AU2015275224B2 - Use of aerosolized levofloxacin for treating cystic fibrosis - Google Patents

Abstract

The invention relates to methods for treating cystic fibrosis comprising administering to a human in need thereof an aerosol solution comprising levofloxacin of ofloxacin and a divalent or trivalent cation. The invention relates in particular to administering the aerosol solution to a human wherein said human has a pulmonary infection comprising P. aeruginosa.

Description

USE OF AEROSOLIZED LEVOFLOXACIN FOR TREATING CYSTIC FIBROSIS 2015275224 14 Jun2017

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/240,092 filed on September 4, 2009, and U.S. Provisional Application No. 61/249,231 filed on October 6, 2009, which are hereby expressly incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] Methods and compositions for treating cystic fibrosis are provided. In particular, compositions and methods for the use of aerosolized levofloxacin for treating cystic fibrosis are provided.

[0002a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

BACKGROUND

[0003] Patients with cystic fibrosis (CF) suffer from chronic infections of the lower respiratory tract that can be caused by bacteria, including Pseudomonas aeruginosa. Pseudomonas aeruginosa has been particularly problematic to eradicate and has been implicated as the major cause of morbidity and mortality in CF patients.

[0004] Aerosol delivery of antibiotics directly to the lungs has the potential to increase the local concentration of antibiotic at the site of infection, thereby, enhancing bacterial killing compared with systemic administration. Currently, tobramycin solution for inhalation is the only aerosol antibiotic approved for the management of CF patients with bacteria such as P aeruginosa. Because of the development of resistance to tobramycin and the limited effect on reducing bacterial density in sputum, there is a need for improved therapies to treat CF patients with pulmonary infections caused by multidrug resistant bacteria, including P aeruginosa.

[0004a] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY

[0004b] According to a first aspect the invention provides the use of an aerosol of a solution for the manufacture of a medicament for the treatment of a pulmonary Pseudomonas aeruginosa infection in a human with cystic fibrosis; wherein the solution comprises - 1 - levofloxacin at a concentration between about 75 mg/ml to about 150 mg/ml, a magnesium cation concentration between about 150 mM to about 250 mM, a pH between about 5 to about 7; and 2015275224 14 Jun2017 an osmolality of between about 300 mOsmol/kg to about 500 mOsmol/kg; and wherein the solution comprises about 240 mg levofloxacin and the aerosol is administered to the human twice daily for about 28 days.

[0004c] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0004d] According to a second aspect the invention provides a method for treating a pulmonary infection in a human having cystic fibrosis, wherein the pulmonary infection comprises Pseudomonas aeruginosa, the method comprising administering via inhalation 240 mg of levofloxacin twice daily for 28 days to the human having cystic fibrosis to treat the Pseudomonas aeruginosa pulmonary infection; wherein the levofloxacin is in an aerosol of a solution comprising levofloxacin at a concentration between about 75 mg/ml to about 150 mg/ml, a magnesium cation concentration between about 150 mM to about 250 mM, a pH between about 5 to about 7; and an osmolality of between about 300 mOsmol/kg to about 500 mOsmol/kg; and wherein the solution comprises about 240 mg levofloxacin and the aerosol is administered to the human twice daily for about 28 days.

[0005] Some embodiments of the present invention relate to compositions, use of such compositions, and methods for treating cystic fibrosis. Some such embodiments - la - include compositions and methods for the use of aerosolized levofloxacin for treating cystic fibrosis. 2015275224 22 Dec 2015 [0006] Some embodiments include methods for treating cystic fibrosis in a human in which the human has a pulmonary infection comprising P. aeruginosa. In some such embodiments, the methods includes administering to said human in need thereof an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation to achieve a reduction in the density of the P. aeruginosa in the sputum of said human by at least 40%, at least 44%, at least 70%, at least 90% and at least 97%. Some such embodiments include achieving a reduction in the density of the P. aeruginosa in the sputum of said human by at least 0.25 logio CFU/g sputum, at least 0.50 logio CFU/g sputum, at least 1.0 logio CFU/g sputum, at least 1.5 logio CFU/g sputum, and at least 1.8 logio CFU/g sputum.

[0007] Some embodiments include methods for treating cystic fibrosis in a human that include administering to the human in need thereof an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation to achieve an increase in FEVi of at least 2% and an increase in FEF 25-75 of at least 5%, an increase in FEVi of at least 5% and an increase in FEF 25-75 of at least 10%, an increase in FEVt of at least 7% and an increase in FEF 25-75 of at least 15%, and an increase in FEVi of at least 10%, and an increase in FEF 25-75 of at least 20%.

[0008] Some embodiments include achieving an increase in FEVi of at least 0.05 L and an increase in FEF 25-75 of at least 0.05 L, an increase in FEVj of at least 0.10 L and an increase in FEF 25-75 of at least 0.10 L, an increase in FEV] of at least 0.15 L and an increase in FEF 25-75 of at least 0.15 L, an increase in FEVi of at least 0.20 L and an increase in FEF 25-75 of at least 0.20 L, and an increase in FEVi of at least 0.25 L and an increase in FEF 25-75 of at least 0.25 L. Some embodiments include achieving an increase in FEF 25-75 of at least 0.27 L.

