SCF, TGF-(3, EGF, VEGF and others) and/or their respective receptors.
  • Some examples of the aforementioned anti-inflammatory antagonists/inhibitors include ABN912 (MCP-1/CCL2, Novartis AG), AMG761 (CCR4, Amgen Inc), Enbrel® (TNF, Amgen Inc, Wyeth), huMAb OX40L GENENTECH (TNF superfamily, Genentech Inc, AstraZeneca PLC), R4930 (TNF superfamily, Roche Holding Ltd), SB683699/Firategrast (VLA4, GlaxoSmithKline PLC), CNT0148 (TNFα, Centocor, Inc, Johnson & Johnson, Schering-Plough Corp); Canakinumab (IL-1β, Novartis); Israpafant MITSUBISHI (PAF/IL-5, Mitsubishi Tanabe Pharma Corporation); IL-4 and IL-4 receptor antagonists/inhibitors: AMG317 (Amgen Inc), BAY169996 (Bayer AG), AER-003 (Aerovance), APG-201 (Apogenix); IL-5 and IL-5 receptor antagonists/inhibitors: MEDI563 (AstraZeneca PLC, MedImmune, Inc), Bosatria® (GlaxoSmithKline PLC), Cinquil® (Ception Therapeutic), TMC120B (Mitsubishi Tanabe Pharma Corporation), Bosatria (GlaxoSmithKline PLC), Reslizumab SCHERING (Schering-Plough Corp); MEDI528 (IL-9, AstraZeneca, MedImmune, Inc); IL-13 and IL-13 receptor antagonists/inhibitors: TNX650 GENETECH (Genetech), CAT-354 (AstraZeneca PLC, MedImmune), AMG-317 (Takeda Pharmaceutical Company Limited), MK6105 (Merck & Co Inc), IMA-026 (Wyeth), IMA-638 Anrukinzumab (Wyeth), MILR1444A/Lebrikizumab (Genentech), QAX576 (Novartis), CNTO-607 (Centocor), MK-6105 (Merck, CSL); Dual IL-4 and IL-13 inhibitors: AIR645/ISIS369645 (ISIS Altair), DOM-0910 (GlaxoSmithKline, Domantis), Pitrakinra/AER001/Aerovant™ (Aerovance Inc), AMG-317 (Amgen), and the like.
  • Suitable steroids include corticosteroids, combinations of corticosteroids and LABAs, combinations of corticosteroids and LAMAs, combinations of corticosteroids with LABAs and LAMAs, and the like.
  • Suitable corticosteroids include budesonide, fluticasone, flunisolide, triamcinolone, beclomethasone, mometasone, ciclesonide, dexamethasone, and the like.
  • Examples of budesonide formulations include Captisol-Enabled Budesonide Solution for Nebulization (AstraZeneca PLC), Pulmicort® (AstraZeneca PLC), Pulmicort® Flexhaler (AstraZeneca Plc), Pulmicort® HFA-MDI (AstraZeneca PLC), Pulmicort Respules® (AstraZeneca PLC), Inflammide (Boehringer Ingelheim GmbH), Pulmicort® HFA-MDI (SkyePharma PLC), Unit Dose Budesonide ASTRAZENECA (AstraZeneca PLC), Budesonide Modulite (Chiesi Farmaceutici S.p.A), CHF5188 (Chiesi Farmaceutici S.p.A), Budesonide ABBOTT LABS (Abbott Laboratories), Budesonide clickhaler (Vestura Group PLC), Miflonide (Novartis AG), Xavin (Teva Pharmaceutical Industries Ltd.), Budesonide TEVA (Teva Pharmaceutical Industries Ltd.), Symbicort® (AstraZeneca K.K., AstraZeneca PLC), VR632 (Novartis AG, Sandoz International GmbH), and the like.
  • Examples of fluticasone propionate formulations include Flixotide Evohaler (GlaxoSmithKline PLC), Flixotide Nebules (GlaxoSmithKline Plc), Flovent® (GlaxoSmithKline Plc), Flovent® Diskus (Glaxo SmithKline PLC), Flovent® HFA (GlaxoSmithKline PLC), Flovent® Rotadisk (GlaxoSmithKline PLC), Advair® HFA (GlaxoSmithKline PLC, Theravance Inc), Advair Diskus® (GlaxoSmithKline PLC, Theravance Inc.), VR315 (Novartis AG, Vectura Group PLC, Sandoz International GmbH), and the like. Other formulations of fluticasone include fluticasone as Flusonal (Laboratorios Almirall, S.A.), fluticasone furoate as GW685698 (GlaxoSmithKline PLC, Thervance Inc.), Plusvent (Laboratorios Almirall, S.A.), Flutiform® (Abbott Laboratories, SkyePharma PLC), and the like.
  • Examples of flunisolide formulations include Aerobid® (Forest Laboratories Inc), Aerospan® (Forest Laboratories Inc), and the like. Examples of triamcinolone formulations include Triamcinolone ABBOTT LABS (Abbott Laboratories), Azmacort® (Abbott Laboratories, Sanofi-Aventis), and the like. Examples of beclomethasone dipropionate formulations include Beclovent (GlaxoSmithKline PLC), QVAR® (Johnson & Johnson, Schering-Plough Corp, Teva Pharmacetucial Industries Ltd), Asmabec clickhaler (Vectura Group PLC), Beclomethasone TEVA (Teva Pharmaceutical Industries Ltd), Vanceril (Schering-Plough Corp), BDP Modulite (Chiesi Farmaceutici S.p.A.), Clenil (Chiesi Farmaceutici S.p.A), Beclomethasone dipropionate TEVA (Teva Pharmaceutical Industries Ltd), and the like. Examples of mometasone include QAB149 Mometasone furoate (Schering-Plough Corp), QMF149 (Novartis AG), Fomoterol fumarate, mometoasone furoate (Schering-Plough Corp), MFF258 (Novartis AG, Merck & Co Inc), Asmanex® Twisthaler (Schering-Plough Corp), and the like. Examples of cirlesonide formulations include Alvesco® (Nycomed International Management GmbH, Sepracor, Sanofi-Aventis, Tejin Pharma Limited), Alvesco® Combo (Nycomed International Management GmbH, Sanofi-Aventis), Alvesco® HFA (Nycomed Intenational Management GmbH, Sepracor Inc), and the like. Examples of dexamethasone formulations include DexPak® (Merck), Decadron® (Merck), Adrenocot, CPC-Cort-D, Decaject-10, Solurex and the like. Other corticosteroids include Etiprednol dicloacetate TEVA (Teva Pharmaceutical Industries Ltd), and the like.
  • Combinations of corticosteroids and LABAs include salmeterol with fluticasone, formoterol with budesonide, formoterol with fluticasone, formoterol with mometasone, indacaterol with mometasone, and the like.
  • Examples of salmeterol with fluticasone include Plusvent (Laboratorios Almirall, S.A.), Advair® HFA (GlaxoSmithKline PLC), Advair® Diskus (GlaxoSmithKline PLV, Theravance Inc), VR315 (Novartis AG, Vectura Group PLC, Sandoz International GmbH) and the like. Examples of vilanterol with fluticasone include GSK642444 with fluticasone and the like. Examples of formoterol with budesonide include Symbicort® (AstraZeneca PLC), VR632 (Novartis AG, Vectura Group PLC), and the like. Examples of formoterol with fluticasone include Flutiform® (Abbott Laboratories, SkyePharma PLC), and the like. Examples of formoterol with mometasone include Dulera®/MFF258 (Novartis AG, Merck & Co Inc), and the like. Examples of indacaterol with mometasone include QAB149 Mometasone furoate (Schering-Plough Corp), QMF149 (Novartis AG), and the like. Combinations of corticosteroids with LAMAs include fluticasone with tiotropium, budesonide with tiotropium, mometasone with tiotropium, salmeterol with tiotropium, formoterol with tiotropium, indacaterol with tiotropium, vilanterol with tiotropium, and the like. Combinations of corticosteroids with LAMAs and LABAs include fluticasone with salmeterol and tiotropium.
  • Other anti-asthma molecules include: ARD111421 (VIP agonist, AstraZeneca PLC), AVE0547 (anti-inflammatory, Sanofi-Aventis), AVE0675 (TLR agonist, Pfizer, Sanofi-Aventis), AVE0950 (Syk inhibitor, Sanofi-Aventis), AVE5883 (NK1/NK2 antagonist, Sanofi-Aventis), AVE8923 (tryptase beta inhibitor, Sanofi-Aventis), CGS21680 (adenosine A2A receptor agonist, Novartis AG), ATL844 (A2B receptor antagonist, Novartis AG), BAY443428 (tryptase inhibitor, Bayer AG), CHF5407 (M3 receptor inhibitor, Chiesi Farmaceutici S.p.A.), CPLA2 Inhibitor WYETH (CPLA2 inhibitor, Wyeth), IMA-638 (IL-13 antagonist, Wyeth), LAS100977 (LABA, Laboratorios Almirall, S.A.), MABA (M3 and β2 receptor antagonist, Chiesi Farmaceutici S.p.A), R1671 (mAb, Roche Holding Ltd), CS003 (Neurokinin receptor antagonist, Daiichi Sankyo Company, Limited), DPC168 (CCR antagonist, Bristol-Myers Squibb), E26 (anti-IgE, Genentech Inc), HAE1 (Genentech), IgE inhibitor AMGEN (Amgen Inc), AMG853 (CRTH2 and D2 receptor antagonist, Amgen), IPL576092 (LSAID, Sanofi-Aventis), EPI2010 (antisense adenosine 1, Chiesi Farmaceutici S.p.A.), CHF5480 (PDE-4 inhibitor, Chiesi Farmaceutici S.p.A.), KI04204 (corticosteroid, Abbott Laboratories), SVT47060 (Laboratorios Salvat, S.A.), VML530 (leukotriene synthesis inhibitor, Abbott Laboratories), LAS35201 (M3 receptor antagonist, Laboratorios Almirall, S.A.), MCC847 (D4 receptor antagonist, Mitsubishi Tanabe Pharma Corporation), MEM1414 (PDE-4 inhibitor, Roche), TA270 (5-LO inhibitor, Chugai Pharmaceutical Co Ltd), TAK661 (eosinophil chemotaxis inhibitor, Takeda Pharmaceutical Company Limited), TBC4746 (VLA-4 antagonist, Schering-Plough Corp), VR694 (Vectura Group PLC), PLD177 (steroid, Vectura Group PLC), KI03219 (corticosteroid+LABA, Abbott Laboratories), AMG009 (Amgen Inc), AMG853 (D2 receptor antagonist, Amgen Inc);
  • AstraZeneca PLC: AZD1744 (CCR3/histamine-1 receptor antagonist, AZD1419 (TLR9 agonist), Mast Cell inhibitor ASTRAZENECA, AZD3778 (CCR antagonist), DSP3025 (TLR7 agonist), AZD1981 (CRTh2 receptor antagonist), AZD5985 (CRTh2 antagonist), AZD8075 (CRTh2 antagonist), AZD1678, AZD2098, AZD2392, AZD3825 AZD8848, AZD9215, ZD2138 (5-LO inhibitor), AZD3199 (LABA);
  • GlaxoSmithKline PLC: GW328267 (adenosine A2 receptor agonist), GW559090 (a4 integrin antagonist), GSK679586 (mAb), GSK597901 (adrenergic β2 agonist), AM103 (5-LO inhibitor), GSK256006 (PDE4 inhibitor), GW842470 (PDE-4 inhibitor), GSK870086 (glucocorticoid agonist), GSK159802 (LABA), GSK256066 (PDE-4 inhibitor), GSK642444 (LABA, adrenergic β2 agonist), GSK64244 and Revolair (fluticasone/vilanterol), GSK799943 (corticosteroid), GSK573719 (mAchR antagonist), and GSK573719.
  • Pfizer Inc: PF3526299, PF3893787, PF4191834 (FLAP antagonist), PF610355 (adrenergic β2 agonist), CP664511 (a4131/VCAM-1 interaction inhibitor), CP609643 (inhibitor of a4131/VCAM-1 interactions), CP690550 (JAK3 inhibitor), SAR21609 (TLR9 agonist), AVE7279 (Th1 switching), TBC4746 (VLA-4 antagonist); R343 (IgE receptor signaling inhibitor), SEP42960 (adenosine A3 antagonist);
  • Sanofi-Aventis: MLN6095 (CrTH2 inhibitor), SAR137272 (A3 antagonist), SAR21609 (TLR9 agonist), SAR389644 (DP1 receptor antagonist), SAR398171 (CRTH2 antagonist), SSR161421 (adenosine A3 receptor antagonist);
  • Merck & Co Inc: MK0633, MK0633, MK0591 (5-LO inhibitor), MK886 (leukotriene inhibitor), BIO1211 (VLA-4 antagonist); Novartis AG: QAE397 (long-acting corticosteroid), QAK423, QAN747, QAP642 (CCR3 antagonist), QAX935 (TLR9 agonist), NVA237 (LAMA).
  • Suitable expectorants include guaifenesin, guaiacolculfonate, ammonium chloride, potassium iodide, tyloxapol, antimony pentasulfide and the like.
  • Suitable vaccines include nasally inhaled influenza vaccines and the like.
  • Suitable macromolecules include proteins and large peptides, polysaccharides and oligosaccharides, and DNA and RNA nucleic acid molecules and their analogs having therapeutic, prophylactic or diagnostic activities. Proteins can include antibodies such as monoclonal antibodies. Nucleic acid molecules include genes, antisense molecules such as siRNAs that bind to complementary DNA, RNAi, shRNA, microRNA, RNA, or ribosomes to inhibit transcription or translation. Preferred macromolecules have a molecular weight of at least 800 Da, at least 3000 Da or at least 5000 Da.
  • Selected macromolecule drugs for systemic applications: Ventavis (Iloprost), Calcitonin, Erythropoietin (EPO), Factor IX, Granulocyte Colony Stimulating Factor (G-CSF), Granulocyte Macrophage Colony, Stimulating Factor (GM-CSF), Growth Hormone, Insulin, Interferon Alpha, Interferon Beta, Interferon Gamma, Luteinizing Hormone Releasing Hormone (LHRH), follicle stimulating hormone (FSH), Ciliary Neurotrophic Factor, Growth Hormone Releasing Factor (GRF), Insulin-Like Growth Factor, Insulinotropin, Interleukin-1 Receptor Antagonist, Interleukin-3, Interleukin-4, Interleukin-6, Macrophage Colony Stimulating Factor (M-CSF), Thymosin Alpha 1, IIb/IIIa Inhibitor, Alpha-1 Antitrypsin, Anti-RSV Antibody, palivizumab, motavizumab, and ALN-RSV, Cystic Fibrosis Transmembrane Regulator (CFTR) Gene, Deoxyribonuclase (DNase), Heparin, Bactericidal/Permeability Increasing Protein (BPI), Anti-Cytomegalovirus (CMV) Antibody, Interleukin-1 Receptor Antagonist, and the like. GLP-1 analogs (liraglutide, exenatide, etc.), Domain antibodies (dAbs), Pramlintide acetate (Symlin), Leptin analogs, Synagis (palivizumab, MedImmune) and cisplatin.
  • Selected therapeutics helpful for chronic maintenance of CF include antibiotics/macrolide antibiotics, bronchodilators, inhaled LABAs, and agents to promote airway secretion clearance. Suitable examples of antibiotics/macrolide antibiotics include tobramycin, azithromycin, ciprofloxacin, colistin, aztreonam and the like. Another exemplary antibiotic/macrolide is levofloxacin. Suitable examples of bronchodilators include inhaled short-acting beta2 agonists such as albuterol, and the like. Suitable examples of inhaled LABAs include salmeterol, formoterol, and the like. Suitable examples of agents to promote airway secretion clearance include Pulmozyme (dornase alfa, Genetech), hypertonic saline, DNase, heparin and the like. Selected therapeutics helpful for the prevention and/or treatment of CF include VX-770 (Vertex Pharmaceuticals) and amiloride.
  • Selected therapeutics helpful for the treatment of idiopathic pulmonary fibrosis include Metelimumab (CAT-192) (TGF-β1 mAb inhibitor, Genzyme), Aerovant™ (AER001, pitrakinra) (Dual IL-13, IL-4 protein antagonist, Aerovance), Aeroderm™ (PEGylated Aerovant, Aerovance), microRNA, RNAi, and the like.
  • In preferred embodiments, the respirable dry powder or respirable dry particle comprises an antibiotic, such as a macrolide (e.g., azithromycin, clarithromycin and erythromycin), a tetracycline (e.g., doxycycline, tigecycline), a fluoroquinolone (e.g., gemifloxacin, levofloxacin, ciprofloxacin and mocifloxacin), a cephalosporin (e.g., ceftriaxone, defotaxime, ceftazidime, cefepime), a penicillin (e.g., amoxicillin, amoxicillin with clavulanate, ampicillin, piperacillin, and ticarcillin) optionally with a β-lactamase inhibitor (e.g., sulbactam, tazobactam and clavulanic acid), such as ampicillin-sulbactam, piperacillin-tazobactam and ticarcillin with clavulanate, an aminoglycoside (e.g., amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, and apramycin), a penem or carbapenem (e.g. doripenem, ertapenem, imipenem and meropenem), a monobactam (e.g., aztreonam), an oxazolidinone (e.g., linezolid), vancomycin, glycopeptide antibiotics (e.g. telavancin), tuberculosis-mycobacterium antibiotics, tobramycin, azithromycin, ciprofloxacin, colistin, and the like. In a further preferred embodiment, the respirable dry powder or respirable dry particle comprises levofloxacin. In a further preferred embodiment, the respirable dry powder or respirable dry particle comprises Cayston®. In a further preferred embodiment, the respirable dry powder or respirable dry particle does not comprise tobramycin. In a further preferred embodiment, the respirable dry powder or respirable dry particle does not comprise levofloxacin. In a further preferred embodiment, the respirable dry powder or respirable dry particle does not comprise Cayston®.
  • If desired the salt formulation can contain an agents that disrupt and/or disperse biofilms. Suitable examples of agents to promote disruption and/or dispersion of biofilms include specific amino acid stereoisomers, e.g. D-leucine, D-methionine, D-tyrosine, D-tryptophan, and the like. (Kolodkin-Gal, I., D. Romero, et al. “D-amino acids trigger biofilm disassembly.” Science 328(5978): 627-629.)
  • Dry powder formulations are prepared with the appropriate particle diameter, surface roughness, and tap density for localized delivery to selected regions of the respiratory tract. For example, higher density or larger particles may be used for upper airway delivery. Similarly, a mixture of different sized particles can be administered to target different regions of the lung in one administration.
  • As used herein, the phrase “aerodynamically light particles” refers to respirable particles having a tap density less than about 0.4 g/cm3. The tap density of particles of a dry powder may be obtained by the standard USP tap density measurement. Tap density is an accepted, approximate measure of the envelope mass density. The envelope mass density of an isotropic particle is defined as the mass of the particle divided by the minimum sphere envelope volume in which it can be enclosed. Features contributing to low tap density include irregular surface texture and porous structure.
  • Dry powder formulations (“DPFs”) with large particle size have improved flowability characteristics, such as less aggregation (Visser, J., Powder Technology 58: 1-10 (1989)), easier aerosolization, and potentially less phagocytosis. Rudt, S, and R. H. Muller. J. Controlled Release, 22: 263-272 (1992); Tabata Y., and Y. Ikada. J. Biomed. Mater. Res. 22: 837-858 (1988). Dry powder aerosols for inhalation therapy are generally produced with mass median aerodynamic diameters primarily in the range of less than 5 microns, although dry powders that have any desired range in aerodynamic diameter can be produced. Ganderton D., J. Biopharmaceutical Sciences, 3:101-105 (1992); Gonda, I. “Physico-Chemical Principles in Aerosol Delivery.” in Topics in Pharmaceutical Sciences 1991, Crommelin, D. J. and K. K. Midha, Eds., Medpharm Scientific Publishers, Stuttgart, pp. 95-115 (1992). Large “carrier” particles (containing no salt formulation) can be co-delivered with therapeutic aerosols to aid in achieving efficient aerosolization among other possible benefits. French, D. L., Edwards, D. A. and Niven, R. W., J. Aerosol Sci. 27: 769-783 (1996). Particles with degradation and release times ranging from seconds to months can be designed and fabricated by established methods in the art.