[0009] Some embodiments include methods for treating cystic fibrosis in a human that include administering to said human in need thereof an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation to achieve a hazard ratio less than 1.0, wherein the hazard ratio is indicative of a decreased need for other anti-pseudomonal antimicrobials. In some such embodiments, the hazard ratio is less than 0.8, less than 0.6, less than 0.4, and less than 0.3. -2- [ΘΘ10] Some embodiments include methods of treating cystic fibrosis in a human who is being administered an agent by inhalation selected from the group consisting of one or more of domase alpha, azithromycin, salbutamol, pancrelipase, sodium chloride, seretide, and ADEK, comprising administering to said human an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation. In some embodiments, the agent is selected from the group consisting of salbutamol, pancrelipase, seretide, and ADEK. In some embodiments, the human has a pulmonary infection comprising P. aeruginosa. Some embodiments include achieving a reduction in the density of the P. aeruginosa in the sputum of said human by at least 0.25 logio CFU/g sputum, at least 0.50 logic CFU/g sputum, and at least 1.0 logio CFU/g sputum. 2015275224 22 Dec 2015 [0011] Some embodiments include methods for treating cystic fibrosis in a human in which the human has a pulmonary infection comprising P. aeruginosa. Some such embodiments include repeatedly administering to said human in need thereof an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation, wherein said repeated administration does not result in a greater than 16-fold increase in minimum inhibitory concentration (MIC) of the P. aeruginosa strain in said human having the highest MIC relative to other P. aeruginosa strains. In some embodiments, the repeated administration does not result in a greater than 8-fold increase in minimum inhibitory concentration (MIC) of the P. aeruginosa strain in said human having the highest MIC relative to other P. aeruginosa strains. In some embodiments, repeated administration does not result in a greater than 4-fold increase in minimum inhibitory concentration (MIC) of the P. aeruginosa strain in said human having the highest MIC relative to other P. aeruginosa strains.

[0012] Some embodiments include methods for treating cystic fibrosis in a human in which the human has a pulmonary infection comprising P. aeruginosa. Some such embodiments include repeatedly administering to said human in need thereof an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation to achieve an increase in a CFQ-R respiratory domain greater than 1, greater than 2, greater than 3, greater than 4, and greater than 5.

[0013] Some embodiments include methods of decreasing small airway resistance in a human with cystic fibrosis, comprising administering to said human in need thereof an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation to achieve an increase in FEF 25-75 of at least 5%, at least 10%, at least -3- 15%, and at least 20%, Some embodiments include, achieving an increase in FEF 25-75 of at least 0.05 L, at least 0.10 L, at least 0.15 L, at least 0.20 L, at least 0.25 L, and at least 0.27 L. 2015275224 22 Dec 2015 [0014] Some embodiments include methods of administering levofloxacin or ofloxacin to a human, comprising repeatedly administering to said human an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation, wherein said repeated administration does not result in an incidence of arthralgia. In some methods, administering is repeated at least once daily for 14 days, at least once daily for 28 days, and at least once daily for 35 days. In some such methods, administering is repeated at least twice daily for 14 days, at least twice daily for 28 days, and at least twice daily for 35 days.

[0015] Some embodiments include methods for treating cystic fibrosis in a human in which the human has a pulmonary infection comprising P. aeruginosa and said Λ human has a body surface area less than 1.5 m , Some such methods include administering to said human in need thereof an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation to achieve a dose-normalized serum AUC at least 20 (ng.h/L)/mg Dose. In some embodiments, the administering is repeated daily for at least 14 days the aerosol comprises an amount of levofloxacin or ofloxacin of at least 80 mg, 120 mg, 180 mg, and 240 mg. Some embodiments include achieving a dose-normalized serum AUC at least 20 (ng.h/L)/mg Dose, at least 40 (ng.h/L)/mg Dose, at least 60 (ng.h/L)/mg Dose, at least 80 (ng.h/L)/mg Dose, and at least 100 (ng.h/L)/mg Dose.

[0016] Some embodiments include methods for treating cystic fibrosis in a human in which the human has a pulmonary infection comprising P. aeruginosa and the human has a body surface area less than 1.5 m2. Some such methods include administering to said human in need thereof an aerosol of a solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation to achieve a dose-normalized serum Cmax greater than 2 pg/L/mg administered dose greater than 4 pg/L/mg administered dose, greater than 6 pg/L/mg administered dose, greater than 8 pg/L/mg administered dose, and greater than 14 pg/L/mg administered dose. In some embodiments, the human is less than 15 years of age, less than 12 years of age, less than 10 years of age. In some embodiments, the human comprises a mass less than 55 kg, less than 45 kg, less than 35 kg, less than 25 kg. -4- [0017] In some of the foregoing embodiments, the solution consists essentially of levofloxacin or ofloxacin and the divalent or trivalent cation. 2015275224 22 Dec 2015 [0018] In some of the foregoing embodiments, the solution comprises no lactose.

[0019] In some of the foregoing embodiments, the solution comprises a divalent or trivalent cation concentration from about 50 mM to about 400 mM, and a levofloxacin or ofloxacin concentration from between about 50 mg/ml to about 200 mg/ml.