  • Generally, salt formulations that are dry powders may be produced by spray drying, freeze drying, spray-freeze drying jet milling, single and double emulsion solvent evaporation, and supercritical fluid-based processes, among others. Preferably, salt formulations are produced by spray drying, which entails preparing a solution containing the salt and other components of the formulation, spraying the solution into a closed chamber, and removing the solvent with a heated gas stream. Suitable spray-drying techniques are described, for example, by K. Masters in “Spray Drying Handbook”, John Wiley & Sons, New York (1984). Generally, during spray-drying, heat from a hot gas such as heated air or nitrogen is used to evaporate a solvent from droplets formed by atomizing a continuous liquid feed. When hot air is used, the moisture in the air is at least partially removed before its use. When nitrogen is used, the nitrogen gas can be run “dry”, meaning that no additional water vapor is combined with the gas. If desired the moisture level of the nitrogen or air can be set before the beginning of spry dry run at a fixed value above “dry” nitrogen. If desired, the spray drying or other instruments, e.g., jet milling instrument, used to prepare the dry particles can include an inline geometric particle sizer that determines a geometric diameter of the respirable dry particles as they are being produced, and/or an inline aerodynamic particle sizer that determines the aerodynamic diameter of the respirable dry particles as they are being produced.
  • Spray dried powders that contain salts with sufficient solubility in water or aqueous solvents, such as calcium chloride and calcium lactate, can be readily prepared using conventional methods. Some salts, such as calcium citrate and calcium carbonate, have low solubility in water and other aqueous solvents. Spray dried powders that contain such salts can be prepared using any suitable method. One suitable method involves combining other more soluble salts in solution and permitting reaction (precipitation reaction) to produce the desired salt for the dry powder formulation. For example, if a dry powder formulation comprising calcium citrate and sodium chloride is desired, a solution containing the high solubility salts calcium chloride and sodium citrate can be prepared. The precipitation reaction leading to calcium citrate is 3 CaCl2+2 Na3Cit→Ca3Cit2+6 NaCl. It is preferable that the sodium salt is fully dissolved before the calcium salt is added and that the solution is continuously stirred. The precipitation reaction can be allowed to go to completion or stopped before completion, e.g., by spray drying the solution, as desired. The resulting solution may appear clear with fully dissolved salts or a precipitate may form. Depending on reaction conditions, a precipitate may form quickly or over time. Solutions that contain a light precipitate that results in formation of a stable homogenous suspension can be spray dried.
  • Alternatively, two saturated or sub-saturated solutions are fed into a static mixer in order to obtain a saturated or supersaturated solution post-static mixing. Preferably, the post-spray drying solution is supersaturated. The two solutions may be aqueous or organic, but are preferably substantially aqueous. The post-static mixing solution is then fed into the atomizing unit of a spray dryer. In a preferable embodiment, the post-static mixing solution is immediately fed into the atomizer unit. Some examples of an atomizer unit include a two-fluid nozzle, a rotary atomizer, or a pressure nozzle. Preferably, the atomizer unit is a two-fluid nozzle. In one embodiment, the two-fluid nozzle is an internally mixing nozzle, meaning that the gas impinges on the liquid feed before exiting to most outward orifice. In another embodiment, the two-fluid nozzle is an externally mixing nozzle, meaning that the gas impinges on the liquid feed after exiting the most outward orifice.
  • Dry powder formulations can also be prepared by blending individual components into the final formulation. For example, a first dry powder that contains a calcium salt can be blended with a second dry powder that contains a sodium salt to produce a dry powder salt formulation that contains a calcium salt and a sodium salt. If desired, additional dry powders that contain excipients (e.g., lactose) and/or other active ingredients (e.g., antibiotic, antiviral) can be included in the blend. The blend can contain any desired relative amounts or ratios of salts, excipients and other ingredients (e.g., antibiotics, antivirals).
  • If desired, dry powders can be prepared using polymers that are tailored to optimize particle characteristics including: i) interactions between the agent (e.g., salt) to be delivered and the polymer to provide stabilization of the agent and retention of activity upon delivery; ii) rate of polymer degradation and thus agent release profile; iii) surface characteristics and targeting capabilities via chemical modification; and iv) particle porosity. Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervatian, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art.
  • The compositions of some preferred salt compositions are presented in Table 1. The compositions disclosed in Table 1 are non-limiting examples of salt compositions that can be administered in accordance with the methods of the invention. The weight percentages of the dry powder formulations are on a dry basis.
  • TABLE 1
    Liquid formulations
    Tonicity
    (1X = CaCl2 CaCl2 NaCl NaCl
    Formulation # isotonic) (% w/v) (M) (% w/v) (M)
    1 2X 1.3 0.12 0.90 0.15
    2 4X 4.2 0.38 0.90 0.15
    3 6X 6.4 0.58 0.90 0.15
    4 8X 9.0 0.81 0.90 0.15
    5 11X  13 1.2 0.90 0.15
    6 2X 2.6 0.23 n.a. n.a.
    7 5X 6.4 0.58 n.a. n.a.
    8 10X  13 1.2 n.a. n.a.
    11 2X 2.4 0.21 0.16 0.027
    12 4X 4.7 0.42 0.31 0.053
    13 8X 9.4 0.85 0.62 0.11
    16 4X 2.6 0.23 1.8 0.31
    17 8X 5.2 0.47 3.6 0.62
    Dry powder formulations
    Formulation composition
    Calcium Sodium
    Leucine Calcium salt Sodium salt
    Formulation # (wt %) salt (wt %) salt (wt %)
    18 50.0 Calcium 29.5 Sodium 20.5
    chloride chloride
    19 50.0 Calcium 33.8 Sodium 16.2
    acetate chloride
    20 50.0 Calcium 37.0 Sodium 13.0
    lactate chloride
    21 50.0 Calcium 22.0 Sodium 28.0
    chloride sulfate
    22 50.0 Calcium 19.5 Sodium 30.5
    chloride citrate
    23 10.0 Calcium 66.6 Sodium 23.4
    lactate chloride
    24 10.0 Calcium 39.6 Sodium 50.4
    chloride sulfate
    25 10.0 Calcium 35.1 Sodium 54.9
    chloride citrate
    26 n.a. Calcium 74.0 Sodium 26.0
    lactate chloride
    27 n.a. Calcium 44.0 Sodium 56.0
    chloride sulfate
    28 n.a. Calcium 39.0 Sodium 61.0
    chloride citrate
    29 20.0 Calcium 75.0 Sodium 5.0
    lactate chloride
    30 37.5 Calcium 58.6 Sodium 3.9
    lactate chloride
    n.a. not applicable
  • Preferred Formulations
  • Respirable dry powder calcium salt formulations are preferred for use in the invention. Respirable dry powders containing dry particles that have the composition of Formulation 29 or Formulation 30, or that are based on Formulation 29 or 30 but are co-formulated with another therapeutic agent, are particularly preferred.
  • In one aspect, the dry powder administered in the methods described herein contains respirable dry particles that contain i) about 20% (w/w) leucine, ii) about 75% (w/w) calcium lactate, and iii) about 5% (w/w) sodium chloride. In another aspect, the dry powder administered in the methods described herein contains respirable dry particles that contain i) about 37.5% (w/w) leucine, ii) about 58.6% (w/w) calcium lactate, and iii) about 3.9% (w/w) sodium chloride. The weight percentages are on a dry basis.
  • If desired, the respirable dry powders and/or dry particles are based on Formulation 29 or 30 and include one or more additional therapeutic agents, such as any of the additional therapeutic agents described herein, e.g., mucoactive or mucolytic agents, surfactants, antibiotics, antivirals, antihistamines, cough suppressants, bronchodilators, anti-inflammatory agents, steroids, vaccines, adjuvants, expectorants, macromolecules, or therapeutics that are helpful for chronic maintenance of cystic fibrosis (CF). The additional agent can be blended with a dry powder of Formulation 29 or 30 or co-spray dried as desired.
  • The preferred dry powders can contain dry particles that contain about 20% (w/w) leucine, about 75% (w/w) calcium lactate, and about 5% (w/w) sodium chloride; about 37.5% (w/w) leucine, about 58.6% (w/w) calcium lactate, and about 3.9% (w/w) sodium chloride (e.g, Formulations 29 and 30 respectively), that are blended with an additional therapeutic agent or co-formulated with an additional therapeutic agent. Such blended or co-formulated preparations can be produced in a variety of ways. For example, respirable dry particles of the invention can be blended with an additional therapeutic agent or the components of Formulation 29 or Formulation 30 can be co-spray dried with an additional therapeutic agent, such as any one or combination of the additional therapeutic agents disclosed herein, to produce a dry powder. Blended dry powders contain particles of Formulation 29 and/or 30 and particles that contain an additional therapeutic agent. Preferred additional therapeutic agents are LABAs (e.g., formoterol, salmeterol), short-acting beta agonists (e.g., albuterol), corticosteroids (e.g., fluticasone), LAMAS (e.g., tiotropium), antibiotics (e.g., levofloxacin), recombinant human deoxyribonuclease I (e.g., dornase alfa, also known as DNAse), sodium channel blockers (e.g., amiloride), and combinations thereof. Particularly preferred additional therapeutic agents are short-acting beta agonists (e.g., albuterol), antibiotics (e.g., levofloxacin), recombinant human deoxyribonuclease I (e.g., dornase alfa, also known as DNAse), sodium channel blockers (e.g., amiloride), and combinations thereof.
  • Dry powders can be prepared by co-spray drying an additional therapeutic agent with the calcium lactate and sodium chloride components, and optionally all or a portion of the leucine component of Formulation 29 or Formulation 30. When it is desirable to retain the relative proportions of calcium lactate, sodium chloride and leucine from Formulation 29 or 30, the additional therapeutic agent can be added to a solution of Formulation 29 or Formulation 30 and the resulting solution spray dried to produce dry particles that contain the additional therapeutic agent. In such particles the amount of calcium lactate, sodium chloride and leucine in the dry particles will each be lower than the amounts in Formulation 29 or 30, due to the addition of the additional therapeutic agent. In one example, the formulation can contain up to about 20% (w/w) additional therapeutic agent, and the amount of each of calcium lactate, sodium chloride and leucine are reduced proportionally, but the ratio of the amounts (wt %) of calcium lactate:sodium chloride:leucine is the same as in Formulation 29 or 30. In another example, the formulation can contain up to about 6% (w/w) additional therapeutic agent. In a further example, the formulation can contain up to about 1% (w/w) additional therapeutic agent.
  • In exemplary embodiments, the dry particles are based on Formulation 29 and contain up to about 6% (w/w) of one or more additional therapeutic agents, about 70% to about 75% (w/w) calcium lactate, about 3% to about 5% (w/w) sodium chloride and about 17% to about 20% (w/w) leucine. In other exemplary embodiments, the dry particles are based on Formulation 30 and contain up to about 6% (w/w) of one or more additional therapeutic agent, about 45% to about 58.6% (w/w) calcium lactate, about 1.9% to about 3.9% (w/w) sodium chloride and about 30% to about 37.5% (w/w) leucine. In further exemplary embodiments, the dry particles are based on Formulation 29 and contain up to about 20% (w/w) of one or more additional therapeutic agents, about 60% to about 75% (w/w) calcium lactate, about 2% to about 5% (w/w) sodium chloride and about 15% to about 20% (w/w) leucine. In other exemplary embodiments, the dry particles are based on Formulation 30 and contain up to about 20% (w/w) of one or more additional therapeutic agent, about 54.6% to about 58.6% (w/w) calcium lactate, about 1.9% to about 3.9% (w/w) sodium chloride and about 34.5% to about 37.5% (w/w) leucine. When the additional therapeutic agent is potent, a small amount may be used such as 0.01% to about 1% (w/w), and the composition of the dry particles is substantially the same as Formulation 29 or 30. The additional therapeutic agent can be any of the additional therapeutic agents described herein. Preferred additional therapeutic agents are LABAs (e.g., formoterol, salmeterol), short-acting beta agonists (e.g., albuterol), corticosteroids (e.g., fluticasone), LAMAs (e.g., tiotropium), antibiotics (e.g., levofloxacin), recombinant human deoxyribonuclease I (e.g., dornase alfa also known as DNAse), sodium channel blockers (e.g., amiloride), and combinations thereof. Particularly preferred additional therapeutic agents are short-acting beta agonists (e.g., albuterol), antibiotics (e.g., levofloxacin), recombinant human deoxyribonuclease I (e.g., dornase alfa, also known as DNAse), sodium channel blockers (e.g., amiloride), and combinations thereof.
  • In some dry powder formulations that contain an additional therapeutic agent, all or a portion of the leucine component in Formulation 29 or 30 is replaced with one or more additional therapeutic agents. This approach is particularly advantageous for additional therapeutic agents that require a higher effective dose, e.g., are not highly potent, and produces dry particles that deliver the beneficial effects of calcium cation in the respiratory tract and of the beneficial effects of the additional therapeutic agent(s). In exemplary embodiments, the dry particles are based on Formulation 29 and contain about 0.01% to about 20% (w/w) of one or more additional therapeutic agent, about 75% (w/w) calcium lactate, about 5% (w/w) sodium chloride and about 20% (w/w) or less leucine. In other exemplary embodiments, the dry particles are based on Formulation 30 and contain about 0.01% to about 37.5% (w/w) of one or more additional therapeutic agents, about 58.6% (w/w) calcium lactate, about 3.9% (w/w) sodium chloride and about 37.5% (w/w) or less leucine. The additional therapeutic agent can be any of the additional therapeutic agents described herein. Preferred additional therapeutic agent are LABAs (e.g., formoterol, salmeterol), short-acting beta agonists (e.g., albuterol), corticosteroids (e.g., fluticasone), LAMAs (e.g., tiotropium), antibiotics (e.g., levofloxacin, tobramycin), recombinant human deoxyribonuclease I (e.g., dornase alfa), and combinations thereof. Particularly preferred additional therapeutic agents are short-acting beta agonists (e.g., albuterol), antibiotics (e.g., levofloxacin), recombinant human deoxyribonuclease I (e.g., dornase alfa, also known as DNAse), sodium channel blockers (e.g., amiloride), and combinations thereof. Particular examples of dry powders that contain additional therapeutic agents are disclosed herein as Formulations X-XX.
  • In the preferred dry powders (e.g., Formulations 29 and 30 or based thereon), the dry particles are preferably small and dispersible. These dry particles are also calcium dense. The dry particles contain a high concentration of calcium salt (i.e., about 40% or more (w/w)) and are considered calcium salt dense. Preferably, the dry particles of the invention have a VMGD, when measured at a dispersion (i.e., regulator) pressure setting of 1 bar, of about 5 microns or less, as measured by laser diffraction using a Spraytec system (particle size analysis instrument, Malvern Instruments) or using a HELOS/RODOS system (laser diffraction system with dry dispensing unit, Sympatec GmbH).
  • Preferably, the respirable dry particles have a VMGD as measured by laser diffraction at the dispersion pressure setting of 1.0 bar using a HELOS/RODOS system of about 5 microns or less (e.g., about 0.1 μm to about 5 μm), about 4 μm or less (e.g., about 0.1 μm to about 4 μm), about 3 μm or less (e.g., about 0.1 μm to about 3 μm), about 1 μm to about 5 μm, about 1 μm to about 4 μm, about 1.5 μm to about 3.5 μm, about 2 μm to about 5 μm, about 2 μm to about 4 μm, or about 2 μm to about 3 μm. In a preferred embodiment, the size is about 1 μm to about 3 μm.
  • If desired, the respirable dry particles of can be large and dispersible, and preferably calcium dense. For example, the respirable dry particles can have a VMGD as measured by HELOS/RODOS at the dispersion pressure setting of 1.0 bar of up to about 30 μm.
  • Surprisingly, the preferred respirable dry powders (e.g., Formulations 29 and 30) have poor bulk flow properties, yet, are highly dispersible. This is surprising because flow properties and dispersibility are both known to be negatively effected by particle agglomeration or aggregation. Thus, it is unexpected that particles that have poor flow characteristics would be highly dispersible.
  • In particular, the preferred respirable dry powders have a Hausner Ratio that is greater than 1.5, and can be 1.6 or higher, 1.7 or higher, 1.8 or higher, 1.9 or higher, 2 or higher, 2.1 or higher, 2.2 or higher, 2.3 or higher, 2.4 or higher, 2.5 or higher, 2.6 or higher or 2.7 or higher, between 2.2 and 2.9, between 2.2 and 2.8, between 2.2 and 2.7, between 2.2 and 2.6, between 2.2 and 2.5, between 2.3 and 2.5, between 2.6 and 2.8, about 2.7, or about 2.4. In some preferred embodiments, the respirable dry powders of have a Hausner Ratio that is 1.4 or higher.
  • In addition to any of the features and properties described herein, in any combination, the respirable dry powders and respirable dry particles can have a heat of solution that is not highly exothermic. Preferably, the heat of solution is determined using the ionic liquid of a simulated lung fluid (e.g. as described in Moss, O. R. 1979. Simulants of lung interstitial fluid. Health Phys. 36, 447-448; or in Sun, G. 2001. Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter. Am J Physiol Lung Cell Mol Physiol. 281, L807-L815) at pH 7.4 and 37° C. in an isothermal calorimeter. For example, the respirable dry powders or respirable dry particles can have a heat of solution that is less exothermic than the heat of solution of calcium chloride dihydrate, e.g., have a heat of solution that is greater than about −10 kcal/mol, greater than about −9 kcal/mol, greater than about −8 kcal/mol, greater than about −7 kcal/mol, greater than about −6 kcal/mol, greater than about −5 kcal/mol, greater than about −4 kcal/mol, greater than about −3 kcal/mol, greater than about −2 kcal/mol, greater than about −1 kcal/mol, about −10 kcal/mol to about 10 kcal/mol, about −8 kcal/mol to about 8 kcal/mol, or about −6 kcal/mol to about 6 kcal/mol. In a preferred aspect of the invention, the heat of solution is between about −7 kcal/mol to about 7 kcal/mol, between about −6 kcal/mol to about 6 kcal/mol, or between about −5 kcal/mol to about 5 kcal/mol.
  • The preferred respirable dry particles (e.g., Formulations 29 and 30 or based thereon) are dispersible, and have 1 bar/4 bar and/or 0.5 bar/4 bar of about 1.5 or less (e.g., about 1.0 to about 1.5). Preferably, the dry particles have 1 bar/4 bar and/or 0.5 bar/4 bar of about 1.0 to about 1.2, and/or about 1.0 to about 1.1.
  • The preferred respirable dry particles (e.g., Formulations 29 and 30 or based thereon) have an MMAD of about 10 microns or less, such as an MMAD of about 0.5 micron to about 10 microns. Preferably, the dry particles of the invention have an MMAD of about 7 microns or less (e.g. about 0.5 micron to about 7 microns), preferably about 1 micron to about 7 microns, or about 2 microns to about 7 microns, or about 3 microns to about 7 microns, or about 4 microns to about 7 microns, about 5 microns to about 7 microns, about 1 micron to about 6 microns, about 1 micron to about 5 microns, about 2 microns to about 5 microns, about 2 microns to about 4 microns, or about 3 microns. In a preferred embodiment, the size is about 1 μm to about 3 μm.
  • The preferred respirable dry powders have an FPF of less than about 5.6 microns (FPF<5.6 μm) of the total dose of at least about 30%, preferably at least about 40%, or between about 40% and about 50%, at least 50%, or between about 50% and about 60%, or at least 60%, or between about 60% and about 70%, or at least about 70%; and preferably have a FPF of less than about 3.4 microns (FPF<3.4 μm) of the total dose of at least about 20%, preferably at least about 25%, or between about 20% and about 30%, or at least 30%, or between 30% and about 40%, or at least 40%, or between 40% and about 50%, or at least 50%.
  • The preferred respirable dry powders and dry particles preferably have a tap density of about 0.5 g/cm3 to about 1.2 g/cm3. For example, the small and dispersible dry particles of Formulation 29 and Formulation 30 can have a tap density of about 0.6 g/cm3 to about 1.0 g/cm3, about 0.7 g/cm3 to about 1.0 g/cm3, or about 0.8 g/cm3 to about 1.0 g/cm3. If desired, powders and particles that have a tap density that is less than about 0.5 g/cc, or less than about 0.4 g/cc can be prepared.
  • The respirable dry powders and dry particles can have a water or solvent content of less than about 25%, less than about 20%, or less than about 15% by weight of the respirable dry powder or particle. For example, the respirable dry particles of the invention can have a water or solvent content of less than about 15% by weight, less than about 13% by weight, less than about 11.5% by weight, less than about 10% by weight, less than about 9% by weight, less than about 8% by weight, less than about 7% by weight, less than about 6% by weight, less than about 5% by weight, less than about 4% by weight, less than about 3% by weight, less than about 2% by weight, less than about 1% by weight or be anhydrous. The respirable dry particles of the invention can have a water or solvent content of less than about 6% and greater than about 1%, less than about 5.5% and greater than about 1.5%, less than about 5% and greater than about 2%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%.
  • The preferred respirable dry powders and dry particles (e.g., Formulations 29 and 30 or based thereon) contain a high percentage of calcium in the composition, and are calcium dense. The respirable dry particles contain at least 10% calcium by weight of the dry powder (wt calcium/wt dry powder), at least 11% calcium by weight of the dry powder, at least 12% calcium by weight of the dry powder; at least 13% calcium by weight of the dry powder, at least 14% calcium by weight of the dry powder, between 10% and 12% calcium by weight of the dry powder, between 12% and 14% calcium by weight of the dry powder, about 11% or about 13% calcium by weight of the dry powder. Additionally, the respirable dry powder and dry particles of Formulation 29 and Formulation 30 contain a high percentage of calcium salt in the composition, and are calcium salt dense. The respirable dry particles contain at least 40% calcium salt by weight of the dry powder (wt calcium salt/wt dry powder), at least 50% calcium salt by weight of the dry powder, at least 55% calcium salt by weight of the dry powder; at least 60% calcium salt by weight of the dry powder, at least 70% calcium salt by weight of the dry powder, between 40% and 90% calcium salt by weight of the dry powder, between 50% and 85% calcium salt by weight of the dry powder, about 55% or about 80% calcium salt by weight of the dry powder.
  • The preferred respirable dry powders and dry particles (e.g., Formulations 29 and 30 or based thereon) contain a low percentage of sodium in the composition. The respirable dry particles contain less than 4% sodium by weight of the dry powder (wt sodium/wt dry powder), preferably 3% or less sodium by weight of the dry powder, or 2% or less sodium by weight of the dry powder. In another aspect of the invention, the respirable dry particles contain less than 6% sodium salt by weight of the dry powder, or about 5% or less sodium salt by weight of the dry powder.
  • The respirable dry particles of Formulations 29 and 30 are characterized by the crystalline and amorphous content of the particles. The respirable dry particles can comprise a mixture of amorphous and crystalline content. For example, calcium lactate can be substantially in the amorphous phase while sodium chloride or leucine can be substantially in the crystalline phase. This provides several advantages. For example, the crystalline phase (e.g., crystalline sodium chloride and/or crystalline leucine) can contribute to the stability of the dry particles in the dry state and to the dispersibility characteristics, whereas the amorphous phase (e.g., amorphous calcium salt) can facilitate rapid water uptake and dissolution of the particle upon deposition in the respiratory tract.
  • The preferred highly dispersible dry powders (e.g., Formulations 29 and 30 or based thereon) can be administered with low inhalation energy. In order to relate the dispersion of powder at different inhalation flow rates, volumes, and from inhalers of different resistances, the energy required to perform the inhalation maneuver can be calculated. Inhalation energy can be calculated from the equation E=R2Q2V where E is the inhalation energy in Joules, R is the inhaler resistance in kPa1/2/LPM, Q is the steady flow rate in L/min and V is the inhaled air volume in L.
  • The preferred respirable dry powders and dry particles (e.g., Formulations 29 and 30 or based thereon) are characterized by a high emitted dose (e.g., CEPM of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) from a dry powder inhaler when a total inhalation energy of less than about 2 Joules or less than aboutl Joule, or less than about 0.8 Joule, or less than about 0.5 Joule, or less than about 0.3 Joule is applied to the dry powder inhaler. For example, an emitted dose of at least 75%, at least 80%, at least 85%, at least 90%, at least 95% CEPM of Formulation 29 or Formulation 30 contained in a unit dose container, containing about 50 mg or about 40 mg of the appropriate formulation, in a dry powder inhaler can be achieved when a total inhalation energy of less than about 1 Joule (e.g., less than about 0.8 Joule, less than about 0.5 Joule, less than about 0.3 Joule) is applied to the dry powder inhaler. An emitted dose of at least about 70% CEPM of respirable dry powder contained in a unit dose container, containing about 50 mg or about 40 mg of the respirable dry powder, in a dry powder inhaler can be achieved when a total inhalation energy of less than about 0.28 Joule is applied to the dry powder inhaler. In a preferred aspect of the invention, the dry powder inhaler is a passive dry powder inhaler. The dry powder can fill the unit dose container, or the unit dose container can be at least 40% full, at least 50% full, at least 60% full, at least 70% full, at least 80% full, or at least 90% full. The unit dose container can be a capsule (e.g. size 000, 00, 0E, 0, 1, 2, 3, and 4, with respective volumetric capacities of 1.37 ml, 950 μl, 770 μl, 680 μl, 480 μl, 360 μl, 270 μl, and 200 μl). Alternatively, the unit dose container can be a blister. The blister can be packaged as a single blister, or as part of a set of blisters, for example, 7 blisters, 14 blisters, 28 blisters, or 30 blisters.
  • Healthy adult populations are predicted to be able to achieve inhalation energies ranging from 2.9 Joules for comfortable inhalations to 22 Joules for maximum inhalations by using values of peak inspiratory flow rate (PIFR) measured by Clarke et al. (Journal of Aerosol Med, 6(2), p. 99-110, 1993) for the flow rate Q from two inhaler resistances of 0.02 and 0.055 kPa1/2/LPM, with a inhalation volume of 2 L based on both FDA guidance documents for dry powder inhalers and on the work of Tiddens et al. (Journal of Aerosol Med, 19(4), p. 456-465, 2006) who found adults averaging 2.2 L inhaled volume through a variety of DPIs.
  • Mild, moderate and severe adult COPD patients are predicted to be able to achieve maximum inhalation energies of 5.1 to 21 Joules, 5.2 to 19 Joules, and 2.3 to 18 Joules respectively. This is again based on using measured PIFR values for the flow rate Q in the equation for inhalation energy. The PIFR achievable for each group is a function of the inhaler resistance that is being inhaled through. The work of Broeders et al. (Eur Respir J, 18, p. 780-783, 2001) was used to predict maximum and minimum achievable PIFR through 2 dry powder inhalers of resistances 0.021 and 0.032 kPa1/2/LPM for each.
  • Similarly, adult asthmatic patients are predicted to be able to achieve maximum inhalation energies of 7.4 to 21 Joules based on the same assumptions as the COPD population and PIFR data from Broeders et al.
  • Healthy adults and children, COPD patients, asthmatic patients ages 5 and above, and CF patients, for example, are capable of providing sufficient inhalation energy to empty and disperse the dry powder formulations of the invention. For example, a 50 mg dose of Formulation 29 or Formulation 30 was found to require only 0.28 Joules to empty more than 70% of the fill weight in a single inhalation. All the adult patient populations listed above were calculated to be able to achieve greater than 2 Joules, 7 times more than the inhalational energy required.
  • An advantage of the invention is that the preferred dry powders disperse well across a wide range of flow rates and are relatively flow rate independent, hence providing a relatively equal dosing across a variety of inspiratory flow rates. The preferred dry particles and powders enable the use of a simple, passive DPI for a wide patient population.
  • In particular aspects, a respirable dry powder containing respirable dry particles of Formulation 29, that comprise i) about 20% (w/w) leucine, ii) about 75% (w/w) calcium lactate, and iii) about 5% (w/w) sodium chloride is used in the methods of the invention. The respirable dry powder or dry particles of Formulation 29 can be characterized by:
  • 1. VMGD at 1 bar as measured using a HELOS/RODOS system between 1 microns and 3 microns, preferably between 1.5 microns and 2.5 microns or between 2 microns and 3 microns;
  • 2. Hausner Ratio of 2.0 or higher, preferably between 2.0 and 3.2, and more preferably between 2.4 and 3.0;
  • 3. 1 bar/4 bar of 1.5 or less, preferably between 1.0 and 1.2;
      • 4. 0.5 bar/4 bar of 1.5 or less, preferably between 1.0 and 1.3;
      • 5. FPF<5.6 of at least 30%, preferably at least 40% and more preferably at least 50%;
  • 6. FPF<3.4 of at least 20%, preferably at least 30%; and/or
  • 7. tap density of about 0.5 g/cm3 to about 1.2 g/cm3, preferably between about 0.7 g/cm3 and about 1.0 g/cm3.
  • The respirable dry powder or dry particles of Formulation 29 can be further characterized by a water content of less then 25% by weight, preferably less than 15% or less than 10% by weight, or less than 5% by weight, and by the presence of a mixture of amorphous and crystalline content, with calcium lactate substantially in the amorphous phase and sodium chloride and/or leucine substantially in the crystalline phase. Alternatively, the calcium lactate and sodium chloride are substantially in the amorphous phase and the leucine is in either the crystalline and/or amorphous phase. In addition, the respirable dry powder or dry particles of Formulation 29 can be further characterized by an emitted dose of at least about 80% of Formulation 29 contained in a unit dose container that contains 40 mg or more, or 50 mg or more, of Formulation 29, in a dry powder inhaler, when a total inhalation energy of less than about 0.5 Joule is applied to the dry powder inhaler; by an emitted dose of at least about 90% of Formulation 29 contained in a unit dose container that contains 40 mg or more, or 50 mg or more, of Formulation 29, in a dry powder inhaler, when a total inhalation energy of less than about 0.5 Joule is applied to the dry powder inhaler; or by an emitted dose of at least about 95% of Formulation 29 contained in a unit dose container containing 40 mg or more, or 50 mg or more of Formulation 29, in a dry powder inhaler, when a total inhalation energy of less than about 1 Joule is applied to the dry powder inhaler.
  • In other particular aspects, a respirable dry powder containing respirable dry particles of Formulation 29, that comprise i) about 20% (w/w) leucine, ii) about 75% (w/w) calcium lactate, and iii) about 5% (w/w) sodium chloride is used in the methods of the invention. The respirable dry powder or dry particles can be characterized by:
  • 1. VMGD at 1 bar as measured using a HELOS/RODOS system between 1 microns and 3 microns, preferably between 1.5 microns and 2.5 microns or between 2 microns and 3 microns;
  • 2. Hausner Ratio of 1.4 or higher;
  • 3. 1 bar/4 bar of 1.5 or less, preferably between 1.0 and 1.2;
  • 4. 0.5 bar/4 bar of 1.5 or less, preferably between 1.0 and 1.3;
  • 5. FPF<5.6 of at least 38%, preferably at least 40% and more preferably at least 50%;
  • 6. FPF<3.4 of at least 20%, preferably at least 30%;
  • 7. tap density of about 0.5 g/cm3 to about 1.2 g/cm3, preferably between about 0.7 g/cm3 and about 1.0 g/cm3; and/or
  • 8. heat of solution of about −8 kcal to about 8 kcal/mol.
  • In other particular aspects, a respirable dry powder containing respirable dry particles of Formulation 30, that comprise i) about 37.5% (w/w) leucine, ii) about 58.6% (w/w) calcium lactate, and iii) about 3.9% (w/w) sodium chloride is used in the methods of the invention. The respirable dry powder or dry particles of Formulation 30 can be characterized by:
  • 1. VMGD at 1 bar as measured using a HELOS/RODOS system between 1.5 microns and 3 microns, preferably between 2 microns and 3 microns;
  • 2. Hausner Ratio of 1.9 or higher, preferably between 2.0 and 3.0, and more preferably between 2.2 and 2.6;
  • 3. 1 bar/4 bar of 1.5 or less, preferably between 1.0 and 1.2;
  • 4. 0.5 bar/4 bar of 1.5 or less, preferably between 1.0 and 1.3;
  • 5. FPF<5.6 of at least 40%, preferably at least 50%;
  • 6. FPF<3.4 of at least 20%, preferably at least 30%; and/or
  • 7. tap density of about 0.5 g/cm3 to about 1.0 g/cm3, preferably between about 0.6 g/cm3 and about 1.0 g/cm3, or about 0.7 g/cm3 and about 1.0 g/cm3.
  • The respirable dry powder or dry particles of Formulation 30 can be further characterized by a water content of less then 25% by weight, preferably less than 15% or less than 10% by weight, or less than 5% by weight, or less than 2% by weight, and by the presence of a mixture of amorphous and crystalline content, with calcium lactate substantially in the amorphous phase and sodium chloride and/or leucine substantially in the crystalline phase. Alternatively, the calcium lactate and sodium chloride are substantially in the amorphous phase and the leucine is in either the crystalline and/or amorphous phase. In addition, the respirable dry powder or dry particles of Formulation 30 can be further characterized by an emitted dose of at least about 70% of Formulation 30 contained in a unit dose container that contains 50 mg or more of Formulation 30, in a dry powder inhaler is achieved when a total inhalation energy of less than about 0.5 Joule is applied to the dry powder inhaler; or by an emitted dose of at least about 80% of Formulation 30 contained in a unit dose containing 50 mg or more of Formulation 30, in a dry powder inhaler is achieved when a total inhalation energy of less than about 1 Joule is applied to the dry powder inhaler.
  • The respirable dry particles and dry powders described herein are suitable for inhalation therapies. The respirable dry particles may be fabricated with the appropriate surface roughness, diameter and tap density for localized delivery to selected regions of the respiratory system such as the deep lung or upper or central airways.
  • EXEMPLIFICATION Example 1 Calcium Chloride Reduces Formation of Particles that Contain Pathogen
  • To test whether changes in the surface viscoelastic properties of mucus mimetic could translate into differences in particle formation, and thus impact transmission, studies using a simulated cough system were conducted. G. Zayas, J. Dimitry, A. Zayas, D. O'Brien, M. King, BMC Pulm Med 5, 11 (2005). The simulated cough system involves passing air, at a defined pressure, through a pneumotachograph and across a model trachea that has been lined with mucus mimetic. A schematic of the system is shown in FIG. 1A. The air pressure passed through the system is such that it will mimic the flow profile and volume of a cough. To test the effect of different aerosols on particle formation, saline or calcium aerosols were topically delivered to the surface of a mucus mimetic (locus bean gum) followed by simulating a cough through the system and collecting the particles with an optical particle counter (CI-500B Climet Instruments, Redlands, Calif.). Exposure of the mimetic to 0.12M CaCl2 in 0.90% sodium chloride reduced the number of particles relative to the control condition by 93% (FIG. 1B; n=4, p<0.01 one-way ANOVA), where as 0.90% sodium chloride treatment had only a modest effect (34% of control, n=4). Next, in order to test whether the reduced particle counts would correlate with a reduction in the number of aerosolized bacteria using the same system, mucus mimetic was mixed with Klebsiella pneumoniae and was added to the model trachea of the cough system. After exposure of the mimetic to 0.12M CaCl2 in 0.90% sodium chloride or leaving the mimetic untreated, a cough was simulated and the particles were collected in liquid broth. Bacteria (particles) collected in the broth were diluted and plated on agar plates to enumerate the number of bacteria in each condition. Exposure of the mimetic to 0.12M CaCl2 in 0.90% sodium chloride before cough simulation suppressed the number of particles formed by 75% compared to the untreated control (FIG. 1C). These results demonstrate that administering salt aerosols topically to mucus surfaces can act to limit airborne spread of pathogens and reduce contagion and spread of disease.
  • Example 2 In Vivo Studies
  • Mouse studies were conducted to assess whether salt formulations are effective in treating pneumonia in vivo.
  • Mouse Model
  • Specific pathogen-free female C57BL/6 mice (6-7 weeks, 16-22 g) were used in these studies. Mice were given access to food and water ad libitum. For infections, S. pneumoniae (Serotype 3; ATCC 6303) were streaked onto blood agar plates and grown at 37° C. plus 5% CO2 overnight. Prior to infection, animals were anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine. Single colonies of S. pneumoniae were resuspended in sterile saline to OD600=0.3 and then diluted 1:4 in saline. Colloidal carbon was added to 1% and 50 μL of the resulting solution (˜1×106 CFU) was instilled into the left lung of anesthetized mice to produce infection. Following infection, the bacterial titer of the inoculum was determined by serial dilution and plating on blood agar plates. After 24 hours, mice were euthanized and the bacterial burden in lungs of infected animals was determined by plating serially diluted lung homogenate on blood agar plates.
  • Salt Formulation Aerosol Delivery Systems
  • A whole-body exposure system using a high output nebulizer was utilized to deliver salt aerosols to a pie-chamber exposure system. Each pie chamber exposure chamber was modified such that a single tube delivered aerosol to a central manifold and ultimately to one of 11 mouse holding chambers via 4 inlet ports in each chamber. The total flow through the system was 11.7 L/min and animals were exposed to cationic aerosols for 15 minutes.
  • Aerosol Characterization
  • Particle sizing of the aerosol generated by the high output nebulizer was performed using an inhaler adaptor set-up on a Sympatec Helos particle size analyzer outfitted with an R3 lens (0.5 to 175 μM size range). The nebulizer was filled with 45 mL isotonic saline (JT Baker, Phillipsburg, N.J.) and the outlet port of the tubing connected to the nebulizer was positioned ˜1 cm from the inhaler adaptor. Each test measurement was taken for 5 seconds (Copt 16.5-29.31%) and the volume median diameter (MMD; x50) and the geometric standard deviation were recorded for each measurement. Flow rates were determined during each test run using a pneumotachometer and a Validyne pressure transducer connected to a voltage amplifier and voltmeter. The system was calibrated such that 1 CFM=1V.
  • Nebulizer output rates were determined by measuring the mass deposition onto collection filters. Filters were weighed immediately before collection and immediately after a 30 second collection period. Three test runs were performed using a fresh solution of isotonic saline for each measurement.
  • Salt Formulation Aerosol Dosing Estimates
  • Estimated CaCl2 dose levels and aerosol concentrations are shown in Table 2.
  • TABLE 2
    Exposure time Dose Level of Aerosol Concentration
    Study (min) CaAB(mg/kg/day) (mg/L)
    Mouse 15 2.3 0.146
    pneumonia
    ABased on the formula presented below.
    BThis estimation of achieved dose assumes 100% deposition within the respiratory tract.
  • Achieved dose levels to animals during the exposure period were estimated using the following formula:
  • D L = E c × R M V × T B W
  • DL=Achieved dose levels (mg/kg/day)
    Ec=Actual aerosol concentration delivered to the animals (mg/L air)
    RMV=Respiratory Minute Volume (L/min.) according to the method of Bide et al.: RMV (L/min.)=0.499×BW (kg) 0.809 (estimated average over exposure period)
    T=Time, duration of daily exposure (min.)
    BW=Mean body weight (kg).
  • Mouse Treatment Study
  • Mice were randomly assigned to different study groups on the day of the infections. Different aerosol exposure times relative to the time of infection were utilized to test the effect of aerosols in both prophylaxis and treatment regimens. For each exposure, mice were loaded into a customized whole-body pie chamber system in which aerosols were delivered to a central manifold and subsequently to each individual animal. Aerosol exposures consisted of a 15 minute exposure of 0.12M calcium chloride in 0.90% sodium chloride, which delivered an estimated dose of 6.4 mg/kg/day of CaCl2. After 24 hours of infection, animals were euthanized by isoflurane inhalation and the lungs were surgically removed and placed in sterile water. Lungs were homogenized using a glass mortar and pestle until no large tissue fragments were visible. Colony forming units (CFU) were enumerated by serially diluting lung homogenates in sterile water and plating on blood agar plates. Plates were incubated overnight at 37° C. plus 5% CO2 and CFU counted the following day. Differences between groups were evaluated by Mann-Whitney U test.
  • 2A. Prophylactic Exposure and Treatment of Mice with Calcium Chloride Formulations Reduces The Bacterial Burden in Murine Lungs
  • A S. pneumoniae mouse model was employed to evaluate the treatment effect of salt on a bacterial infection. As shown in FIG. 2A, treatment (2.3 mg Ca/kg deposited dose) by whole body exposure of 0.12M calcium chloride in 0.90% sodium chloride two hours post-infection led to significantly lower bacterial burden 24 hours later (n=15) relative to untreated controls (n=15). Prophylaxis in the mouse pneumonia model was evaluated by pretreating mice (n=12) with salt solutions 2 hours before installation and subsequently infecting with S. pneumoniae by intratracheal installation. FIG. 2B demonstrates that relative to untreated controls (n=12) and infected animals treated 2 hours post infection (n=15), bacterial burden 24 hours post infection was statistically lower in the prophylactic group compared to any other.
  • 2B. Prophylactic Treatment of Bacterial Pneumonia is Driven by Calcium Chloride Specifically and not Divalent Cations in General
  • The role of the nature of the aerosolized cation was evaluated by repeating treatment studies in the mouse pneumonia model by treating animals two hours before infection with salt solutions of 2.0% magnesium chloride and 0.90% sodium chloride (n=12), as well as with 0.90% sodium chloride alone (n=12). Animals treated with the MgCl2 formulation (FIG. 3A) and saline formulation (FIG. 3B) solutions had similar bacterial burdens as the untreated controls 24 hours post-infection, demonstrating that the efficacy of the salt formulations in treating bacterial pneumonia was specific to CaCl2 containing formulations and not general to cations (whether monovalent or divalent).
  • 2C. Therapeutic Activities of Calcium:Sodium Formulations in Treating Bacterial Infections
  • In this example, the therapeutic activities of formulations comprising calcium chloride and sodium chloride in treating bacterial infections were examined using a mouse model. The data showed that the calcium:sodium formulations were effective in treating Streptococcus pneumoniae infection in the mouse model.