[0020] In some of the foregoing embodiments, the solution comprises a divalent or trivalent cation concentration from about 100 mM to about 300 mM, and a levofloxacin or ofloxacin concentration from between about 75 mg/ml to about 150 mg/ml.

[0021] In some of the foregoing embodiments, the solution comprises a divalent or trivalent cation concentration from about 150 mM to about 250 mM, and a levofloxacin or ofloxacin concentration from between about 90 mg/ml to about 125 mg/ml, [0022] In some of the foregoing embodiments, wherein the solution comprises an osmolality from about 300 mOsmol/kg to about 500 mOsmol/kg, and a pH from about 5 to about 8.

[0023] In some of the foregoing embodiments, the solution comprises an osmolality from about 350 mOsmol/kg to about 425 mOsmol/kg, and a pH from about 5 to about 6.5.

[0024] In some of the foregoing embodiments, the solution comprises a pH from about 5.5 to about 6.5.

[0025] In some of the foregoing embodiments, the divalent or trivalent cation is selected from magnesium, calcium, zinc, copper, aluminum, and iron.

[0026] In some of the foregoing embodiments, the solution comprises magnesium chloride.

[0027] In some of the foregoing embodiments, the solution comprises a levofloxacin or ofloxacin concentration between about 90 mg/ml to about 110 mg/ml, a magnesium chloride concentration between about 175 mM to about 225 mM, a pH between about 5 to about 7; an osmolarity of between about 300 mOsmol/kg to about 500 mOsmol/kg, and lacks lactose. -5- [Θ028] In some of the foregoing embodiments, the aerosol comprises a mass median aerodynamic diameter from about 2 microns to about 5 microns with a geometric standard deviation less than or equal to about 2.5 microns. 2015275224 22 Dec 2015 [0029] In some of the foregoing embodiments, the aerosol comprises a mass median aerodynamic diameter from about 2.5 microns to about 4.5 microns with a geometric standard deviation less than or equal to about 1.8 microns.

[0030] In some of the foregoing embodiments, the aerosol comprises a mass median aerodynamic diameter from about 2.8 microns to about 4.3 microns with a geometric standard deviation less than or equal to about 2 microns.

[0031] Some of the foregoing embodiments also include producing the aerosol with a vibrating mesh nebulizer. In some such embodiments, the vibrating mesh nebulizer is a PARI E-FLOW® nebulizer.

[0032] In some of the foregoing embodiments, the amount of levofloxacin or ofloxacin administered to the lung is at least about 20 mg, at least about 100 mg, at least about 125 mg, and at least about 150 mg [0033] In some of the foregoing embodiments, at least about 100 mg the aerosol is administered to the lung in less than about 10 minutes, less than about 5 minutes, less than about 3 minutes, less than about 2 minutes, [0034] Some of the foregoing embodiments also include co-administering an additional active agent selected from the group consisting of antibiotics, bronchodilators, anticholinergics, glucocorticoids, eicosanoid inhibitors, and combinations thereof.

[0035] In some embodiments, the antibiotic can include tobramycin, aztreonam, ciprofloxacin, azithromycin, tetracycline, quinupristin, linezolid, vancomycin, and chloramphenicol, colisitin and combinations thereof. In some embodiments, the bronchodilator can include salbutamol, levosaibuterol, terbutaline, fenoterol, terbutlaine, pirbuterol, procaterol, bitolterol, rimiterol, carbuterol, tulobuterol, reproterol, salmeterol, formoterol, arformoterol, bambuterol, clenbuterol, indacterol, theophylline, roflumilast, cilomilast, and combinations thereof. In some embodiments, the anticholinergic can include ipratropium, tiotropium, and combinations thereof. In some embodiments, the glucocorticoid can inlcude prednisone, fluticasone, budesonide, mometasone, ciclesonide, beclomethasone, and combinations thereof. In some embodiments, the eicosanoid can include montelukast, pranlukast, zafirlukast, zileuton, ramatroban, seratrodast, and -6- combinations thereof. In some embodiments, co-administering comprises inhaling the additional active agent. 2015275224 22 Dec 2015 [0036] Some of the foregoing embodiments include administering the aerosol once daily.

[0037] Some of the foregoing embodiments administering the aerosol twice daily.

[0038] In some of the foregoing embodiments, the pulmonary infection further comprises one or more bacteria that can include Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, and Pseudomonas putida, Stenotrophomonas maltophilia, Aeromonas hydrophilia, Escherichia coli, Citrobacter fi-eundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aero genes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholera, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Burkholderia cepacia, Francisella tularensis, Kingella, and Moraxella.

[0039] In some of the foregoing embodiments, the pulmonary infection further comprises a gram-negative anaerobic bacteria.

[0040] In some of the foregoing embodiments, the pulmonary infection further comprises one or more of the bacteria that can include Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, and Bacteroides splanchnicus.

[0041] In some of the foregoing embodiments, the pulmonary infection further comprises a gram-positive bacteria. .7.

[0042] In some of the foregoing embodiments, the pulmonary infection further comprises one or more of the bacteria that can include Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus milleri; Streptococcus (Group G); Streptococcus (Group C/F); Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus saccharolyticus. 2015275224 22 Dec 2015 [0043] In some of the foregoing embodiments, the pulmonary infection further comprises a gram-positive anaerobic bacteria.