  • Bacteria were prepared by growing cultures on tryptic soy agar (TSA) blood plates overnight at 37° C. plus 5% CO2. Single colonies were resuspended to an OD600˜0.3 in sterile PBS and subsequently diluted 1:4 in sterile PBS [2×107 Colony forming units (CFU)/mL]. Mice were infected with 50 μL of bacterial suspension (˜1×106 CFU) by intratracheal instillation while under anesthesia.
  • C57BL6 mice were exposed to aerosolized liquid formulations in a whole-body exposure system using either a high output nebulizer or Pari LC Sprint nebulizers connected to a pie chamber cage that individually holds up to 11 animals. Treatments were performed 2 hours before infection with Serotype 3 Streptococcus pneumoniae. Unless otherwise stated, exposure times were 3 minutes in duration. Twenty-four hours after infection mice were euthanized by pentobarbital injection and lungs were collected and homogenized in sterile PBS. Lung homogenate samples were serially diluted in sterile PBS and plated on TSA blood agar plates. CFU were enumerated the following day.
  • Results: Calcium: Sodium Formulations (Ca2+:Na+ at 8:1 Molar Ratio) Reduced Bacterial Burden in a Dose Responsive Manner
  • The therapeutic activities of the calcium:sodium formulations were evaluated in the same model and over a wide dose range. With dosing time held constant, different calcium doses were delivered by using formulations consisting of different concentrations of Ca2+:Na+ and therefore different tonicities. es. The formulations containing Ca2+:Na+ at an 8:1 molar ratio reduced bacterial burden in a dose responsive manner, with the greatest reduction observed at lower doses of calcium (about a 4-fold reduction at a dose of 0.32 mg Ca2+/kg and tonicity of 0.5× (Formulation 9, Table 1), and about a 5-fold reduction at a dose of 0.72 mg Ca2+/kg and tonicity of 1.0×) (Formulation 10, Table 1) (FIG. 4A). Interestingly, these reductions were comparable to the reduction seen for Formulation A (1.29% CaCl2 and 0.9% NaCl), however at significantly lower doses. The 2× tonicity formulation (Formulation 11, Table 1), which is equivalent to Formulation A in tonicity, had a relatively modest effect on reducing bacterial titers (˜1.6 fold reduction) when administered at a dose of 1.58 mg Ca2+/kg.
  • Increasing Dose Through Longer Nebulizations Did not Significantly Affect the Therapeutic Activities of the Calcium:Sodium Formulations
  • FIG. 4A showed that that calcium:sodium formulations at an 8:1 ratio of calcium to sodium reduced the severity of bacterial infections at doses of less than 1.58 mg Ca2+/kg. Specifically, the 1× formulation (Formulation 10, ˜0.72 mg Ca2+/kg) was the most highly effective. The study whose results were presented in FIG. 4A tested a dose time of 3 minutes. To further examine the effect of dosage, a dose range of Ca2+ was tested by increasing the duration of dosing. Animals were treated with a Ca2+:Na+ formulation (1× tonicity=isotonic; 8:1 molar ratio) for different amounts of time (1.5 minutes to 12 minutes). These dose times resulted in Ca2+ dosages oft approximately 0.36, 0.72, 1.44, and 2.88 mg Ca2+/kg for the 1.5, 3, 6, and 12 minutes dosing times, respectively. As shown in FIG. 4B, at the shortest dosing time, no decrease in bacterial titer was observed as compared to control animals (which were dosed 3 minutes with saline), whereas the 3, 6, and 12 minutes doses each reduced bacterial titers to statistically significant levels.
  • 2D. Synergistic Activities of Calcium and Ampicillin in Treating Bacterial Infections
  • Mice (C57BL6) were exposed to nebulized solutions of Ca:Na Formulation 10 (1× tonicity; 8:1 molar ratio of Ca2+:Na+, delivered dose ˜0.72 mg Ca/kg), ampicillin in saline (96.75 mg/mL in 0.9% NaCl, delivered dose ˜3 mg/kg), or ampicillin (96.75 mg/mL) dissolved in the 1× (Formulation 10) using whole body exposure chambers. Mice were exposed to each formulation 2 h before infection with S. pneumonia. Both the 1× Ca:Na formulation and the ampicillin alone reduced bacterial burden in the lungs of infected mice to the saline control (p<0.001 Mann-Whitney U test). The 1× formulation reduced bacterial titers approximately 4.5-fold and the ampicillin reduced titers 33-fold. Unexpectedly, the combination of the two therapies resulted in an even greater reduction in bacterial titers (333-fold) than either single treatment showing a therapeutic benefit to delivering inhaled antibiotics in the calcium formulations described herein. (FIG. 4C)
  • Dry Powders
  • Materials used in the following Examples and their sources are listed below. Calcium lactate pentahydrate, sodium chloride, and L-leucine were obtained from Sigma-Aldrich Co. (St. Louis, Mo.), Spectrum Chemicals (Gardena, Calif.), or Merck (Darmstadt, Germany). Ultrapure (Type II ASTM) water was from a water purification system (Millipore Corp., Billerica, Mass.), or equivalent.
  • Methods:
  • Geometric or Volume Diameter. Volume median diameter (x50 or Dv50), which may also be referred to as volume median geometric diameter (VMGD), was determined using a laser diffraction technique. The equipment consisted of a HELOS diffractometer and a RODOS dry powder disperser (Sympatec, Inc., Princeton, N.J.). The RODOS disperser applies a shear force to a sample of particles, controlled by the regulator pressure (typically set at 1.0 bar with maximum orifice ring pressure) of the incoming compressed dry air. The pressure settings may be varied to vary the amount of energy used to disperse the powder. For example, the dispersion energy may be modulated by changing the regulator pressure from 0.2 bar to 4.0 bar. Powder sample is dispensed from a microspatula into the RODOS funnel. The dispersed particles travel through a laser beam where the resulting diffracted light pattern produced is collected, typically using an R1 lens, by a series of detectors. The ensemble diffraction pattern is then translated into a volume-based particle size distribution using the Fraunhofer diffraction model, on the basis that smaller particles diffract light at larger angles. Using this method, geometric standard deviation (GSD) for the volume diameter was also determined.
  • Volume median diameter can also be measured using a method where the powder is emitted from a dry powder inhaler device. The equipment consisted of a Spraytec laser diffraction particle size system (Malvern, Worcestershire, UK), “Spraytec”. Powder formulations were filled into size 3 HPMC capsules (Capsugel V-Caps) by hand with the fill weight measured gravimetrically using an analytical balance (Mettler Tolerdo XS205). A capsule based passive dry powder inhalers (RS-01 Model 7, High resistance Plastiape S.p.A.) was used which had specific resistance of 0.036 kPa1/2 LPM−1. Flow rate and inhaled volume were set using a timer controlled solenoid valve with flow control valve (TPK2000, Copley Scientific). Capsules were placed in the dry powder inhaler, punctured and the inhaler sealed to the inlet of the laser diffraction particle sizer. The steady air flow rate through the system was initiated using the TPK2000 and the particle size distribution was measured via the Spraytec at lkHz for the durations at least 2 seconds and up to the total inhalation duration. Particle size distribution parameters calculated included the volume median diameter (Dv50) and the geometric standard deviation (GSD) and the fine particle fraction (FPF) of particles less than 5 micrometers in diameter. At the completion of the inhalation duration, the dry powder inhaler was opened, the capsule removed and re-weighed to calculate the mass of powder that had been emitted from the capsule during the inhalation duration (capsule emitted powder mass or CEPM).
  • The previous description of the use of the Spraytec was for what is described as its “closed bench configuration”. Alternatively, the Spraytec can be used in its “open bench configuration”. In the open bench configuration, capsules were placed in the dry powder inhaler, punctured and the inhaler sealed inside a cylinder. The cylinder was connected to a positive pressure air source with steady air flow through the system again measured with a mass flow meter and its duration controlled with a timer controlled solenoid valve. The exit of the dry powder inhaler was exposed to room pressure and the resulting aerosol jet passed through the laser of the diffraction particle sizer (Spraytec) in its open bench configuration before being captured by a vacuum extractor. The steady air flow rate through the system was initiated using the solenoid valve and the particle size distribution was measured via the Spraytec at lkHz for the duration of the single inhalation maneuver with a minimum of 2 seconds, as in the closed bench configuration. When data are reported in the examples as being measured by the Spraytec, they are from the open bench configuration unless otherwise noted.
  • Fine Particle Fraction. The aerodynamic properties of the powders dispersed from an inhaler device were assessed with an Mk-II 1 ACFM Andersen Cascade Impactor (Copley Scientific Limited, Nottingham, UK). The instrument was run in controlled environmental conditions of 18 to 25° C. and relative humidity (RH) between 20 and 40%. The instrument consists of eight stages that separate aerosol particles based on inertial impaction. At each stage, the aerosol stream passes through a set of nozzles and impinges on a corresponding impaction plate. Particles having small enough inertia will continue with the aerosol stream to the next stage, while the remaining particles will impact upon the plate. At each successive stage, the aerosol passes through nozzles at a higher velocity and aerodynamically smaller particles are collected on the plate. After the aerosol passes through the final stage, a filter collects the smallest particles that remain, called the “final collection filter”. Gravimetric and/or chemical analyses can then be performed to determine the particle size distribution. A short stack cascade impactor, also referred to as a collapsed cascade impactor, is also utilized to allow for reduced labor time to evaluate two aerodynamic particle size cut-points. With this collapsed cascade impactor, stages are eliminated except those required to establish fine and coarse particle fractions.
  • The impaction techniques utilized allowed for the collection of two or eight separate powder fractions. The capsules (HPMC, Size 3; Shionogi Qualicaps, Madrid, Spain or Capsugel Vcaps, Peapack, N.J.) were hand filled with powder to a specific weight and placed in a hand-held, breath-activated dry powder inhaler (DPI) device, the high resistance RS-01 DPI (Plastiape, Osnago, Italy). The capsule was punctured and the powder was drawn through the cascade impactor operated at a flow rate of 60.0 L/min for 2.0 s. At this flowrate, the calibrated cut-off diameters for the eight stages are 8.6, 6.5, 4.4, 3.3, 2.0, 1.1, 0.5 and 0.3 microns and for the two stages used with the short stack cascade impactor, the cut-off diameters are 5.6 microns and 3.4 microns. The fractions were collected by placing filters in the apparatus and determining the amount of powder that impinged on them by gravimetric measurements or chemical measurements on an HPLC, as labeled in the tables. The fine particle fraction of the total dose of powder (FPFTD) less than or equal to an effective cut-off aerodynamic diameter was calculated by dividing the powder mass recovered from the desired stages of the impactor by the total particle mass in the capsule. Results are reported as the fine particle fraction of less than 5.6 microns (FPFTD<5.6 microns) and the fine particle fraction of less than 3.4 microns (FPFTD<3.4 microns). The fine particle fraction can alternatively be calculated relative to the recovered or emitted dose of powder by dividing the powder mass recovered from the desired stages of the impactor by the total powder mass recovered.
  • Aerodynamic Diameter. Mass median aerodynamic diameter (MMAD) was determined using the information obtained by the Andersen Cascade Impactor. The cumulative mass under the stage cut-off diameter is calculated for each stage and normalized by the recovered dose of powder. The MMAD of the powder is then calculated by linear interpolation of the stage cut-off diameters that bracket the 50th percentile.
  • Fine Particle Dose. The fine particle dose was determined using the information obtained by the ACI. The cumulative mass deposited on the final collection filter, and stages 6, 5, 4, 3, and 2 for a single dose of powder actuated into the ACI is equal to the fine particle dose less than 4.4 microns (FPD<4.4 μm).
  • Emitted Geometric or Volume Diameter. The volume median diameter (Dv50) of the powder after it emitted from a dry powder inhaler, which may also be referred to as volume median geometric diameter (VMGD), was determined using a laser diffraction technique via the Spraytec diffractometer (Malvern, Inc.). Powder was filled into size 3 capsules (V-Caps, Capsugel) and placed in a capsule based dry powder inhaler (RS01 Model 7 High resistance, Plastiape, Italy), or DPI, which was connected airtightly to the inhaler adapter of the Spraytec. A steady airflow rate was drawn through the DPI typically at 60 L/min for a set duration, typically of 2 seconds controlled by a timer controlled solenoid (TPK2000, Copley, Scientific, UK). Alternatively, the airflow rate drawn through the DPI was sometimes run at 15 L/min, 20 L/min, or 30 L/min. The outlet aerosol then passed perpendicularly through the laser beam as an internal flow. The resulting geometric particle size distribution of the aerosol was calculated from the software based on the measured scatter pattern on the photodetectors with samples typically taken at 1000 Hz for the duration of the inhalation. The Dv50, GSD, FPF<5.0 μm measured were then averaged over the duration of the inhalation.
  • Capsule Emitted Powder Mass. A measure of the emission properties of the powders was determined by using the information obtained from the Andersen Cascade Impactor tests or emitted geometric diameter by Spraytec. The filled capsule weight was recorded at the beginning of the run and the final capsule weight was recorded after the completion of the run. The difference in weight represented the amount of powder emitted from the capsule (CEPM or capsule emitted powder mass). The CEPM was reported as a mass of powder or as a percent by dividing the amount of powder emitted from the capsule by the total initial particle mass in the capsule. While the standard CEPM was measured at 60 L/min, it was also measured at 15 L/min, 20 L/min, or 30 L/min.
  • Tap Density. Tap density was measured using a modified method requiring smaller powder quantities, following USP <616> with the substitution of a 1.5 cc microcentrifuge tube (Eppendorf AG, Hamburg, Germany) or a 0.3 cc section of a disposable serological polystyrene micropipette (Grenier Bio-One, Monroe, N.C.) with polyethylene caps (Kimble Chase, Vineland, N.J.) to cap both ends and hold the powder. Instruments for measuring tap density, known to those skilled in the art, include but are not limited to the Dual Platform Microprocessor Controlled Tap Density Tester (Vankel, Cary, N.C.) or a SOTAX Tap Density Tester model TD2 (Horsham, Pa.). Tap density is a standard, approximated measure of the envelope mass density. The envelope mass density of an isotropic particle is defined as the mass of the particle divided by the minimum spherical envelope volume within which it can be enclosed.
  • Bulk Density. Bulk density was estimated prior to tap density measurement procedure by dividing the weight of the powder by the volume of the powder, as estimated using the volumetric measuring device.
  • Hausner Ratio. The Hausner ratio, a dimensionless number that is correlated to the flowability of a powder, was calculated by dividing the tap density by the bulk density.
  • Liquid Feedstock Preparation for Spray Drying. Spray drying homogenous particles requires that the ingredients of interest be solubilized in solution or suspended in a uniform and stable suspension. Calcium lactate and sodium chloride are sufficiently water-soluble to prepare suitable spray drying solutions. The solubility for each of these two salts is listed in Table 3.
  • TABLE 3
    Calcium Lactate and Sodium Chloride Solubility in Water
    Salt Solubility in Water (at 20-30° C., 1 bar)
    Salt Water solubility (g/L)
    Calcium lactate 1051
    Sodium chloride 3601
    1Perry, Robert H., Don W. Green, and James O. Maloney. Perry's Chemical Engineers' Handbook. 7th ed. New York: McGraw-Hill, 1997. Print.
  • The ratios used for formulations were based on the molecular weight of the anhydrous salts. For certain salts, hydrated forms are more readily available than the anhydrous form. This required an adjustment in the ratios originally calculated, using a multiplier to correlate the molecular weight of the anhydrous salt with the molecular weight of the hydrate. An example of this calculation is included below. The weight percent of calcium ion in calcium lactate and calcium lactate pentahydrate is listed in Table 4.
  • TABLE 4
    Weight Percent of Ca2+ in Calcium Lactate
    Weight % of
    MW Ca2+ in
    Salt Formula (g/mole) molecule
    Calcium lactate Ca(C3H5O3)2 218.2 18.3
    Calcium lactate Ca(C3H5O3)2•5H2O 308.2 13.0
    pentahydrate
  • Spray Drying Using Niro Spray Dryer. Dry powders were produced by spray drying utilizing a Niro Mobile Minor spray dryer (GEA Process Engineering Inc., Columbia, Md.) with powder collection from a cyclone, a product filter or both. Atomization of the liquid feed was performed using a co-current two-fluid nozzle either from Niro (GEA Process Engineering Inc., Columbia, Md.) or a Spraying Systems (Carol Stream, Ill.) two-fluid nozzle with gas cap 67147 and fluid cap 2850SS, although other two-fluid nozzle setups can also be utilized in a similar manner. In some embodiments, the two-fluid nozzle can be in an internal mixing setup or an external mixing setup. Additional atomization techniques include rotary atomization or a pressure nozzle. The liquid feed was fed using gear pumps (Cole-Parmer Instrument Company, Vernon Hills, Ill.) directly into the two-fluid nozzle or into a static mixer (Charles Ross & Son Company, Hauppauge, N.Y.) immediately before introduction into the two-fluid nozzle. An additional liquid feed technique includes feeding from a pressurized vessel. Nitrogen or air may be used as the drying gas, provided that moisture in the air is at least partially removed before its use. Pressurized nitrogen or air can be used as the atomization gas feed to the two-fluid nozzle. The process gas inlet temperature can range from 70° C. to 300° C. and outlet temperature from 50° C. to 120° C. with a liquid feedstock rate of 10 mL/min to 100 mL/min. The gas supplying the two-fluid atomizer can vary depending on nozzle selection and for the Niro co-current two-fluid nozzle can range from 5 kg/hr to 50 kg/hr or for the Spraying Systems two-fluid nozzle with gas cap 67147 and fluid cap 2850SS can range from 30 g/min to 150 g/min. The atomizing gas rate can be set to achieve a certain gas to liquid mass ratio, which directly affects the droplet size created. The pressure inside the drying drum can range from +3 ″WC to −6 ″WC. Spray dried powders can be collected in a container at the outlet of the cyclone, onto a cartridge or baghouse filter, or from both a cyclone and a cartridge or baghouse filter.
  • Spray Drying Using Büchi Spray Dryer. Dry powders were prepared by spray drying on a Büchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG, Flawil, Switzerland) with powder collection from either a standard or High Performance cyclone. The system used the Büchi B-296 dehumidifier to ensure stable temperature and humidity of the air used to spray dry. Furthermore, when the relative humidity in the room exceeded 30% RH, an external LG dehumidifier (model 49007903, LG Electronics, Englewood Cliffs, N.J.) was run constantly. Atomization of the liquid feed was accomplished utilizing a Büchi two-fluid nozzle with a 1.5 mm diameter. Inlet temperature of the process gas can range from 100° C. to 220° C. and outlet temperature from 60° C. to 120° C. with a liquid feedstock flowrate of 3 mL/min to 10 mL/min. The two-fluid atomizing gas ranges from 25 mm to 45 mm (300 LPH to 530 LPH) and the aspirator rate from 70% to 100% (28 m3/hr to 38 m3/hr).
  • Table 5 provides feedstock formulations used in preparation of some dry powders described herein.
  • TABLE 5
    Feedstock Formulations
    Formula-
    tion Feedstock Composition (w/w)
    29 20.0% leucine, 75.0% calcium lactate, 5.0% sodium chloride
    30 37.5% leucine, 58.6% calcium lactate, 3.9% sodium chloride
    III 39.4% leucine, 58.6% calcium lactate, 2.0% sodium chloride
    IV 10.0% leucine, 58.6% calcium lactate, 31.4% sodium chloride
    V 30.0% leucine, 65.6% calcium lactate, 4.4% sodium chloride
    VI 20.0% leucine, 77.4% calcium lactate, 2.6% sodium chloride
    VII 20.0% leucine, 70.6% calcium lactate, 9.4% sodium chloride
    VIII 33.6% leucine, 58.6% calcium lactate, 3.1% sodium chloride
    IX 25.7% leucine, 58.6% calcium lactate, 6.2% sodium chloride
    Cation Contribution
    Formulation % Ca2+ (w/w) % Na+ (w/w)
    29 13.8 2.0
    30 10.8 1.5
    III 10.8 0.8
    IV 10.8 12.4
    V 12.0 1.7
    VI 14.2 1.0
    VII 13.0 3.7
    VIII 10.8 3.1
    IX 10.8 6.2
  • The dry powders exemplified herein are referred to by formulation number and have the chemical composition disclosed in Table 5. Dry powders produced using different solution preparations, equipment, or process parameters, but that have the same chemical composition, are referred to using differing capital letters. For example, Formulation I-A and I-B have the same chemical composition but were produced using different equipment and/or process parameters.
  • Description of Placebo Used for In Vivo Experiments
  • Placebo formulations comprising either 100 wt % leucine (Placebo-A and Placebo-C) or 98 weight percent leucine with 2 weight percent sodium chloride (Placebo-B) were produced by spray drying. An aqueous phase was prepared for a batch process by dissolving leucine or leucine and sodium chloride in ultrapure water with constant agitation until the materials were completely dissolved in the water at room temperature. The solution was then spray dried using a Niro Mobile Minor spray dryer (GEA Process Engineering Inc., Columbia, Md.). The total liquid feedstock solids concentration was 15 g/L. Atomization of the liquid feed used a co-current two-fluid nozzle from Niro (GEA Process Engineering Inc., Columbia, Md.) for Placebo-A and a Spraying Systems (Carol Stream, Ill.) two-fluid nozzle with gas cap 67147 and fluid cap 2850SS for Placebo-B and Placebo-C. The liquid feed was fed using gear pumps (Cole-Parmer Instrument Company, Vernon Hills, Ill.) into a static mixer (Charles Ross & Son Company, Hauppauge, N.Y.) immediately before introduction into the two-fluid nozzle. Nitrogen was used as the drying gas. Process parameters are shown in Table 4, where the process gas inlet temperature, two-fluid atomization gas, process gas flowrate and liquid feedstock flowrate were controlled and the outlet temperature recorded. The pressure inside the drying chamber was controlled at −2 ″WC. Spray dried powders were collected from a product collection filter.
  • TABLE 6
    Placebo Spray Drying Process Conditions
    Formulation
    Process parameter Placebo-A Placebo-B, C
    Process gas inlet 282 264
    temperature (° C.)
    Process gas outlet 98 99
    temperature (° C.)
    Process gas flowrate 85 80
    (kg/hr)
    Atomization gas 242 80
    flowrate (g/min)
    Liquid feedstock 70 66
    flowrate (mL/min)
  • Example 3 Manufacturing of Liquid Feed into Dry Powder Comprised of Dry Particles
  • This example describes the preparation of dry powders using an aqueous feedstock, and the characteristics of the manufactured dry powder comprising of dry particles.
  • The feedstock was prepared as a batch by dissolving leucine in ultrapure water, then sodium chloride, and finally calcium lactate pentahydrate. The solution was kept agitated throughout the process until the materials were completely dissolved in the water at room temperature. Details on a selection of the liquid feedstock preparation are shown in Table 7, where the total solids concentration is reported as the total of the dissolved anhydrous material weights. Formulations 29-A and 30-A were prepared with separate feedstocks utilizing a Niro spray dryer.
  • TABLE 7
    Summary of liquid feedstock preparations
    of Formulations 29-A and 30-A.
    Formulation: 29-A 30-A
    Liquid feedstock mixing Batch Batch
    mixed mixed
    Total solids concentration 20 g/L 15 g/L
    Total solids 400 g 300 g
    Total volume water 20.0 L 20.0 L
    Amount leucine in 1 L 4.00 g 5.625 g
    Amount sodium chloride in 1 L 1.00 g 0.585 g
    Amount calcium lactate pentahydrate 21.19 g 12.420 g
    in 1 L
  • Formulation 29-A and 30-A dry powders were produced by spray drying on the Niro Mobile Minor spray dryer (GEA Process Engineering Inc., Columbia, Md.) with powder collection from a product filter. Atomization of the liquid feed used a Spraying Systems (Carol Stream, Ill.) two-fluid nozzle with gas cap 67147 and fluid cap 2850SS. The liquid feed was fed using gear pumps (Cole-Parmer Instrument Company, Vernon Hills, Ill.) directly into the two-fluid nozzle. Nitrogen was used as the drying gas. No humidification of the nitrogren drying gas was performed. Process parameters are shown in Table 8, where the process gas inlet temperature, two-fluid atomization gas, process gas flowrate and liquid feedstock flowrate were controlled and the outlet temperature recorded. The pressure inside the drying chamber was controlled at −2 ″WC. Spray dried powders were collected from a product collection filter.
  • TABLE 8
    Formulation Spray Drying Process Conditions
    Formulation
    Process Parameter I-A II-A III-A IV-A, B, C VI-B
    Liquid feedstock 20 15 15 15 15
    solids concen-
    tration: (g/L)
    Process gas inlet 192 212 220 265 220
    temperature: (° C.)
    Process gas outlet 89 100 99 99 99
    temperature: (° C.)
    Process gas 85 85 85 80 85
    flowrate: (kg/hr)
    Atomization gas 47 48 45 80 45
    flowrate: (g/min)
    Liquid feedstock 38 40 45 66 45
    flowrate: (mL/min)
  • Formulations 29-B and 30-B dry powders were produced by spray drying on the Büchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG, Flawil, Switzerland) with powder collection on a 60 mL glass vessel from a High Performance cyclone. The system used the Büchi B-296 dehumidifier and an external LG dehumidifier (model 49007903, LG Electronics, Englewood Cliffs, N.J.) was run constantly. Atomization of the liquid feed utilized a Büchi two-fluid nozzle with a 1.5 mm diameter. The two-fluid atomizing gas was set at 40 mm and the aspirator rate to 90%. Room air was used as the drying gas. For Formulation 29-B, inlet temperature of the process gas was 180° C. and outlet temperature at approximately 88° C. with a liquid feedstock flow rate of approximately 4.9 mL/min. The liquid feedstock solids concentration was 10 g/L in ultrapure water. For Formulation 30-B inlet temperature of the process gas was from 96° C. to 105° C. to target an outlet temperature of approximately 97° C. to 100° C. with a liquid feedstock flowrate of approximately 4.9 mL/min. The liquid feedstock solids concentration for all of these runs was 5 g/L.
  • Physical properties of the particles obtained for formulations 29-A and 30-A are summarized in Table 9. Values with ± indicates standard deviation of the value reported.
  • TABLE 9
    Chemical, Physical, and Aerosol Properties
    of Powders and Particles
    Formula- Formula-
    tion 29-A tion 30-A
    Chemical properties
    Calcium content of dry powder: 0.13 ± 0.00 0.11 ± 0.00
    (mg calcium/mg of powder; i.e.,
    %/100 w/w)
    Sodium content of dry powder: 0.02 ± 0.00 0.02 ± 0.00
    (mg sodium/mg of powder; i.e.,
    %/100 w/w)
    Physical properties
    Tap Density: (g/cc) 0.85 ± 0.01 0.71 ± 0.03
    Bulk density: (g/cc) 0.37 ± 0.19 0.29 ± 0.03
    Hausner Ratio 2.3 2.4
    Water content by K F (Karl Fischer) 2.4 ± 0.1 1.4 ± 0.0
    coulometric titration with oven:
    (with standard deviation): (% water)
    Aerosol properties
    Emitted Dose: (percentage of dose 93.8  93.8 
    emitting from inhaler/dose in
    capsule placed in inhaler): (%)
    FPF (fine particle fraction) on 25.0 ± 0.4  26.0 ± 3.1 
    ACI (Andersen Cascade Impactor)
    2-stage: (% less than 3.4 μm)
    FPFTD on ACI 2-stage: 41.7 ± 0.9  44.8 ± 2.6 
    (% less than 5.6 μm)
    MMAD (mass median aerodynamic 5.22 ± 0.21 6.29 ± 0.32
    diameter) on ACI 8-stage: (um,
    with standard deviation)
    GSD (geometric standard deviation) 2.05 ± 0.01 1.93 ± 0.03
    of MMAD on ACI 8-stage:
    Dv50 (volume median geometric 3.3 ± 1.0 2.4 ± 0.0
    diameter) on Spraytec: (μm)
    GSD of Dv50 on Spraytec: 4.1 ± 0.2 2.8 ± 0.0
    Dv50 on HELOS/RODOS at 0.2 bar: (μm) 2.54 2.7 
    Dv50 on HELOS/RODOS at 0.5 bar: (μm) 2.35 2.61
    Dv50 on HELOS/RODOS at 1.0 bar: (μm) 2.35 2.38
    GSD on HELOS/RODOS at 1.0 bar: (μm) 2.72 2.79
    Dv50 on HELOS/RODOS at 2.0 bar: (μm) 2.32 2.31
    Dv50 on HELOS/RODOS at 4.0 bar: (μm) 2.23 2.22
    Dispersibility on HELOS/RODOS at 1.05 1.07
    1 bar/4 bar:
    Dispersibility on HELOS/RODOS at 1.05 1.18
    0.5 bar/4 bar:
  • As shown in Table 9, Formulation 29 has a HELOS/RODOS dispersibility ratio at 1/4 bar and 0.5/4 bar dispersion energies of 1.05 each, while Formulation 30 has HELOS/RODOS dispersibility ratios at 1/4 bar and 0.5/4 bar dispersion energies of 1.07 and 1.18, respectively. Values that are close to 1.0, as these values are, are considered indicative that the powders are highly dispersible.
  • Example 4 Dispersibility: Emitted Mass and Particle Size of Formulations 29 and 30 as a Function of Inhaled Energy
  • This example demonstrates the dispersibility of dry powder formulations comprising calcium lactate powders when delivered from a dry powder inhaler over a range of inhalation flow rate and volumes.
  • The dispersibility of various powder formulations was investigated by measuring the geometric particle size and the percentage of powder emitted from capsules when applying an inhalation maneuver to a dry powder inhaler with flow rates representative of patient use. The particle size distribution and weight change of the filled capsules were measured for multiple powder formulations as a function of flow rate and inhaled volume and fill weight in a passive dry powder inhaler.
  • Powder formulations were filled into size 3 HPMC capsules (Capsugel V-Caps) by hand with the fill weight measured gravimetrically using an analytical balance (Mettler Toledo XS205). Fill weights of 50 mg were filled for Formulations 29-A and 30-A. A capsule based passive dry powder inhaler (RS-01 Model 7, High Resistance, Plastiape S.p.A.) was used which had specific resistances of 0.036 kPa1/2 LPM−1. Flow rate and inhaled volume were set using a timer controlled solenoid valve with flow control valve with an inline mass flow meter (TSI model 3063). Capsules were placed in the dry powder inhaler, punctured and the inhaler sealed inside a cylinder, exposing the outlet of the DPI to the laser diffraction particle sizer (Spraytec, Malvern), also referred to herein as the Spraytec laser diffraction system (SLDS), in its open bench configuration. The steady air flow rate through the system was initiated using the solenoid valve and the particle size distribution was measured via the Spraytec at lkHz for the duration of the single inhalation maneuver with a minimum of 2 seconds. Particle size distribution parameters calculated utilizing the laser diffraction particle sizer included the volume median diameter using the SLDS and called Dv50SLDS so as to avoid confusion with the HELOS/RODOS determined Dv50 and the geometric standard deviation, called GSDSLDS so as to avoid confusion with the GSD of the HELOS/RODOS Dv50 data, and the fine particle fraction of particles less than 5 micrometers in diameter, using the SLDS, and called FPFSLDS (<5.0 μm) so as to avoid confusion with the FPF as determined on the Andersen Cascade Impactor. At the completion of the inhalation duration, the dry powder inhaler was opened, the capsule removed and re-weighed to calculate the mass of powder that had been emitted from the capsule during the inhalation duration. At each testing condition, 5 replicate capsules were measured and the results of Dv50SLDS and FPFSLDS, and capsule emitted powder mass (CEPM) were averaged.
  • In order to relate the dispersion of powder at different flow rates, volumes, and from inhalers of different resistances, the energy required to perform the inhalation maneuver was calculated and the particle size and dose emission data plotted against the inhalation energy Inhalation energy was calculated as E=R2Q2V where E is the inhalation energy in Joules, R is the inhaler resistance in kPa1/2/LPM, Q is the steady flow rate in L/min and V is the inhaled air volume in L.
  • Emitted mass of Formulations 29-A and 30-A at a capsule fill weight of 50 mg using the high resistance RS-01 dry powder inhaler were determined. For each powder, a 2 L inhalation was used at the high flow rate condition of 60 LPM, corresponding to the highest energy condition of 9.2 Joules. Three other flow rates, 30, 20 and 15 LPM, were tested using an inhalation volume of 1 L. The entire mass of powder filled into the capsule emptied out of the capsule in a single inhalation for Formulation 29-A and 30-A at the highest energy condition tested. For Formulations 29-A, capsule dose emission dropped below 80% of the fill weight at 0.29 Joules. For Formulation 30-A, capsule dose emission dropped below 80% of the fill weight at 0.51 Joules.
  • The particle size distributions of the emitted powder of Formulations 29-A and 30-A are listed in Table 10, characterized by the Dv50SLDS and GSDSLDS as a function of the applied flow rate and inhalation energy. Consistent values of Dv50SLDS at decreasing energy values indicate that the powder is well dispersed since additional energy does not result in additional deagglomeration of the emitted powder. The Dv50SLDS values are consistent for all 4 Formulations with the mean Dv50SLDS increasing by less than 2 micrometers from the highest inhalation energy condition (and hence most dispersed state) down to inhalation energies of 0.29 Joules. For Formulation 29-A, the mean Dv50SLDS did not increase from baseline by 2 micrometers over the whole tested range with the maximum increase of 1.4 micrometers (from 2.1 to 3.5 micrometers) for a decrease of inhalation energy from 9.2 Joules to 0.29 Joules. In these ranges, the Dv50SLDS is not significantly increased in size, which would be expected if the emitting powder contained a lot of agglomerates and was not well dispersed.
  • TABLE 10
    Dv50SLDS, GSDSLDS and FPFSLDS as a function of Inhaled Energy
    Inhaled Energy (J), E = R2Q2V
    9.2 1.1 0.5 0.3
    Flow Rate (LPM)
    60 30 20 15
    Formu- Dv50SLDS 2 ± 0.2 3 ± 0.2 3.6 ± 0.1 5 ± 0.3
    lation (μm)
    30-A GSDSLDS 5.6 ± 0.2 4.3 ± 1 3.7 ± 0.5 3.6 ± 0.2
    FPFSLDS <  70 ± 0.8  65 ± 2.3  62 ± 1.7  50 ± 2.3
    5 μm
    Formu- Dv50SLDS 2.1 ± 0.4 2.1 ± 0.1 2.8 ± 0.1 3.5 ± 0.1
    lation (μm)
    29-A GSDSLDS 5.2 ± 0.3 4.3 ± 0.2 3.3 ± 0.3 3.3 ± 0.3
    FPFSLDS <  74 ± 1.8  74 ± 0.5  71 ± 1.2  63 ± 0.8
    5 μm
  • Example 5 Emitted Dose for Formulations 29 and 30
  • This example demonstrates that the emitted dose of dry powder formulations comprising calcium lactate powders when delivered from a dry powder inhaler is repeatable and high (i.e., greater than 80%), indicating that the powders tested readily emit from the capsule and the dry powder inhaler.
  • The uniformity of the emitted dose of four powder formulations was measured by determining the mass of calcium which exited the dry powder inhaler (DPI) and was collected in a cylindrical sampling tube. The sampling tube was 120 mm long and 35 mm in diameter based on that specified in the United States Pharmacopeia <601> and contained a 47 mm glass microfiber filter (1820-047, Whatman) at the end to collect the aerosolized powder. At the entrance of the sampling tube, a cylindrical cap, or mouthpiece adapter was connected to make an airtight seal to the DPI. Powder formulations were filled into size 3 HPMC capsules (V-Caps, Capsugel) by hand with the fill weight measured gravimetrically using an analytical balance (Mettler Toledo X5205). A fill weight of 50 mg was filled for Formulation 30-A, and a fill weight of 40 mg was filled for Formulation 29-A. A reloadable, capsule based passive dry powder inhaler (RS-01 Model 7, High Resistance, Plastiape, Osnago, Italy) was used to disperse the powder into the sampling tube. Two capsules were used for each measurement, with two actuations of 2 L of air at 60 LPM drawn through the dry powder inhaler (DPI) for each capsule. The flow rate and inhaled volume were set using a timer controlled solenoid valve with flow control valve (TPK2000 Copley Scientific). Ten replicate emitted dose measurements were performed for Formulations 29-A and 30-A. The inner surfaces of the sampling tube, mouthpiece adapter and 47 mm glass microfiber filter were rinsed with 75 mL of water and the rinse solutions assayed by HPLC for calcium ion concentration. The measured emitted calcium mass was divided by the calcium present in the filled capsules (calculated from the measured fill weight of the individual capsules and the known calcium content of the powder formulation) to give the emitted dose as a percentage. The average emitted dose and standard deviation for each formulation is shown in Table 11. The nominal calcium content of the filled doses was 11 mg of calcium for both formulations with Formulation 30-A having 2 capsules per dose with 50 mg of powder in each capsule and 10.8% calcium (w/w) in the formulation, and Formulation 29-A having 2 capsules per dose with 40 mg of powder in each capsule and 13.8% calcium (w/w) in the formulation.
  • TABLE 11
    Average emitted doses for Formulations 29 and 30
    Formulation Formulation
    29-A (n = 10) 30-A (n = 10)
    Average ED 93.8% 93.8%
    Std 0.84% 1.0%
    Ca2+ 13.8% 10.8%
    content
  • Both powders were found to have repeatable emitted doses as illustrated by the low standard deviations. High emitted dose of powder formulations from a DPI is important for minimizing the amount of powder needed to load into a DPI, both for cost of goods concerns and for maximizing the number of doses that can be contained in a given inhaler. The average emitted dose for the 2 formulations tested was 93.8% of the filled powder, which indicates that the powders are efficiently aerosolized and delivered out of the DPI with minimal residual powder being left in the DPI or capsule.
  • Example 6 Tap and Bulk Densities and Hausner Ratio for Formulations 29 and 30
  • Bulk and tapped densities were determined using a SOTAX Tap Density Tester model TD2 (Horsham, Pa.). For any given run, the entire sample was introduced to a tared 0.3 cc section of a disposable serological polystyrene micropipette (Grenier Bio-One, Monroe, N.C.) using a funnel made with weighing paper (VWR International, West Chester, Pa.) and the pipette section was plugged with polyethylene caps (Kimble Chase, Vineland, N.J.) to hold the powder. The powder mass and initial volume (V0) were recorded and the pipette was attached to the anvil and run according to the USP method for determining tap density. For the first pass, the pippette was tapped using Tap Count 1 (500 taps) and the resulting volume Va was recorded. For the second pass, Tap Count 2 was used (750 taps) resulting in the new volume Vb1. If Vb1>98% of Va, the test was complete, otherwise Tap Count 3 was used (1250 taps) iteratively until Vbn>98% of Vbn-1. Bulk density was estimated prior to tap density measurement by dividing the weight of the powder by the volume of the powder, as estimated using the volumetric measuring device. Calculations were made to determine the powder bulk density (dB), tap density (dT), and Hausner Ratio (H), which is the tap density divided by the bulk density.
  • Results for the density tests for Formulations 29 (29-A) and 30 (30-A) are shown in Table 12. The tap densities for all formulations are high (greater than 0.7 g/cc), especially for Formulation 29 (0.89 g/cc). The bulk densities are such that the Hausner ratio is quite high for Formulations 29 and 30. These Hausner Ratios are described in the art as being characteristic of powders with extremely poor flow properties (See, e.g., USP <1174>). USP <1174> notes that dry powders with a Hausner ratio greater than 1.35 are classified as poor flowing powders. Flow properties and dispersibility are both negatively affected by particle agglomeration or aggregation. It is therefore unexpected that powders with Hasuner Ratios of 1.4 to 3.2 would be highly dispersible and possess good aerosolization properties.
  • TABLE 12
    Densities and Hausner Ratio for Formulations 29 and 30
    Bulk ρ (g/cc) Tapped ρ (g/cc) Hausner
    Formulation Ave St Dev Ave St Dev Ratio
    29-A 0.37 0.19 0.85 0.01 2.3
    30-A 0.29 0.03 0.71 0.03 2.4
  • Example 7 Aerodynamic Particle Sizes for Formulations 29 and 30
  • This example demonstrates that the aerodynamic size distribution of dry powder formulations comprising calcium lactate powders when delivered from a dry powder inhaler is in a range appropriate for deposition in the respiratory tract.
  • The aerodynamic particle size distributions of four powder formulations were measured by characterizing the powders with an eight stage Anderson cascade impactor (ACI). Powder formulations were filled into size 3 HPMC capsules (Capsugel V-Caps) by hand with the fill weight measured gravimetrically using an analytical balance (Mettler Toledo X5205). A fill weight of 50 mg was filled for Formulation 30-A, and a fill weight of 40 mg was filled for Formulation 29-A. A reloadable, capsule based passive dry powder inhaler (RS-01 Model 7, High Resistance, Plastiape, Osnago, Italy) was used to disperse the powder into the cascade impactor. Two capsules were used for each measurement, with two actuations of 2 L of air at 60 LPM drawn through the dry powder inhaler (DPI) for each capsule. The flow rate and inhaled volume were set using a timer controlled solenoid valve with flow control valve (TPK2000 Copley Scientific). Three replicate ACI measurements were performed. The impactor stages, induction port (IP), entrance cone (EC) and after filter (F) were rinsed with measured volumes of water and the rinse solutions assayed by HPLC for calcium ion concentration. The size distribution, MMAD, GSD and fine particle dose <4.4 micrometers (FPD<4.4 μm) of the emitted powder was averaged across the replicates and are tabulated in Table 13. For the Formulation 30-A, the dose filled was two capsules of 50 mg powder fill weight which corresponded to 10.8 mg of Ca2+ filled into the capsules. For Formulation 29-A, the two capsules of 40 mg of powder filled contained the same 10.8 mg of Ca2+ due to that formulation's higher Ca2+ content.