[0044] In some of the foregoing embodiments, the pulmonary infection further comprises one or more bacteria that can include Clostridium difficile, Clostridium perfringens, Clostridium tetini, and Clostridium botulinum.

[0045] In some of the foregoing embodiments, the pulmonary infection further comprises an acid-fast bacteria.

[0046] In some of the foregoing embodiments, the pulmonary infection further comprises one or more bacteria that can include Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium leprae.

[0047] In some of the foregoing embodiments, the pulmonary infection further comprises an atypical bacteria.

[0048] In some of the foregoing embodiments, the pulmonary infection further comprises one or more bacteria that can include Chlamydia pneumoniae and Mycoplasma pneumoniae.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figures 1A and IB show graphs using linear axes and semi-logarithmic axes, respectively, of the arithmetic mean serum concentrations of levofloxacin after administration of single 180 mg doses as a 50 mg/ml and 100 mg/ml solution for inhalation and after administration of 240 mg as a 100 mg/ml solution for inhalation once daily for 7 days to patients with CF.

[0050] Figures 2A and 2B show graphs using linear axes and semi-logarithmic axes, respectively, of the arithmetic mean sputum concentrations of levofloxacin after administration of single 180 mg doses as a 50 mg/ml and 100 mg/ml solution for -8- inhalation and after administration of 240 mg as a 100 mg/ml solution for inhalation once daily for 7 days to patients with CF. 2015275224 22 Dec 2015 [0051] Figure 3 shows a graph of the mean change in P. aeruginosa (loglO CFU/g sputum) over time for various treatment arms in an EE patient population. Patients were administered: levofloxacin 120 mg QD, levofloxacin 240 mg QD, levofloxacin 240 mg BID, or placebo.

[0052] Figure 4 shows a graph of change in P. aeruginosa (log 10 CFU/g sputum) over time for various treatment arms in a MITT patient population. Patients were administered: levofloxacin 120 mg QD, levofloxacin 240 mg QD, levofloxacin 240 mg BID, or placebo.

[0053] Figure 5 shows a graph of survival distribution function over time for various treatment groups in an EE population.

[0054] Figure 6 shows a graph of survival distribution function over time for various treatment groups in a MITT population.

[0055] Figure 7 shows a graph of percent change in FEV i (L) at Day 28 for the EE population treated with placebo, 120 mg QD, 240 mg QD, or 240 mg BID.

[0056] Figure 8 shows a graph of percent change in FEV| (L) vs. placebo at Day 28 for the EE population treated with 120 mg QD, 240 mg QD, or 240 mg BID. *p=0.0102.

[0057] Figure 9 shows a graph of the categorical change in percent predicted FEVi at Day 28 for the EE population treated with 120 mg QD, 240 mg QD, or 240 mg BID. *p=0.0370, **p=0.0037.

[0058] Figure 10 shows a graph of dose normalized serum AUC (ng.h/L/mg Dose) vs, pediatric CF patient body weight ( 180 mg (n=7); ♦ 240 mg (n=18)). The mean value is shown for serum levofloxacin AUC in adult CF patients studied in another clinical trial (not shown) and based on a mean weight of 71 kg (A; +/- standard deviation).

[0059] Figure 11A shows a graph of dose normalized serum AUC (ng.h/L/mg Dose) vs. pediatric CF patient age ( 180 mg; ♦ 240 mg). Figure 1 IB shows a graph of dose normalized serum AUC (ng.h/L/mg Dose) vs. BSA (body surface area) (a 180 mg; ♦ 240 mg). ). Figure 11C shows a graph of dose normalized serum Cmax (pg/L/mg administered dose) vs. pediatric CF patient body weight (a 180 mg; ♦ 240 mg). Figure -9- 11D shows a graph of dose normalized serum Croax (pg/L/mg administered dose) vs. pediatric CF patient age ( 180 mg; ♦ 240 mg). Figure 11E shows a graph of dose normalized serum Cmax (pg/L/mg administered dose) vs. BSA ( 180 mg; ♦ 240 mg). 2015275224 22 Dec 2015

DETAILED DESCRIPTION

[0060] The present invention relates to the use of particular formulations of levofloxacin solutions for inhalation and particular dosage and administration regimens for the treatment of cystic fibrosis. In some embodiments, treatment of cystic fibrosis includes treatment of pulmonary infections associated with cystic fibrosis, such as P. aeruginosa, S. pneumoniae, H. influenzae, Burkholderia sp., Staphylococcus aureus, Stenotrophomonas sp., and M. catarrhalis. Methods described herein for treating cystic fibrosis can include administering formulations of levofloxacin. In some embodiments, methods for treating cystic fibrosis can also include achieving a reduction in the density of particular pathogens in the lungs of a subject. In some embodiments, methods for treating cystic fibrosis can also include improving pulmonary characteristics of a subject that can be measured with parameters such as FEVi, FEF 25-72, and the like.

[0061] The present invention provides several advantages. For example, aerosol levofloxacin provides high doses of drug directly to the lung. High doses are advantageous in reducing the development of resistant strains. In addition, the present invention provides formulations with reduced adverse effects typically associated with fluoroquinolones, such as arthralgia. Some embodiments provide methods for treating cystic fibrosis that decrease the risk of acute exacerbations in CF patients at risk for exacerbations. More embodiments provide methods for increasing the airflow in the lungs of CF patients. In various embodiments, the above methods are achieved by administering aerosolized levofloxacin or ofloxacin in dosages and administration schedules sufficient to achieve the recited result.

Definitions [0062] The term “administration” or “administering” refers to a method of giving a dosage of an antimicrobial pharmaceutical composition to a vertebrate. The preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, the site of the potential or actual bacterial infection, the microbe involved, and the severity of an actual microbial infection. In some -10- embodiments, administration can include loading an instrument to deliver a drug to a subject. In some such embodiments, administering can include loading a nebulizer with a drug to be delivered to a patient. Thus, the dosage administered may be the dosage loaded into the instrument (e.g,, nebulizer). 2015275224 22 Dec 2015 [0063] A “carrier” or “excipient” is a compound or material used to facilitate administration of the compound, for example, to increase the solubility of the compound. Solid carriers include, e.g., starch, lactose, dicalcium phosphate, sucrose, and kaolin. Liquid carriers include, e.g., sterile water, saline, buffers, non-ionic surfactants, and edible oils such as oil, peanut and sesame oils. In addition, various adjuvants such as are commonly used in the art may be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway, NJ. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, incorporated by reference herein in its entirety.

[0064] A “diagnostic” as used herein is a compound, method, system, or device that assists in the identification and characterization of a health or disease state. The diagnostic can be used in standard assays as is known in the art.

[0065] The term “mammal” is used in its usual biological sense. Thus, it specifically includes humans, cattle, horses, dogs, and cats, but also includes many other species.

[0066] The term “microbial infection” refers to the undesired proliferation or presence of invasion of pathogenic microbes in a host organism. This includes the excessive growth of microbes that are normally present in or on the body of a mammal or other organism. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host mammal. Thus, a microbial infection exists when excessive numbers of a microbial population are present in or on a mammal’s body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of a mammal.

[0067] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in -11- the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. 2015275224 22 Dec 2015 [0068] The term “pharmaceutically acceptable salt” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which are not biologically or otherwise undesirable. In many cases, the compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, naphtoic acid, oleic acid, palmitic acid, pamoic (emboic) acid, stearic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, ascorbic acid, glucoheptonic acid, glucuronic acid, lactic acid, lactobioic acid, tartaric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiaiy amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, histidine, arginine, lysine, benethamine, N-methyl-glucamine, and ethanolamine. Other acids include dodecylsufuric acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, and saccharin.

[0069] Solvate” refers to the compound formed by the interaction of a solvent and fluoroquinolone antimicrobial, a metabolite, or salt thereof. Suitable solvates are pharmaceutically acceptable solvates including hydrates.

[0070] In the context of the response of a microbe, such as a bacterium, to an antimicrobial agent, the term “susceptibility” refers to the sensitivity of the microbe for -12- the presence of the antimicrobial agent. So, to increase the susceptibility means that the microbe will be inhibited by a lower concentration of the antimicrobial agent in the medium surrounding the microbial cells. This is equivalent to saying that the microbe is more sensitive to the antimicrobial agent, In most cases the minimum inhibitory concentration (MIC) of that antimicrobial agent will have been reduced, The MIC90 can include the concentration to inhibit growth in 90% of organisms. 2015275224 22 Dec 2015 [0071] By “therapeutically effective amount” or “pharmaceutically effective amount” is meant a fluoroquinolone antimicrobial agent, as disclosed for this invention, which has a therapeutic effect. The doses of fluoroquinolone antimicrobial agent which are useful in treatment are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount means those amounts of fluoroquinolone antimicrobial agent which produce the desired therapeutic effect as judged by clinical trial results and/or model animal infection studies. In particular embodiments, the fluoroquinolone antimicrobial agent are administered in a pre-determined dose, and thus a therapeutically effective amount would be an amount of the dose administered. This amount and the amount of the fluoroquinolone antimicrobial agent can be routinely determined by one of skill in the art, and will vary, depending on several factors, such as the particular microbial strain involved. This amount can further depend upon the patient’s height, weight, sex, age and medical history. For prophylactic treatments, a therapeutically effective amount is that amount which would be effective to prevent a microbial infection.

[0072] A “therapeutic effect” relieves, to some extent, one or more of the symptoms of the infection, and includes curing an infection, “Curing” means that the symptoms of active infection are eliminated, including the total or substantial elimination of excessive members of viable microbe of those involved in the infection to a point at or below the threshold of detection by traditional measurements. However, certain longterm or permanent effects of the infection may exist even after a cure is obtained (such as extensive tissue damage). As used herein, a “therapeutic effect” is defined as a statistically significant reduction in bacterial load in a host, emergence of resistance, or improvement in infection symptoms as measured by human clinical results or animal studies.