  • TABLE 13
    Andersen Cascade Impactor Distributions, Fine Particle Dose, and
    Mass Median Aerodynamic Diameters for Formulations 29 and 30
    ACI Stage Formulation 29-A Formulation 30-A
    IP (+EC) (mg Ca2+) 2.64 ± 0.06 1.89 ± 0.17
    −1  (mg Ca2+) 1.27 ± 0.14 2.06 ± 0.35
    0 (mg Ca2+) 1.31 ± 0.04 2.01 ± 0.1 
    1 (mg Ca2+) 1.31 ± 0.07 1.66 ± 0.08
    2 (mg Ca2+) 0.88 ± 0.08 0.86 ± 0.01
    3 (mg Ca2+) 1.03 ± 0.09 0.86 ± 0.08
    4 (mg Ca2+) 0.56 ± 0.06 0.52 ± 0.03
    5 (mg Ca2+) 0.22 ± 0.01 0.27 ± 0.03
    6 (mg Ca2+) 0.09 ± 0.01 0.09 ± 0.01
    F (mg Ca2+)  0.1 ± 0.03 0.14 ± 0.01
    FPD < 4.4 μm (mg Ca2+) 2.88 ± 0.1  2.72 ± 0.08
    MMAD (mm) 5.22 ± 0.21 6.29 ± 0.32
    GSD 2.05 ± 0.01 1.93 ± 0.03
  • Both formulations were found to have repeatable size distributions as illustrated by the low standard deviations for all the tabulated values. All replicates had greater than 85% of the Ca2+ that was filled into the two capsules recovered in the cascade impactor. This both shows that the dosing of the formulations from the DPI is consistent and has low and consistent powder retention in the capsules and DPI as well as shows that the measured size distributions are characteristic of the full dose delivered and not just a sample of the dose. All four formulations have respirable doses as indicated in this test by the fine particle dose <4.4 micrometers that are a significant portion of the filled dose, with fine particle doses ranging from 2.0 mg to 5.4 mg of the filled 10.8 mg of calcium. With a maximum GSD of 2.1 for the four formulations, the polydispersity of the size distributions is relatively small relative to typical dry powder formulations for inhalation.
  • Example 8 Solid State Analysis Spray Drying to Generate Formulations for Solid-State Analysis
  • Formulations 29-C and 30-C were produced in a Buchi Mini Spray-Dryer B-290. For solid-state analysis, the batches were made with the process conditions in Table 14.
  • TABLE 14
    Batches of Formulation 29 and 30 manufactured
    Spray Calcium Sodium
    Drying Leucine lactate chloride Ca2+/Na+
    Formulation Conditions (% w/w) (% w/w) (% w/w) ratio
    29-C A 20.0 75.0 5.0 4:1
    30-C A 37.5 58.6 3.9 4:1
  • Materials. The raw materials utilized are listed in Table 15.
  • TABLE 15
    Materials used in the solution preparation, (Merck (Darmstadt,
    Germany) and Hovione (Sete Casas, Portugal).
    Material Supplier
    Deionized water Hovione
    Calcium lactate Merck
    L-Leucine Merck
    Sodium chloride Merck
  • Solution preparation. For each of the formulations, the following excipients, and respective quantities were used are shown in Table 16.
  • TABLE 16
    Solution composition
    Formulation 29 30
    Leucine (g) 2 3.75
    Calcium lactate•5H2O (g) 10.6 8.28
    Sodium chloride (g) 0.5 0.39
    Added water (g) 486.9 487.58
    Total solid materials (g)* 10 10
    Ca:Na ratio 4:1 4:1
    Solids concentration (% w/w) 2 2
    *Considering the water from calcium lactate.
  • Spray Drying Conditions and Post Process Characterization. The spray drying conditions and the post process results for all batches are shown in Table 17. The Dv50, also referred to as Dv(50), was measured with a Malvern Mastersizer (Worcestershire, UK) which similar to the HELOS/RODOS, measures bulk powder geometric size without the need for an inhaler to disperse the powder.
  • TABLE 17
    Summary tables of the Spray Drying conditions
    and the post process results for Formulation 29
    Formulation
    29-A 30-A
    Components
    Leucine/Calcium Lactate/Sodium Chloride
    Feed properties and spray drying parameters
    Excipients ratio % w/w 20/75/5 37.5/58.6/3.9
    (anhydrous)
    Solids (anhydrous) g 10.01 10.009
    Water g 487 488
    Solution g 500.11 500.42
    Spray-drying A A
    condition
    C_feed (1) % w/w 2.001 1.998
    Inlet temperature ° C. 136 ± 1  144 ± 1 
    (T_in)
    Outlet temperature ° C. 90 ± 1 90 ± 1
    (T_out)
    Rotameter mm 44 44
    F_atomiz (2) ml/min 16 16
    F_feed (3) ml/min 3.85 3.71
    Atomization ratio (4) 4 4
    Drying time min 130 135
    Process throughput and yield
    F_solids (5) g/min 0.08 0.07
    Yield (1st cyclone) g 6.65 7.88
    Yield (1st cyclone) % w/w 66.43 78.73
    Yield (2nd cyclone) g 0.46 not determined
    Yield (2nd cyclone) % w/w 75.9 not determined
    Post-Process Results
    Dv(50) μm 1.7 1.7
    Water by KF % w/w 1.7 1.5
    Dv(50) μm 0.7 not determined
    (2nd cyclone)
    (1) C_feed—concentration feed
    (2) F_atomiz—atomization flow
    (3) F_feed—Feed flow
    (4) Atomization ratio = F_atomiz/F_feed
    (5) F_solids—solids flow

    Modulated Differential Scanning Calorimetry (mDSC):
  • mDSC experiments were performed utilizing a DSCQ200 System from TA Instruments Inc. Approximately 10 mg of samples were placed inside hermetically sealed pans. The mDSC conditions utilized were: (i) equilibration at 0° C. and modulation with a heating rate of 2° C./min, and (ii) amplitude of 0.32° C. and period of 60 s until 250° C. Glass transition temperatures were determined by the inflection point of the step change in the reversible heat flow versus temperature curve. Using this method, the Tg of Formulation 29-C was determined to be approximately 107° C. and Formulation 30-C approximately 91° C.
  • X-Ray Powder Diffraction Data
  • Formulations 29-C and 30-C were analyzed for amorphous/crystalline content and polymorphic form using high resolution X-ray powder diffraction (XRPD). For XRPD, phase identification was performed to identify any crystalline phases observed in each XRPD pattern. XRPD patterns were collected using a PANalytical X'Pert Pro diffractometer (Almelo, The Netherlands). The specimen was analyzed using Cu radiation produced using an Optix long fine-focus source. An elliptically graded multilayer mirror was used to focus the Cu Kα X-rays of the source through the specimen and onto the detector. The specimen was sandwiched between 3-micron thick films, analyzed in transmission geometry, and rotated to optimize orientation statistics. A beam-stop was used to minimize the background generated by air scattering. Soller slits were used for the incident and diffracted beams to minimize axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) located 240 mm from the specimen. Scans were obtained over 3-60° with a step size of 0.017° and a step time of 70 s. Peaks at approximately 6, 19, 24, 31 and 33° characteristic of leucine (leucine scan not shown) were observed in the diffractogram for Formulation 30-C, indicating the presence of crystalline leucine in this powder. No crystallinity peaks characteristic of either calcium lactate pentahydrate or sodium chloride were observed in the diffractograms for either Formulations 29-C or 30-C, indicating that these components are likely present in an amorphous form in these powders.
  • Scanning Electron Microscopy (SEM)
  • SEM images were obtained of Formulation 29-B. SEM was performed using a FEI Quanta 200 Scanning Electron Microscope equipped with an Everhart Thornley (ET) detector. Images were collected and analyzed using xTm (v. 2.01) and XT Docu (v. 3.2) software. The magnification was verified using a NIST traceable standard. The sample was prepared for analysis by placing a small amount of specimen on a carbon adhesive tab supported on an aluminum mount. The sample was then sputter coated twice with Au/Pd using a Cresington 108 auto Sputter Coater at approximately 20 mA and 0.13 mbar (Ar) for 75 seconds. The samples was observed under high vacuum using a beam voltage of 5 kV. The SEM image showed that Formulation 29-B is composed of partially collapsed spherical particles with sizes ranging from approximately 0.5 to 5 μm.
  • Example 9 Efficacy of Formulations 29 and 30 on Inflammation in the TS Mouse, a Model of COPD
  • Animal models of tobacco smoke (TS) exposure have been used to study the mechanisms of TS induced COPD. Chronic exposure models (up to 6 months) generally result in mild emphysema similar to the human disease. Shorter models have been established to facilitate the testing of novel therapeutics and to evaluate acute airway inflammation following TS exposure. (Fox, J. C., S. Bolton, et al. (2007). Identification of Tobacco Smoke Models to Evaluate Acute Airway Inflammation Versus Airway Remodeling. American Thoracic Society, San Francisco, Calif., Medicherla, S., M. F. Fitzgerald, et al. (2008). “p38alpha-selective mitogen-activated protein kinase inhibitor SD-282 reduces inflammation in a subchronic model of tobacco smoke-induced airway inflammation.” J Pharmacol Exp Ther 324(3): 921-9.)
  • A. Formulation 30 Reduces COPD-Associated Inflammation
  • A study was performed to evaluate the efficacy of dry powder Formulation 30 on the pulmonary inflammation induced by TS exposure. A 4-day TS exposure model was used. Mice (C57BL6/J) were exposed to TS for up to 45 minutes per day on four successive days by whole body exposure. On each day of TS exposure, mice were treated with Formulation 30-A 1 h before and 6 h after TS exposure. Formulations 30-A dosing was performed using a whole body exposure system and a capsule based delivery system. A Placebo dry powder of 100% leucine (Placebo-C) was used as a control powder. A p38 MAP kinase inhibitor ADS110836 was used as a reference agent (WO2009/098612 Example 11) and was administered by an intranasal route.
  • Different doses of calcium were delivered by increasing the number of capsules used. Doses were calculated by collecting samples from the pie cage system onto a glass fiber filter at 1 LPM. The aerosol collected onto the filter was recovered and the calcium concentration was determined by HPLC. This data was used to calculate the aerosol concentration (Ec) of calcium ion, which was subsequently used to determine the estimated dose level. The estimated dose level (DL) is given by the equation:

  • DL=Ec·RMV·T/BW
  • where RMV is the respiratory minute volume of the animal (0.21 LPM), T is the exposure time, and BW is the body weight of the animal in kg. The resulting estimated dose is then adjusted for the respirable fraction of the aerosol, which is determined based on the fine particle fraction (FPF; % mass less than 5.6 μm).
  • Animals were euthanized by intra-peritoneal barbiturate anaesthetic overdose 24 h after the final exposure to either air (sham) or TS on day 5. A bronchoalveolar lavage (BAL) was performed using 0.4 ml of phosphate buffered saline (PBS). Cells recovered from the BAL were enumerated and differential cell counts carried out using cytospin prepared slides.
  • Inflammatory cell counts in the BAL fluid of animals exposed to TS for 4 days were determined. TS exposed animals were exposed to Formulation 30-A or a control dry powder of 100% leucine. The leucine treated animals exposed to TS exhibited a 10-fold increase in total cell counts compared to air treated animals that were also administered the control powder. The magnitude of this increase demonstrates the degree of inflammation observed after 4-days of TS exposure. Additional groups of animals were exposed to Formulation 30-A. Formulation 30-A was give b.i.d. at two different doses (3 capsules of dry powder and 6 capsules of dry powder, where each capsule contained 150 mg of dry powder). The results are summarized in Table 18.
  • TABLE 18
    Reduction of inflammation with treatment using Formulation
    30-A and a p38 Inhibitor as a positive control
    Compound
    Formulation 30-A
    3 capsules dry 6 capsules dry P38
    powder exposure powder exposure 0.1 mg/kg
    Treatment b.i.d. b.i.d. i.n. q.d.
    Inhibition % % %
    Total cells 45 58 54
    Macrophages 40 53 51
    Epithelial cells 28 49 46
    Neutrophils 67 78 68
    Eosinophils Not significantly increased following TS-exposure
    Lymphocytes 64 74 70
    All p values were <0.001 except for Formulation 30-A, 3 capsules dry powder exposure b.i.d. for the Epithelial cells, which had a p value of 0.01.
  • The data show a dose responsive result for Formulation 30-A whereby doubling the dose from 3 capsules b.i.d. to 6 capsule b.i.d. causes an increased inhibition of total inflammatory cells, macrophages, epithelial cells, neutrophils, and lymphocytes.
  • B. Formulation 29 Reduces COPD-Associated Inflammation Both Prophylactically and Therapeutically.
  • A study was performed to evaluate the efficacy of Formulation 29 on inflammation caused by tobacco smoke when administered not only prophylactically, but also therapeutically. An 11 day TS exposure model was used in which mice (C57BL6/J) were exposed to TS by whole body exposure for up to 45 minutes per day. For prophylactic dosing, mice (n=10) were treated once daily with Formulation 29 (˜1.6 mg Ca/kg) by whole body exposure 1 hour before TS exposure beginning on day 0 and continuing to day 11. For therapeutic dosing, mice (n=10) were treated as in the prophylactic regimen except that treatments were not started until day 5 (5 days after TS exposure) and continued until the end of the study (day 11). As a positive control, mice were administered a p38 MAPK inhibitor (+control; 100 μg/kg) intranasally once a day beginning on day 0. Control mice were treated with a dry powder comprised of 100% leucine. Data were analyzed by one-way ANOVA *p<0.001. TS exposure significantly increased the number of macrophages, neutrophils and lymphocytes compared to air treated animals (FIG. 5A-C). Prophylactic or therapeutic dosing with Formulation 29 significantly reduced the number of all three cell types to statistically significant levels (FIG. 5A-C). Of note, Formulation 29, when administered therapeutically (after the mice had already been exposed to TS for several days) was equally as effective at reducing macrophage, neutrophil and lymphocyte levels as the p38 MAPK inhibitor administered prophylactically from day 0.
  • Together, the data suggested that aerosol delivery of dry powder formulations comprised of calcium and sodium salts can effectively limit inflammation. The magnitude of the effect was comparable to other drugs that are known to be effective in the TS model and to the p38 MAP kinase inhibitor reference compound used in the studies. A combination therapy that includes a calcium and sodium dry powder formulation with the p38 MAPK inhibitor would likely provide enhanced efficacy and an even greater reduction in inflammation. Similarly, a combination of a calcium and sodium-containing dry powder with other drugs used for the treatment of respiratory diseases characterized by inflammation like COPD, asthma and CF would likely also provide an enhanced benefit. These other drugs include ICS, bronchodiolators (LABA/LAMA), p38 MAPK inhibitors, PDE4 inhibitors, antibody therapies, NF-κB inhibitors, and others. (Barnes, P. J. (2008). “Future treatments for chronic obstructive pulmonary disease and its co-morbidities.” Proc Am Thorac Soc 5(8): 857-64.)
  • Example 10 Dry Powders Reduce the Expression of Inflammatory Chemokines/Cytokines
  • In diseases like allergic asthma and COPD, the influx of inflammatory cells like eosinophils, macrophages, neutrophils into the airway lumen in response to environmental insult is due to cellular release of cytokines and/or chemokines. These cytokines/chemokines signal to induce the chemotaxis of inflammatory cells to the airway lumen. Using the previously described tobacco smoke (TS) mouse model of COPD, studies were undertaken to determine if the calcium-containing dry powders both reduced inflammation and modulated inflammatory cytokine/chemokine expression. Mice were exposed to TS for 4 consecutive days and treated with Formulation IV-A or Formulation 30-A once daily 1 hour before TS exposure. Control animals were exposed to a dry powder formulation of 100% leucine and a second control group was treated with leucine, but not exposed to TS. A p38 MAP kinase inhibitor ADS110836 was used as a reference agent (WO2009/098612 Example 11) and was administered by an intranasal route. At euthanasia, bronchoalveolar lavages (BAL) were performed and BAL samples were assayed for a panel of 13 different cytokines and chemokines that have a role in the inflammation. Protein levels were assessed in a multiplex assay using Luminex technology and concentrations of each protein were determined from standard curves. Data were analyzed by one-way ANOVA and the p values are shown below each group relative to the vehicle group * p<0.05. KC and MIP2 represent two key neutrophil chemokines and perform a function analogous to IL-8 in humans. KC and MIP2 expression is upregulated by exposure to TS (see FIGS. 6A-B, Leu Air versus Leu bars). Treatment with either Formulation IV-A or 30-A reduced the BAL levels of KC (FIG. 6A) and MIP2 (FIG. 6B) compared to leucine treated animals. The data were similar to the effects of these same formulations on neutrophil chemotaxis to the lung in the same animals and suggested that one mechanism by which the formulations reduce neutrophilic inflammation is through the reduction of chemokine levels that recruit these cells to the lung. These data further suggested that treatment with calcium-containing formulations modulates the biochemical and biological response of the airway epithelium and airway macrophages.
  • Example 11 Efficacy of Formulation 29 in a Mouse Model of Bacterial Pneumonia for Both Prophylaxis and Treatment
  • A mouse model of bacterial pneumonia was used to evaluate the efficacy of calcium lactate dry powder formulations in vivo. Bacteria (Streptococcus) were prepared by growing cultures on tryptic soy agar (TSA) blood plates overnight at 37° C. plus 5% CO2. Single colonies were re-suspended in sterile PBS to an optical density at 600 nm (OD600) of 0.3 in sterile PBS and subsequently diluted 1:2 in sterile PBS [˜4×107 Colony forming units (CFU)/M1]. Mice were infected with 504 of bacterial suspension (˜2×106 CFU) by intratracheal instillation while under anesthesia.
  • C57BL6 mice were treated with either Placebo-C (100% Leucine) dry powder or Formulation 29-B for in a whole-body exposure system. Dry powder aerosol was generated using a capsule based system connected to a top-loading pie chamber cage that individually holds up to 11 animals. All dry powder treatments were delivered at 10 psi and 7 scfh (≈2.8 L/min). Treatments were performed either 2 h before infection with Serotype 3 S. or 4 hours after infections. Twenty-four hours after infection mice were euthanized by pentobarbital injection and lungs were collected and homogenized in sterile PBS. Lung homogenate samples were serially diluted in sterile PBS and plated on TSA blood agar plates. Agar plates were incubated overnight at 37° C. and CFU were enumerated the following day for quantification of bacterial burden in lungs.
  • Mice treated with Formulation 29-B using either dosing regimen had reduced bacterial counts in lung homogenate samples compared to animals treated with a control dry powder (Table 19). This suggests that such formulations may be beneficial as both a preventative treatment prior to pathogen exposure or alternatively as a therapeutic after the onset of infection.
  • TABLE 19
    Prophylaxis and Treatment with Formulation 29
    Prophylaxis Treatment
    Placebo-C Formulation 29-B Placebo-C Formulation 29-B
    Mean 7.41 6.98** 7.25 6.80*
    Log10
    CFU/lung
    N = 5/group. Data were analyzed by Student t-test;
    *p < 0.05,
    **p < 0.01.
  • Example 12 Calcium-Containing Dry Powders do not Cause Airway Hyperreactivity
  • In respiratory diseases and conditions, the inhalation of foreign particles can often have adverse effects on the small airway of the lung. This can result in airway constriction leading to increased airway resistance, work of breathing and, in extreme cases, a considerable risk to the health of a patient. Thus, it is vital that inhaled therapies, particularly in the setting of inflamed or hyper reactive airways, do not result in any unintended consequences such as bronchoconstriction. Accordingly, a study was undertaken to determine whether a calcium-sodium formulation (Formulation 29) would have an adverse effect on airway bronchoconstriction. Airway resistance was assessed utilizing dual chamber plethysmography. Briefly, mice were constrained in a conical restrainer and placed in a device that consists of two sealed chambers; one encompassing the head and the other encompassing the body with an airtight seal between the two. Pneumotachs measured airflow in each individual chamber and specific airway resistance (sRaw), a direct measure of airway caliber, was calculated as a function of the time delay between flow signals. In order to precisely determine the influence of Formulation 29 on sRaw, 5 minutes of baseline sRaw measurements were obtained and the mice were subsequently exposed to a high dose of Formulation 29 (0.90 mg Ca2+/kg). Exposure of the mice to the dry powder was accomplished through the use of a whole body exposure chamber using a capsule-based dry powder inhaler system. Following treatment, 5 minutes of post-treatment sRaw measurements were obtained. Mice were then exposed to escalating doses of methacholine chloride (MCh) in 0.9% sodium chloride for inhalation via nebulization into the head chamber for 10 seconds. The experimental procedure is shown below.