[0073] “Treat,” “treatment,” or “treating,” as used herein refers to administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. The term “prophylactic treatment” refers to treating a patient who is not yet infected, but -13- who is susceptible to, or otherwise at risk of, a particular infection such that there is a reduced onset of infection. The term “therapeutic treatment” refers to administering treatment to a patient already suffering from an infection. Thus, in preferred embodiments, treating is the administration to a mammal (either for therapeutic or prophylactic purposes) of therapeutically effective amounts of a fluoroquinolone antimicrobial agent. 2015275224 22 Dec 2015 [0074] Pharmacokinetics (PK) is concerned with the time course of antimicrobial concentration in the body. Pharmacodynamics (PD) is concerned with the relationship between pharmacokinetics and the antimicrobial efficacy in vivo. PK/PD parameters correlate antimicrobial exposure with antimicrobial activity. The rate of killing by antimicrobial is dependent on antimicrobial mode of action and is determined by either the length of time necessary to kill (time-dependent) or the effect of increasing concentrations (concentration-dependent). To predict the therapeutic efficacy of antimicrobials with diverse mechanisms of action different PK/PD parameters may be used. PK/PD parameters may be used to determine the bioavailability of antimicrobial compositions, for example, bioavailability of a composition in the pulmonary system, and/or bioavailability of a composition in plasma/serum.

[0075] “AUC/MIC ratio” is one example of a PK/PD parameter. AUC is defined as the area under the plasma/serum or site-of-infection concentration-time curve of an antimicrobial agent in vivo (in animat or human). For example, the site of infection and/or the site where concentration is measured can include portions of the pulmonary system, such as bronchial fluid and/or sputum. Accordingly, AUC may include serum AUC, and pulmonary AUC. AUQo-t) can include the area under curve for time zero to a specific time ‘t.’ AUC(o.jnf) can include the area under curve from time zero to infinity. AUC/MIC ratio is determined by dividing the 24-hour-AUC for an individual antimicrobial by the MIC for the same antimicrobial determined in vitro. Activity of antimicrobials with the dose-dependent killing (such as fluoroquinolones) is well predicted by the magnitude of the AUC/MIC ratio.

[0076] “Cmax:MIC” ratio is another PK:PD parameter. It describes the maximum drug concentration in plasma or tissue relative to the MIC. Fluoroquinolones and aminoglycosides are examples where Cmax:MIC may predict in vivo bacterial killing where resistance can be suppressed. -14- [0077] “Time above MIC” (T>MIC) is another PK/PD parameter. It is expressed a percentage of a dosage interval in which the plasma or site-of-infection level exceeds the MIC. Activity of antimicrobials with the time-dependent killing (such as beta-lactams or oxazolidinones) is well predicted by the magnitude of the T>MIC ratio. 2015275224 22 Dec 2015 [0078] The term “dosing interval” refers to the time between administrations of the two sequential doses of a pharmaceutical’s during multiple dosing regimens. For example, in the case of orally administered ciprofloxacin, which is administered twice daily (traditional regimen of 400 mg b.i.d) and orally administered levofloxacin, which is administered once a day (500 mg or 750 mg q.d.), the dosing intervals are 12 hours and 24 hours, respectively.

[0079] As used herein, the “peak period” of a pharmaceutical’s in vivo concentration is defined as that time of the pharmaceutical dosing interval when the pharmaceutical concentration is not less than 50% of its maximum plasma or site-of-infection concentration. In some embodiments, “peak period” is used to describe an interval of antimicrobial dosing.

[0080] The “respirable delivered dose” is the amount of drug inhaled during the inspiratory phase of the breath simulator that is equal to or less than 5 microns using a simulator programmed to the European Standard pattern of 15 breaths per minute, with an inspiration to expiration ratio of 1:1.

Pharmaceutical Corn-positions [0081] For purposes of the method described herein, a fluoroquinolone antimicrobial agent formulated with a divalent or trivalent cation having improved pulmonary bioavailability may be administered using an inhaler. In some embodiments, a fluoroquinolone antimicrobial disclosed herein is produced as a pharmaceutical composition suitable for aerosol formation, good taste, storage stability, and patient safety and tolerability. In some embodiments, the isoform content of the manufactured fluoroquinolone may be optimized for tolerability, antimicrobial activity and stability.

[0082] Formulations can include a divalent or trivalent cation. The divalent or trivalent cation can include, for example, magnesium, calcium, zinc, copper, aluminum, and iron. In some embodiments, the solution comprises magnesium chloride, magnesium sulfate, zinc chloride, or copper chloride. In some embodiments, the divalent or trivalent cation concentration can be from about 25 mM to about 400 mM, from about 50 mM to -15- about 400 mM, from about 100 mM to about 300 mM, from about 100 raM to about 250 mM, from about 125 mM to about 250 mM, from about 150 mM to about 250 mM, from about 175 mM to about 225 mM, from about 180 mM to about 220 mM, and from about 190 mM to about 210 mM. In some embodiments, the magnesium chloride, magnesium sulfate, zinc chloride, or copper chloride can have a concentration from about 5% to about 25%, from about 10% to about 20%, and from about 15% to about 20%. In some embodiments, the ratio of fluoroquinolone to divalent or trivalent cation can be 1:1 to 2:1 or 1:1 to 1:2. 2015275224 22 Dec 2015 [0083] Non-limiting fluoroquinolones for use as described herein include levofloxacin, ofloxacin, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, lomefloxacin, moxifloxacin, norfloxacin, pefloxacin, sparfloxacin, garenoxacin, sitafloxacin, and DX-619.

[0084] The formulation can have a fluoroquinolone concentration, for example, levofloxacin or ofloxacin, greater than about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, and about 200 mg/ml. In some embodiments, the formulation can have a fluoroquinolone concentration, for example, levofloxacin or ofloxacin, from about 50 mg/ml to about 200 mg/ml, from about 75 mg/ml to about 150 mg/ml, from about 80 mg/ml to about 125 mg/ml, from about 80 mg/ml to about 120 mg/ml, from about 90 mg/ml to about 125 mg/ml, from about 90 mg/ml to about 120 mg/ml, and from about 90 mg/ml to about 110 mg/ml.

[0085] The formulation can have an osmolality from about 300 mOsmol/kg to about 500 mOsmol/kg, from about 325 mOsmol/kg to about 450 mOsmol/kg, from about 350 mOsmol/kg to about 425 mOsmol/kg, and from about 350 mOsmol/kg to about 400 mOsmol/kg. In some embodiments, the osmolality of the formulation is greater than about 300 mOsmol/kg, about 325 mOsmol/kg, about 350 mOsmol/kg, about 375 mOsmol/kg, about 400 mOsmol/kg, about 425 mOsmoJ/kg, about 450 mOsmol/kg, about 475 mOsmol/kg, and about 500 mOsmol/kg.

[0086] The formulation can have a pH from about 4.5 to about 8.5, from about 5.0 to about 8.0, from about 5.0 to about 7.0, from about 5.0 to about 6.5, from about 5.5 to about 6.5, and from 6.0 to about 6.5. -16- [0087] The formulation can comprise a conventional pharmaceutical carrier, excipient or the like (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like), or auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like). In some embodiments, the formulation can lack a conventional pharmaceutical carrier, excipient or the like. Some embodiments include a formulation lacking lactose. Some embodiments comprise lactose at a concentration less than about 10%, 5%, 1%, or 0.1%. In some embodiments, the formulation can consist essentially of levofloxacin or ofloxacin and a divalent or trivalent cation, 2015275224 22 Dec 2015 [0088] In some embodiments, a formulation can comprise a levofloxacin concentration between about 75 mg/ml to about 150 mg/ml, a magnesium chloride concentration between about 150 mM to about 250 mM, a pH between about 5 to about 7; an osmolality of between about 300 mOsmol/kg to about 500 mOsmol/kg, and lacks lactose.

[0089] In some embodiments, a formulation comprises a levofloxacin concentration about 100 mg/ml, a magnesium chloride concentration about 200 mM, a pH about 6.2 an osmolality about 383 mOsmol/kg, and lacks lactose. In some embodiments, a formulation consists essentially of a levofloxacin concentration about 100 mg/ml, a magnesium chloride concentration about 200 mM, a pH about 6.2 an osmolality about 383 mOsmol/kg, and lacks lactose. In some embodiments, a formulation consists of a levofloxacin concentration about 100 mg/ml, a magnesium chloride concentration about 200 mM, a pH about 6.2 an osmolality about 383 mOsmol/kg, and lacks lactose.

[0090] In some embodiments, the aerosol fluoroquinolone therapy may be administered as a treatment or prophylaxis in combination or alternating therapeutic sequence with other aerosol, oral or parenteral antibiotics. By non-limiting example this may include tobramycin and/or other aminoglycoside, aztreonam, carumonam and tigemonam and/or other beta or mono-bactam, ciprofloxacin and/or other fluoroquinolones, azithromycin and/or other macrolides or ketolides, tetracycline and/or other tetracyclines, quinupristin and/or other streptogramins, linezolid and/or other oxazolidinones, vancomycin and/or other glycopeptides, and chloramphenicol and/or -17- other phenicols, and colisitin and/or other polymyxins. In more embodiments, the antibiotic can include quinolones, tetracyclines, glycopeptides, aminoglycosides, β-lactams, rifamycins, macrolides/ketolides, oxazolidinones, coumermycins, chloramphenicol, streptogramins, trimethoprim, sulfamethoxazole, or polymyxins. In particular embodiments, an antibiotic of the above classes can be, for example, one of the following. In some embodiments, any of the foregoing antibiotics can be administered by any acceptable method or route, for example, by aerosol, orally or parenterally. 2015275224 22 Dec 2015

Beta-Lactam Antibiotics [0091] Beta-lactam antibiotics include, but are not limited to, imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefmetazole, cefoxitin, cefotetan, azthreonam, carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000, and LY206763.

Macrolides [0092] Macrolides include, but are not limited to, azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramicin, roxithromycin, and troleandomycin.

Ketolides [0093] Ketolides include, but are not limited to, telithromycin and cethrimycin. -18-

Quinolones 2015275224 22 Dec 2015 [0094] Quinolones include, but are not limited to, amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, moxifloxacin; gemifloxacin; garenofloxacin; PD 131628, PD138312, PD 140248, Q-35, AM-1155, NM394, T-3761, rufloxacin, OPC-17116, DU-6859a (see, e.g, Sato, K. et al, 1992, Antimicrob Agents Chemother. 37:1491-98), and DV-7751a (see, e.g., Tanaka, M. et al, 1992, Antimicrob, Agents Chemother, 37:2212-18).