  • Figure US20130164338A1-20130627-C00001
  • After each subsequent dose of MCh (0, 6.25, 12.5 25, and 50 mg/ml) the head chamber was cleared and an additional 5 minutes of sRaw was taken. The average sRaw for each 5 minute period was calculated for each animal and normalized to baseline sRaw. This was repeated for two additional groups of mice, whereby the first group was treated with 100% leucine dry power in place of Formulation 29, and the second group received a sham treatment consisting of dry air only.
  • Surprisingly, treatment with Formulation 29 (and leucine) resulted in little change in sRaw and, instead, was statistically indistinguishable from the sham treatment. In fact, when the animals were exposed to nebulized saline for inhalation (0 mg/ml MCh), the magnitude increase in sRaw was higher than that which was seen during dry powder treatment. In each group, sRaw increased with escalating MCh dose; however, at no point was there a significant difference in sRaw between treatment groups.
  • Overall, the data demonstrated that calcium dry powder treatment had little influence on sRaw in healthy non-challenged airways and that a calcium dry powder does not adversely influence airway response during periods of bronchoconstriction. Unexpectedly, 0.9% sodium chloride solution for inhalation, a widely utilized diluent for inhaled drug therapies, resulted in a larger magnitude increase in sRaw than did Formulation 29. These results clearly demonstrated that calcium-containing dry powders are not likely to inadvertently constrict small airways like some currently accepted therapies (e.g., mannitol inhalation therapy for cystic fibrosis) and could serve as a safe and effective therapy for conditions like COPD, asthma and CF.
  • Example 13 Calcium-Containing Formulations Enhance Mucociliary Clearance In Vivo
  • A liquid and a dry powder formulation were evaluated in an established sheep mucociliary clearance (MCC) model. MCC was evaluated in four healthy sheep by measurement of the clearance of pulmonary Tc99m-labeled sulfur colloid aerosols that were delivered by inhalation. Immediately following the treatment aerosol exposures, the radio-labeled sulfur colloid aerosol was delivered to each of the sheep via the same aerosol delivery system and MCC determined via the collection of serial images.
  • A Pari LC jet nebulizer operating with a single sheep exposure system was used to deliver Formulation 13-A (which is 9.4% CaCl2 (w/v), 0.62% NaCl (w/v) in water, at a concentration resulting in a tonicity factor of 8 times isotonic). The nebulizer was connected to a dosimeter system consisting of a solenoid valve and a source of compressed air (20 psi). The output of the nebulizer is connected to a T-piece, with one end attached to a respirator (Harvard Apparatus Inc., Holliston, Mass.). The system was activated for 1 second at the onset of the inspiratory cycle of the respirator, which was set at an inspiratory/expiratory ratio of 1:1 and a rate of 20 breaths/minute. A tidal volume of 300 ml was used to deliver the nebulized fomulation. The nebulizer was filled with 4 mL of Formulation 13-A and run to dryness. A dry powder, Formulation 29, was delivered with a similar exposure system but with a rotating brush generator (RBG1000, Palas) used to generate the dry powder aerosol instead of the nebulizer. A 15 minute dose of the dry powder Formulation 29 was delivered with the aerosol continuously generated by the RBG.
  • The same aerosol exposure system as the liquid treatment was used to deliver aerosolized technetium labeled sulfur colloid (99 mTC-SC) immediately after treatment. Animals were conscious, supported in a mobile restraint, intubated with a cuffed endotracheal tube and maintained conscious for the duration of the study.
  • After 99 mTC-SC nebulization, the animals are immediately extubated and positioned in their natural upright position underneath a gamma camera (Dyna Cam, Picker Corp., Nothford, Conn.) so that the field of image was perpendicular to the animals' spinal cord. After acquisition of a baseline image, serial images were obtained at 5 min intervals for the first hour. All images were obtained and stored in the computer for analysis. An area of interest was traced over the image corresponding to the right lung of the animals, and counts were recorded. The left lung was excluded from analysis because its corresponding image was superimposed over the stomach and counts could be affected by swallowed radiolabeled mucus. The counts were corrected for decay and clearance expressed as the percentage reduction of radioactivity present from the baseline image.
  • The dose delivered for both formulations was measured in-vitro with a breathing simulator system drawing the inspiratory flow through filter samples collected at the distal end of a tracheal tube. For the Formulation 29 dry powder, 10 filter samples of 1.5 minutes each were assayed for deposited calcium by HPLC and the average rate of calcium deposition was determined, From this the dose delivered in 15 minutes to a 50 kg sheep was calculated to be 0.5 mg Ca2+/kg. For the liquid Formulation 13-A, 1.5 minute filter samples were again assayed for calcium content by HPLC and the dose delivered when running the 4 mL solution to dryness was calculated for a 50 kg sheep to be 0.5 mg Ca2+/kg. These measured doses correspond to the dose delivered from the distal end of the tracheal tube to the sheep during treatment.
  • Each formulation was tested on 4 different sheep. The sheep mucociliary clearance model is a well established model with vehicle clearance typically measuring approximately 5-10% at 60 minutes after delivery of the radioactive aerosol (see for example Coote et al, 2009, DEPT 329:769-774). It is known in the art that average clearance measurements greater than about 10% at 60 minutes post baseline indicate enhanced clearance in the model. Both the dry powder Formulation 29 and the liquid Formulation 13-A show enhanced mucociliary clearance in the sheep model, with average clearances ±standard error at 60 minutes post baseline of 16.7%±2.7% and 18.9%±1.2% of baseline radioactivity respectively.
  • The rate of mucociliary clearance was found to increase over the 60 minute period post dosing. The timecourse of clearance observed for Formulations 29 and 13-A are shown in FIG. 7 for n=4 sheep for each data point. Data points indicate average values of clearance from baseline at each 5 minute interval for the four sheep per group (Mean±SEM). Mucociliary clearance was generally increasing throughout the 60 minute interval for both formulations with clearance at 20 minutes of 4.6±2.8% of baseline and 9.4±1.8% of baseline, and clearance at 40 minutes of 12.1±2.5% of baseline and 13.6±0.1% of baseline for Formulations 29 and 13-A respectively.
  • The data presented herein show that calcium salt based dry powder and hypertonic liquid formulations can be used to increase mucociliary clearance.
  • Example 14 Calcium-Containing Dry Powders that Also Contain Additional Therapeutic Agents A. Powder Preparation.
  • Feedstock solutions were prepared and used to manufacture dry powders comprised of neat, dry particles containing calcium lactate, sodium chloride, optionally leucine, and other therapeutic agents. Table 20 lists the components of the feedstock formulations used in preparation of the dry powders comprised of dry particles. Weight percentages are given on a dry basis.
  • TABLE 20
    Feedstock compositions of calcium-
    salt with other therapeutic agents
    Formula-
    tion Feedstock Composition (w/w)
    X 75.0% calcium lactate, 5.0% sodium chloride, 18.96%
    leucine, 0.91% fluticasone propionate (FP), 0.13%
    salmeterol xinafoate (SX)
    XI 75.0% calcium lactate, 5.0% sodium chloride, 15.42%
    leucine, 4.0% fluticasone propionate, 0.58% salmeterol
    xinafoate
    XII 75.0% calcium lactate, 5.0% sodium chloride, 15.31%
    leucine, 4.0% fluticasone propionate, 0.58% salmeterol
    xinafoate, 0.113% tiotropium bromide (TioB)
    XIII 75.0% calcium lactate, 5.0% sodium chloride, 18.85%
    leucine, 0.91% fluticasone propionate, 0.13% salmeterol
    xinafoate, 0.113% tiotropium bromide
    XIV 75.0% calcium lactate, 5.0% sodium chloride, 19.89%
    leucine, 0.113% tiotropium bromide
    XV 75.0% calcium lactate, 5.0% sodium chloride, 16.0%
    leucine, 4.0% fluticasone propionate
    XVI 75.0% calcium lactate, 5.0% sodium chloride, 15.89%
    leucine, 4.0% fluticasone propionate, 0.113% tiotropium
    bromide
    XVII 75.0% calcium lactate, 5.0% sodium chloride, 20%
    levofloxacin (Levo)
    XVIII 75.0% calcium lactate, 5.0% sodium chloride, 17.5%
    leucine, 2.5% Immunoglobulin G (IgG)
    XIX 75.0% calcium lactate, 5.0% sodium chloride, 19.9%
    leucine, 0.1% formoterol fumarate (FF)
    XX 75.0% calcium lactate, 5.0% sodium chloride, 18.92%
    leucine, 1.08% albuterol sulfate (AS)
  • The feedstock solutions were made according to the parameters in Table 21
  • TABLE 21
    Formulation Conditions
    Formulation:
    X XI XII XIII XIV XV
    Total solids (g) 4 5 4 4 3 4
    Total volume water 0.4 0.5 0.4 0.4 0.3 0.4
    (L)
    Amount leucine in 1.9 1.541 1.53 1.89 1.99 1.6
    1 L (g)
    Amount FP in 1 L (g) 0.091 0.4 0.4 0 0 0.4
    Amount SX in 1 L (g) 0.013 0.058 0.058 0 0 0
    Amount TioB in 1 L 0 0 0.0113 0.0113 0.0113 0
    (g)
    Amount Levo in 1 L 0 0 0 0 0 0
    (g)
    Amount IgG in 1 L (g) 0 0 0 0 0 0
    Amount FF in 1 L (g) 0 0 0 0 0 0
    Amount AS in 1 L (g) 0 0 0 0 0 0
    Formulation:
    XVI XVII XVIII XIX XX
    Total solids (g) 4 5 5 4 4
    Total volume water (L) 0.4 0.5 0.5 0.4 0.4
    Amount leucine in 1 L (g) 1.59 0 1.75 1.99 1.892
    Amount FP in 1 L (g) 0.091 0 0 0 0
    Amount SX in 1 L (g) 0 0 0 0 0
    Amount TioB in 1 L (g) 0.0113 0 0 0 0
    Amount Levo in 1 L (g) 0 2 0 0 0
    Amount IgG in 1 L (g) 0 0 0.25 0 0
    Amount FF in 1 L (g) 0 0 0 0.01 0
    Amount AS in 1 L (g) 0 0 0 0 0.108
    For all formulations, the liquid feedstock was batch mixed, the total solids concentration was 10 g/L, the amount of sodium chloride in 1 liter was 0.5 g, and the amount of calcium lactate pentahydrate in 1 liter was 10.6 g.
  • Formulation X through XX dry powders were produced by spray drying on the Büchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG, Flawil, Switzerland) with powder collection on a 60 mL glass vessel from a High Performance cyclone. The system used the Büchi B-296 dehumidifier and an external LG dehumidifier (model 49007903, LG Electronics, Englewood Cliffs, N.J.) was run constantly. Atomization of the liquid feed utilized a Büchi two-fluid nozzle with a 1.5 mm diameter. The two-fluid atomizing gas was set at 40 mm and the aspirator rate to 90%. Air was used as the drying gas and the atomization gas. Table 22 below includes details about the spray drying conditions.
  • TABLE 22
    Spray Drying Process Conditions
    Process Formulation
    Parameters X XI XII XIII XIV XV XVI XVII XVIII XIX XX
    Liquid 10 10 10 10 10 10 10 10 10 10 10
    feedstock
    solids
    concentration
    (g/L)
    Process gas 180 180 180 180 180 180 180 179-180 100 180 180
    inlet
    temperature
    (° C.)
    Process gas 87-90 73-75 73-75 74-75 84-93 76-79 76-80 91-95 55-57 80 74-78
    outlet
    temperature
    (° C.)
    Process gas 667 667 667 667 667 667 667 667 667 667 667
    flowrate
    (liter/hr,
    LPH)
    Atomization 35 28 28 28 28 28 28 35 32 28 28
    gas flowrate
    (meters3/hr)
    Liquid 9.5 10 10 10 5.2 10 9.8 5.7 2.7 5.7 5.7
    feedstock
    flowrate
    (mL/min)
  • B. Powder Characterization.
  • Powder physical and aerosol properties are summarized in Tables 23-26. Values with ±indicates standard deviation of the value reported. Table 23 shows that all formulations had an FPFTD<3.4 μm greater than 18%. Formulations X, XI, XIV, XV, XVI, XVII, XVIII, and XIX each had an FPFTD<3.4 μm greater than 25%. Formulations X, XI, XV, and XVI each had FPFTD<3.4 μm greater than 30%. All formulations had an FPFTD<5.6 μm greater than 40%. Formulations X, XI, XIV, XV, XVI, XVII, XVIII and XIX had an FPFTD<5.6 μm greater than 50%. Formulation XV had an FPFTD<5.6 μm greater than 60%. All formulations had a tapped density greater than 0.45 g/cc. Formulations X, XII, XIII, XIV, XV, XVII, XVIII, XIX, and XX each had tapped densities greater than 0.5 g/cc. Formulations X, XIII, XIV, XVII, XVIII, XIX and XX each had tapped densities greater than 0.65 g/cc. All formulations had a Hausner Ratio greater than 1.8. Formulations XII, XIV, XV, XVI, XVIII, and XIX each had a Hausner Ratio greater than 2.0. Formulations XV, XVI, and XIX each had a Hausner Ratio equal to or greater than 2.4.
  • TABLE 23
    Aerodynamic and density properties
    ACI-2 Density
    FPFTD < 3.4 μm FPFTD < 5.6 μm Bulk Tapped
    Form. % % g/cc g/cc H.R.
    X 30.48% ± 0.66% 56.85% ± 0.17% 0.34 ± 0.01 0.66 ± 0.03 1.93
    XI 30.77% ± 0.54% 56.37% ± 0.24% N/A ± N/A N/A ± N/A N/A
    XII 18.64% ± 0.79% 45.30% ± 0.29% 0.25 ± 0.09 0.51 ± 0.02 2.05
    XIII 18.37% ± 0.65% 41.29% ± 1.14% 0.36 ± 0.01 0.69 ± 0.01 1.93
    XIV 28.25% ± 1.01% 53.19% ± 0.23% 0.36 ± 0.01 0.86 ± 0.03 2.38
    XV 36.15% ± 0.55% 62.62% ± 1.83% 0.23 ± 0.02 0.58 ± 0.04 2.46
    XVI 31.34% ± 0.37% 59.34% ± 0.21% 0.18 ± 0.01 0.48 ± 0.03 2.65
    XVII 25.16% ± 1.02% 52.17% ± 1.14% 0.34 ± 0.08 0.68 ± 0.02 1.98
    XVIII 27.18% ± 1.31% 52.38% ± 1.47% 0.36 ± 0.01 0.77 ± 0.02 2.15
    XIX 27.84% ± 9.09% 52.59% ± 8.34% 0.37 ± 0.00 0.90 ± 0.09 2.40
    XX 23.78% ± 0.92% 47.71% ± 0.60% 0.40 ± 0.07 0.79 ± 0.02 1.99
    Form. = Formulation;
    H.R. = Hausner Ratio
  • Table 24 shows that all formulations had geometic diameters (Dv50) of less than 3.5 μm at a dry powder inhaler flowrate of 60 LPM. Formulations X, XIII, XIV, XV, XVI, XVII, XVIII, XIX and XX had Dv50 of less than 2.5 μm at 60 LPM. All formulations had a Dv50 of less than 6.0 μm at 15 LPM. Formulations X, XIII, XIV, XV, XVII, XVIII, XIX and XX had a Dv50 of less than 4.6 μm at 15 LPM. Formulations XIV, XV, XVII, XVIII, XIX and XX had a Dv50 of less than 4.0 μm at 15 LPM.
  • TABLE 24
    Dispersibility properties (Spraytec geometric diameters)
    Dispersibility - Spraytec
    @ 60 LPM @ 15 LPM
    Formulation Dv50 (μm) GSD Dv50 (μm) GSD
    X 2.10 ± 0.08 4.15 ± 0.45 4.38 ± 0.15 3.88 ± 0.24
    XI 2.76 ± 0.11 4.18 ± 0.50 4.93 ± 0.14 2.49 ± 0.50
    XII 3.09 ± 0.32 4.68 ± 0.16 5.95 ± 0.31 3.39 ± 0.15
    XIII 2.23 ± 0.11 4.15 ± 0.40 4.58 ± 0.12 4.19 ± 0.18
    XIV 1.92 ± 0.17 6.04 ± 0.42 2.51 ± 0.11 3.07 ± 0.40
    XV 1.95 ± 0.06 5.47 ± 0.24 3.78 ± 0.08 3.25 ± 0.16
    XVI 2.18 ± 0.08 5.24 ± 0.47 4.72 ± 0.14 3.00 ± 0.19
    XVII 2.01 ± 0.13 6.12 ± 0.45 2.83 ± 0.24 2.61 ± 0.42
    XVIII 1.80 ± 0.11 6.07 ± 0.22 2.23 ± 0.21 3.16 ± 0.55
    XIX 2.11 ± 0.12 5.38 ± 0.67 2.60 ± 0.05 3.04 ± 0.19
    XX 2.13 ± 0.08 5.83 ± 0.20 2.56 ± 0.04 3.22 ± 0.20
  • Table 25 shows that all formulations had a capsule emitted particle mass (CEPM) of greater than 94% at 60 LPM. Formulations X, XI, XII, XIV, XV, XVI, XVII, XVIII, XIX and XX each had a CEPM of greater than 97% at 60 LPM. All formulations had a CEPM of greater than 80% at 15 LPM, except XI. Formulations XII, XIV, XV, XVI, XVIII, XIX and XX each had a CEPM of greater than 90% at 15 LPM.
  • TABLE 25
    Dispersitibilty properties (CEPM)
    Dispersibility - CEPM
    @ 60 LPM @ 15 LPM
    Formulation CEPM CEPM
    X 97.48% ± 0.49% 80.33% ± 4.27%
    XI 99.09% ± 0.24%  59.92% ± 27.96%
    XII 97.19% ± 0.25% 93.15% ± 3.90%
    XIII 94.80% ± 1.53% 82.46% ± 4.61%
    XIV 97.83% ± 0.45% 95.99% ± 0.32%
    XV 98.05% ± 0.39% 92.22% ± 3.48%
    XVI 103.32% ± 2.01%  101.23% ± 2.07% 
    XVII 99.57% ± 0.00% 80.41% ± 0.32%
    XVIII 99.71% ± 0.16% 98.08% ± 0.57%
    XIX 100.22% ± 0.22%  98.06% ± 0.47%
    XX 99.87% ± 0.22% 98.10% ± 0.21%
  • Table 26 shows that all measured formulations had a Dv50 using the RODOS at its 1.0 bar setting of less than 2.5 μm. Formulations X, XIII, XIV, XV, XVI, XVII, and XVIII each had a Dv50 of less than 2.2 μm. Formulations X, XIII, XV, XVI, and XVII each had a Dv50 of less than 2.0 μm. All measured formulations had a RODOS Ratio for 0.5/4 bar of less than 1.2. All measured formulations had a RODOS Ratio for 1/4 bar of less than 1.1.
  • TABLE 26
    Dispersibility properties (Geometric diameter using RODOS)
    RODOS
    0.5 bar 1.0 bar 4.0 bar 0.5/4 1/4
    Formulation Dv50 (μm) GSD Dv50 (μm) GSD Dv50 (μm) GSD bar bar
    X 1.92 2.15 1.78 2.12 1.67 2.04 1.15 1.07
    XI N/A N/A N/A N/A N/A N/A N/A N/A
    XII 2.64 2.21 2.40 2.15 2.24 2.17 1.18 1.07
    XIII 1.87 2.12 1.95 2.17 2.36 2.13 0.79 0.83
    XIV 2.01 2.16 2.12 2.22 1.99 2.19 1.01 1.07
    XV 2.12 2.16 1.84 2.15 1.92 2.16 1.10 0.96
    XVI 2.13 2.15 1.83 2.14 1.87 2.18 1.14 0.98
    XVII 1.93 2.23 1.83 2.24 1.69 2.17 1.14 1.08
    XVIII 2.08 2.12 2.03 2.09 1.95 2.15 1.07 1.04
    XIX 2.13 2.14 2.26 2.20 2.15 2.25 0.99 1.05
    XX 2.24 2.14 2.22 2.19 2.23 2.22 1.00 1.00

    C. Anti-Inflammatory Efficacy of a Co-Formulation of a Calcium Salt with Fluticasone Propionate and Salmeterol Xinafoate (Formulation XI) in an OVA Mouse Model of Allergic Asthma
  • Formulation XI was evaluated in a mouse model of allergic asthma using ovalbumin (OVA) as an allergen. In this model, mice were sensitized to OVA over a period of two weeks, on Day 0, 7, and 14, and subsequently challenged on days 27, 28, and 29, via a liquid aerosol, with OVA. This challenge induced lung inflammation and increased airway hyperreactivity in response to an airway challenge. The principle change in inflammation was an increase in the number of eosinophils in the lungs. Similar changes in lung inflammation and pulmonary function have been observed in humans with asthma.
  • Balb/c mice were sensitized and challenged to OVA, as per the sensitization protocol described above. Mice were treated with Placebo-B dry powder (98% leucine, 2% NaCl, w/w on a dry basis), Formulation 14-A (30% leucine, 65.4% NaCl, 4.0% fluticasone propionate and 0.13% salmeterol xinafoate, w/w on a dry basis), and Formulation XI (75.0% calcium lactate, 15.31% leucine, 5.0% NaCl, 4.0% fluticasone propionate and 0.58% salmeterol xinafoate, w/w on a dry basis). Treatments were made in a whole body exposure chamber using a capsule based dry powder inhaler system. Treatment was administered BID and took place on days 27, 28, 29, and 30. On the final day of the study (day 31), mice were euthanized and bronchoalveolar lavages (BAL) were performed. The total number of cells per BAL was determined. In addition, the percentage and total number of eosinophils, neutrophils, macrophages, and lymphocytes were determined by differential staining.
  • The effect of Formulation XI on inflammation was assessed. Based on the literature, such as, (Ohta, S. et al. (2010), “Effect of tiotropium bromide on airway inflammation and remodeling in a mouse model of asthma”, Clinical and Experimental Allergy 40:1266-1275), and Riesenfeld, E. P. (2010), “Inhaled salmeterol and/or fluticasone alters structure/function in a murine model of allergic airways disease”, Respiratory Research, 11:22), fluticasone propionate (FP) is known to reduce eosinophilic cells and total cellularity in the mouse OVA model.
  • What was unknown in the art was the effect of co-formulating FP with a calcium salt formulation. Therefore, Formulation XI was tested. The results in Table 27 show that for a similar dose (mg FP/kg mouse body weight), Formulation XI was equally as efficacious in reducing eosinophilic cells and total cellularity as when the FP was formulated without the calcium salt (Formulation 14-A).
  • TABLE 27
    Formulation XI reduces eosinophilic and total cellular inflammation
    in a murine model of allergic asthma
    Placebo-B Formulation 14-A Formulation XI
    cells * cells * cells *
    106/ml Std Dev 106/ml Std Dev 106/ml Std Dev
    Eosinophils 0.55 0.27 0.11 0.10 0.11 0.09
    Total cells 1.38 .50 0.49 0.20 0.71 0.91
    (Cellularity)
  • D. Effect of Co-Formulations of a Calcium Salt and Salmeterol Xinafoate and Tiotropium Bromide (Formulations XI and XVII, Respectively) on Specific Airway Resistance in a Mouse OVA Model
  • The sensitization of mice with OVA and subsequent challenging of mice with OVA was achieved, as described in Example 14(C). In addition to changes in inflammation, mice sensitized and challenged with OVA exhibit increased airway hyperreactivity, which can be measured as changes in airway resistance following bronchoprovocation. Pulmonary function testing was conducted one hour following treatment on day 30. This involved measuring the specific airway resistance (sRaw) in the mice. Baseline sRaw measurements were taken for 5 minutes. The mice subsequently underwent a methacholine (MCh) challenge for assessing pulmonary function with escalating concentrations of MCh delivered via nebulization in a head chamber using doses of MCh of 0 mg/ml, 50 mg/ml or 100 mg/ml.
  • The mice were challenged to test their pulmonary function according to the methods described in Example 12. It was known from the literature, for example, (Schutz, N. (2004), “Prevention of bronchoconstriction in sensitized guinea pigs: efficacy of common prophylactic drugs”, Respir Physiol Neurobiol 141(2): 167-178), and Ohta, S. et al. (2010), “Effect of tiotropium bromide on airway inflammation and remodeling in a mouse model of asthma”, Clinical and Experimental Allergy 40:1266-1275), that both salmeterol xinafoate (SX) and tiotropium bromide (TioB) enhanced pulmonary function, resulting in lower sRaw values, for animals and human beings challenged with methacholine chloride (MCh) in 0.9% sodium chloride for inhalation.
  • While the effects of SX and TioB on sRaw were known from the literature, the effect of co-formulating SX and TioB formulations with a calcium salt were unknown. Formulations XI (75.0% calcium lactate, 15.31% leucine, 5.0% NaCl, 4.0% fluticasone propionate and 0.58% salmeterol xinafoate, w/w on a dry basis), XIV (75.0% calcium lactate, 19.89% leucine, 5.0% NaCl, and 0.113% tiotropium bromide, w/w on a dry basis), 14-A (30% leucine, 65.4% NaCl 4.0% fluticasone propionate and 0.13% salmeterol xinafoate, w/w on a dry basis), and 14-B (34.47% leucine, 65.42% NaCl and 0.113% tiotropium bromide, w/w on a dry basis) were tested. Non-calcium containing Formulations 14-A and 14-B were tested in order to contrast the efficacies of the calcium-containing Formulations XI and XIV, respectively. Results from pulmonary function testing are shown in FIG. 8 and FIG. 9 for Formulations XI and XIV, respectively. These data show that calcium-containing Formulation XIV matched the positive control, Formulation 14-B, and completely eliminates airway hyperreactivity in response to methacholine challenge in an OVA model of allergic asthma. Treatment with Formulation XI did not match the reduction in sRaw that Formulation 14-A achieved, however, the variability within the group treated with Formulation XI overlapped that of Formulation 14-A and the mean reduction was lower than that observed with Placebo-B.
  • E. Efficacy of Co-Formulations of a Calcium Salt with Fluticasone Propionate and Salmeterol Xinafoate (Formulation 29) in an LPS Mouse Model of Acute Lung Injury
  • In this study, a mouse model of acute lung injury was used to study the effects of calcium and sodium formulations combined with other therapeutics on pulmonary inflammation. Mice were exposed to aerosolized lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa. This challenge resulted in lung inflammation and caused changes in pulmonary function. The principle change in inflammation was an increase in the number of neutrophils in the lungs. Similar changes in lung inflammation and pulmonary function were observed in humans suffering from acute lung injury.
  • Mice were exposed to whole body exposure with nebulized LPS, 1.12 mg/ml, for 30 minutes. Treatment with dry powder Formulations XI (75.0% calcium lactate, 15.31% leucine, 5.0% NaCl, 4.0% fluticasone propionate and 0.58% salmeterol xinafoate, w/w on a dry basis) was performed 1 hour following LPS exposure using a whole body exposure chamber using a capsule based dry powder inhaler system. Animals were treated with 2, 90 mg capsules corresponding to approximately 0.32 mg Ca2+/kg delivered to the lung. To compare the influence of formulations with and without calcium salt, an additional group of animals was exposed to an equivalent amount (i.e. mg of fluticasone/kg of body mass) of an additional powder consisting of Formulation 14-A (30% leucine, 65.4% NaCl, 4.0% fluticasone propionate and 0.13% salmeterol xinafoate). A separate group of animals was treated with 2, 30 mg capsules of Placebo-B control powder (98% leucine, 2% NaCl). Three hours following dry powder treatment all mice were euthanized and underwent whole lung lavage for determination of total and differential cell counts.
  • As shown in Table 28, treatment of mice with Formulation XI significantly reduced total cell counts and neutrophils in the BAL fluid when compared with animals exposed to Placebo-B and reduced inflammatory cells to a greater extent than the calcium-free Formulation 14-A. Thus, treatment of mice with Formulation XI significantly reduced lung inflammation in an LPS model of acute lung injury.
  • TABLE 28
    Formulation XI reduces inflammation in a rodent model
    of acute lung injury
    Placebo-B Formulation 14-A Formulation XI
    cells * Std Std cells * Std
    106/ml Dev cells * 106/ml Dev 106/ml Dev
    Neutrophils 1.80 0.69 1.27 0.47 1.01 0.46
    Total cells 1.94 0.71 1.37 0.52 1.12 0.47
    (Cellularity)

    F. Anti-Bacterial Efficacy of Co-Formulations of a Calcium Salt and Levofloxacin in a Pseudomonas aeruginosa Mouse Model
  • A mouse model of bacterial infection was used to evaluate the efficacy of Formulation XVII in vivo. Neutropenia was induced by injection of cyclophosphamide (100 mg/Kg) on days −4 and −1. Bacteria (Pseudomonas aeruginosa) were grown overnight in 2 ml of Luria Bertani broth at 37 C and approximately 5000 CFU were delivered per mouse via intranasal administration in 50 μA of PBS. Four hours following infection the animals were treated with Placebo-B powder (98% leucine, 2% NaCl), Formulation 14-C (27% leucine, 52% NaCl and 20% levofloxacin), and Formulation XVII (75.0% calcium lactate, 5.0% NaCl, 20% levofloxacin) using a whole body exposure chamber using a capsule based dry powder inhaler system. The next day, animals were euthanized and the lungs and the spleen were harvested and homogenized to determine lung bacterial load and systemic bacterial load, respectively. Homogenates were serially diluted on tryptin-soyagar plates and allowed to incubate overnight at 37° C. The following day, colony forming units were counted and CFU/ml for each the lung and the spleen was calculated.
  • The results are shown in Table 29. It was seen that Formulations XVII and 14-C significantly reduced bacterial burden in the lung by more than 5 log10 CFU and in the spleen by almost 100-fold compared to the Placebo-B treated animals. Thus, treatment of mice with Formulation XVII significantly reduced lung and systemic bacterial burden during Pseudomonas aeruginosa infection. It was observed from these data that the presence of calcium in levofloxacin dry powder formulations did not have a deleterious effect on the efficacy of levofloxacin. This is a surprising result given the literature which says that magnesium and calcium based antacids deleteriously affect the bioavailability of levofloxacin taken through the gastrointestinal tract. (Flor, S. et al. (1990), “Effects of Magnesium-Aluminum Hydroxide and Calcium Carbonate Antacids on Bioavailability of Ofloxacin”, Antimicrobial Agents and Chemotherapy 34(12): 2436-2438), and (Pai, M P. et al. (2006), “Altered steady state pharmacokinteics of levofloxacin in adult cystic fibrosis patients receiving calcium carbonate”, J. Cyst. Fibros., August; 5(3):153-7). (Ofloxacin is a racemic mixture, which consists of 50% levofloxacin, which is known to be biologically active, and 50% of its enantiomer.)
  • TABLE 29
    Formulation XII reduces bacterial burden during
    Pseudomonas aeruginosa infection
    Placebo Formulation 14-C Formulation XVII
    CFU/ml Std Dev CFU/ml Std Dev CFU/ml Std Dev
    Lung 2.85 × 2.88 × 108 2.08 × 3.87 × 104 9.22 × 1.78 × 103
    108 104 103
    Spleen 1.57 × 1.78 × 105 2.16 × 6.81 × 102 2.53 × 2.41 × 103
    105 103 103
  • G. Co-Formation of a Calcium Salt and a Protein (Formulation XVIII) Provides for Delivery of the Protein Both Locally in the Lungs and Systemically
  • In this study, Formulation XVIII (75.0% calcium lactate, 17.5% leucine, 5.0% sodium chloride, 2.5% bovine immunoglobulin G (IgG), w/w on a dry basis) was used to determine if calcium containing dry powder formulations can be used to deliver proteins to the lung and if this dry powder can be used to deliver proteins systemically.
  • In this study, mice were treated with Formulation XVIII using a whole body exposure chamber using a capsule based dry powder inhaler system. Animals were then treated with 2, 4 or 6 capsules of Formulation XVIII with another group of animals were treated with 6 capsules of Placebo-B control powder (98% leucine, 2% NaCl). The placebo controls were run to ensure that there was no cross reactivity with the bovine IgG assay and native mouse proteins in either the serum or the broncho-alveolar lavage (BAL). Immediately following DP treatment the animals were euthanized, underwent BAL and serum was collected. Lavage fluid and serum were then assayed for bovine IgG using a commercially available ELISA kit.
  • The results are shown in Table 30. Placebo-B (n=3 animals, data not reported in table) was below the detectable range of the assay, which was indicative that there was no cross reactivity between the bovine IgG and the native mouse protein in either the serum or the BAL. It can be seen that IgG delivered to the lung increases stepwise with increasing number of capsules delivered to the animals. Furthermore, while treatment with 2 or 4 capsules of Formulation XVIII resulted in slight increases in serum IgG content that were in the range of the detection limit of the ELISA kit, treatment with 6 capsules IgG resulted in an increase to approximately 100 ng/ml IgG. Assuming an approximate serum volume of 2 ml, this would suggest that, on average, 200 ng of IgG was delivered systemically with 6 capsules of Formulation XVII treatment. This demonstrated that calcium-containing dry powders can be utilized to deliver proteins systemically.
  • TABLE 30
    Calcium containing, inhaled dry powders can be utilized
    to deliver proteins to the lungs and systemically
    Lung IgG Serum IgG
    IgG (ng) Std Dev IgG (ng/ml) Std Dev
    Form. XVIII (2 capsules) 100.61 39.45 3.68 6.05
    Form. XVIII (4 capsules) 148.32 28.90 6.63 10.58
    Form. XVIII (6 capsules) 274.73 72.52 107.41 49.41
    n = 6 animals each for the 2, 4, and 6 capsule groups
  • Example 15 In Vitro Biofilms Studies 3A. Biofilm Prevention Activity of Calcium Containing Dry Powder Formulations
  • An in vitro model of biofilm formation on cultured epithelial cells is used to study the effect of calcium dry powder formulations on Pseudomonas aeruginosa biofilm formation. (Anderson, G. G., S. Moreau-Marquis, et al. (2008). “In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells.” Infect and Immunology 76(4): 1423-33.) The epithelial cell cultures are derived from CF-subjects and harbor a defined mutation in CFTR. In these assays, human epithelial cell cultures are cultured on 12-well or 24-well Transwell plates under air-liquid interface conditions and treated topically with calcium dry powder formulations. The calcium dry powder formulations comprise of a calcium salt (calcium lactate), a sodium salt (sodium chloride), and leucine as an excipient. Treatment is initiated approximately 16-20 hours before the addition of bacteria to the system and a second treatment is initiated approximately 1-3 hours before the addition of Pseudomonas aeruginosa. Pseudomonas aeruginosa is added to each culture at t=0 hours and biofilm formation is monitored over time. Biofilm formation is assayed by fluorescence microscopy and by determining colony counts from wells at selected time points. Wells treated with the calcium dry powder formulations are compared to untreated wells and to leucine control treated wells. The leucine control comprises of L-leucine and does not comprise of a calcium salt.
  • 3B. Biofilm Dispersion Activity of Calcium Dry Powder Formulations
  • The ability of calcium dry powder formulations to disperse or modulate the structure of established biofilms is assessed in a similar system to that described in Example 3A. Biofilms are established on cultured epithelial cells and subsequently treated with calcium dry powder formulations. Treatments are made at multiple doses and at different time points after biofilm formation. Biofilm stability is assayed by fluorescence microscopy and by determining colony counts from wells at selected time points. As with Example 3A, the calcium dry powder formulations comprise of a calcium salt (calcium lactate), a sodium salt (sodium chloride), and leucine as an excipient. Wells treated with calcium dry powder formulations are compared to untreated wells and to leucine control treated wells. The leucine control comprises of L-leucine and does not comprise of a calcium salt.
  • In selected experiments, calcium dry powder formulations are made with D-leucine and compared to calcium dry powders formulations made with L-Leucine, as well as with a control comprising of L-leucine. In other experiments, one or both of these hypertonic calcium dry powder formulations are made with and without antibiotics.
  • The entire teachings of all documents cited herein are hereby incorporated herein by reference.
  • While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (22)

What is claimed is:
1. A method for treating cystic fibrosis, comprising administering to an individual with cystic fibrosis an effective amount of a respirable dry powder containing respirable dry particles that comprise, on a dry basis, about 20% (w/w) leucine, about 75% (w/w) calcium lactate, about 5% (w/w) sodium chloride.
2. A method for treating cystic fibrosis, comprising administering to an individual with cystic fibrosis an effective amount of a respirable dry powder containing respirable dry particles that comprise, on a dry basis,
A) about 60% to about 75% (w/w) calcium lactate, about 2% to about 5% (w/w) sodium chloride, about 15% to about 20% (w/w) leucine, and up to about 20% (w/w) of one or more additional therapeutic agents;
B) about 54.6% to about 58.6% (w/w) calcium lactate, about 1.9% to about 3.9% (w/w) sodium chloride, about 34.5% to about 37.5% (w/w) leucine, and up to about 20% (w/w) of one or more additional therapeutic agent;
C) about 75% (w/w) calcium lactate, about 5% (w/w) sodium chloride, about 0.01% to about 20% (w/w) of one or more additional therapeutic agents, and about 20% (w/w) or less leucine; or
D) about 58.6% (w/w) calcium lactate, about 3.9% (w/w) sodium chloride, about 0.01% to about 37.5% (w/w) of one or more additional therapeutic agents, and about 37.5% (w/w) or less leucine.
3. The method of claim 2 wherein the respirable dry particles are administered as an aerosol to the respiratory tract of said individual
4. The method of claim 1, wherein the respirable dry particles have a volume median geometric diameter (VMGD) of 5 microns or less as measured at the one bar dispersion setting on the HELOS/RODOS laser diffraction system.
5. The method of claim 2, wherein the respirable dry particles have a volume median geometric diameter (VMGD) between 2 and 5 microns as measured at the one bar dispersion setting on the HELOS/RODOS laser diffraction system.
6. The method of claim 2, wherein the respirable dry particles have a volume median geometric diameter (VMGD) between 1 and 3 microns as measured at the one bar dispersion setting on the HELOS/RODOS laser diffraction system.
7. The method of claim 2, wherein the respirable dry powder has a Hausner ratio of at least 1.4.
8. The method of claim 2, wherein the respirable dry powder has a dispersibility ratio at 1 bar/4 bar of less than 1.5, as measured at the 1 bar and 4 bar dispersion settings on the HELOS/RODOS laser diffraction system.
9. The method of claim 2, wherein the respirable dry powder has a dispersibility ratio at 1 bar/4 bar between 1.0 and 1.2 as measured by laser diffraction (HELOS/RODOS system).
10. The method of claim 1, wherein the respirable dry powder has a dispersibility ratio at 0.5 bar/4 bar of less than 1.5 as measured by laser diffraction (HELOS/RODOS system).
11. (canceled)
12. The method of claim 2, wherein the respirable dry powder has a Fine Particle Fraction (FPF) of less than 3.4 microns of at least 20%.
13. The method of claim 2, wherein the respirable dry powder has a Fine Particle Fraction (FPF) of less than 5.6 microns of at least 40%.
14. The method of claim 2, wherein a CEPM of at least about 80% of said respirable dry powder contained in a unit dose container, that contains 50 mg of said dry powder, in a dry powder inhaler is achieved when a total inhalation energy of less than about 1 Joule is applied to said dry powder inhaler.
15. The method of claim 1, wherein the respirable dry powder further comprises one or more additional therapeutic agents.
16. The method of claim 15, wherein the one or more additional therapeutic agents are selected from the group consisting of LABAs, short-acting beta agonists, corticosteroids, LAMAs, antibiotics, recombinant human deoxyribonuclease I, and combinations thereof.
17. The method of claim 16, where the LABA is selected from the group consisting of formoterol, salmeterol and combinations thereof; the short-acting beta agonists is albuterol; the corticosteroid is fluticasone; the LAMA is tiotropium; the antibiotic is levofloxacin; and the recombinant human deoxyribonuclease I is dornase alfa.
18. The method of claim 2, wherein the one or more additional therapeutic agents are selected from the group consisting of LABAs, short-acting beta agonists, corticosteroids, LAMAs, antibiotics, recombinant human deoxyribonuclease I, and combinations thereof.
19. The method of claim 18, where the LABA is selected from the group consisting of formoterol, salmeterol and combinations thereof; the short-acting beta agonists is albuterol; the corticosteroid is fluticasone; the LAMA is tiotropium; the antibiotic is levofloxacin, and the recombinant human deoxyribonuclease I is dornase alfa.
20. A method of reducing the formation of a biofilm in a cystic fibrosis patient comprising administering to an individual with cystic fibrosis an effective amount of a dry powder according to claim 2.
21. A method of disrupting or dispersing a biofilm in a cystic fibrosis patient comprising administering to an individual with cystic fibrosis an effective amount of a dry powder according to claim 2.
22. A method for treating or preventing an acute exacerbation of cystic fibrosis comprising administering to the respiratory tract of a patient in need thereof an effective amount of a dry powder according to claim 2.
US13/504,279 2010-08-30 2011-08-26 Treatment of cystic fibrosis using calcium lactate, leucine and sodium chloride in a respiraple dry powder Abandoned US20130164338A1 (en)

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