Tetracyclines. Glvcvlcvclines and Oxazolidinones [0095] Tetracyclines, glycylcyclines, and oxazolidinones include, but are not limited to, chiortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline, linezolide, and eperozolid.

Aminoglycosides [0096] Aminoglycosides include, but are not limited to amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, and tobramycin.

Lincosamides [0097] Lincosamides include, but are not limited to, clindamycin and lincomycin.

Streptogramins [0098] Streptogramins include, but are not limited to quinupristin.

Glvcopeptides [0099] Glycopeptides include, but are not limited to vancomycin.

Polymyxins [0100] Polymyxins include but are not limited to colisitin.

[0101] More examples include fosfomycin, penicillins, cephalosporins, carbapenems, penems, and carbacephems.

[0102] In some embodiments, a formulation can include a fluoroquinolone in combination with an additional active agent. As discussed herein, some such additional active agents can include antibiotics. More additional active agents can include bronchodilators, anticholinergics, glucocorticoids, eicosanoid inhibitors, and combinations thereof. Examples of bronchodilators include salbutamol, levosalbuterol, -19- terbutaline, fenoterol, terbutlaine, pirbuterol, procaterol, bitolterol, rimiterol, carbuterol, tulobuterol, reproterol, salmeterol, formoterol, arformoterol, bambuterol, clenbuterol, indacterol, theophylline, roflumilast, cilomilast. Examples of anticholinergics include ipratropium, and tiotropium. Examples of glucocorticoids include prednisone, fluticasone, budesonide, mometasone, ciclesonide, and beclomethasone. Examples of eicosanoids include montelukast, pranlukast, zafirlukast, zileuton, ramatroban, and seratrodast More additional active agents can include pulmozyme, hypertonic saline, agents that restore chloride channel function in CF, inhaled beta-agonists, inhaled antimuscarinic agents, inhaled corticosteroids, and inhaled phosphodiesterase inhibitors. In some embodiments, the aerosol antibiotic therapy administered as a treatment or prophylaxis may be used in combination or alternating therapeutic sequence with an additional active agent. In more embodiments, the additional active agent may be administered as a treatment, alone, co-formulated, or administered with the aerosol antibiotic therapy 2015275224 22 Dec 2015

Administration [0103] The fluoroquinolone antimicrobials formulated with divalent or trivalent cations may be administered at a therapeutically effective dosage, e.g., a dosage sufficient to achieve the outcomes described herein. Similarly, the manner and schedule of administration may be selected to achieve the outcomes described herein. For example, in some embodiments, the respirable dose administered to the lungs can be from about 20 mg to about 170 mg, from about 30 mg to about 160 mg, from about 40 mg to about 150 mg, from about 50 mg to about 140 mg, and from about 65 mg to about 130 mg. In some embodiments, the loaded dose can be from about 80 mg to about 280 mg from about 90 mg to about 270 mg from about 100 mg to about 260 mg from about 110 mg to about 250 mg, and from about 120 mg to about 240 mg. In particular embodiments a dose can be administered daily, or twice daily. In some embodiments, therapy is administered for at least 28 days.

[0104] Administration of the fluoroquinolone antimicrobial agents disclosed herein or the pharmaceutically acceptable salts thereof can be via any of the accepted modes of administration for agents that serve similar utilities including, but not limited to, aerosol inhalation. Methods, devices and compositions for delivery are described in U.S. Patent Application Publication No. 2006-0276483, incorporated by reference in its -20- entirety. 2015275224 22 Dec 2015 [0105] Pharmaceutically acceptable compositions include solid, semi-solid, liquid and aerosol dosage forms, such as, for example, powders, liquids, suspensions, complexations, liposomes, particulates, or the like. Preferably, the compositions are provided in unit dosage forms suitable for single administration of a precise dose.

[0106] The fluoroquinolone antimicrobial agent can be administered either alone or in some alternatives, in combination with a conventional pharmaceutical carrier, excipient or the like (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like). If desired, the pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like). Generally, depending on the intended mode of administration, the pharmaceutical formulation will contain about 0.005% to 95%, preferably about 0.5% to 50% by weight of a compound of the invention. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0107] In one preferred embodiment, the compositions will take the form of a unit dosage form such as vial containing a liquid, solid to be suspended, dry powder, Iyophilate, or other composition and thus the composition may contain, along with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate, or the like; a lubricant such as magnesium stearate or the like; and a binder such as starch, gum acacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives or the like.

[0108] Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as defined above and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension. Solutions to be aerosolized can be prepared in conventional forms, either as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to aerosol production and inhalation. The percentage of active compound contained in such aerosol compositions is highly dependent on the specific nature thereof, -21- as well as the activity of the compound and the needs of the subject. However, percentages of active ingredient of 0.01% to 90% in solution are employable, and will be higher if the composition is a solid, which will be subsequently diluted to the above percentages. In some embodiments, the composition will comprise 1,0%-50.0% of the active agent in solution. 2015275224 22 Dec 2015 [0109] In some embodiments of the methods, compositions, and uses provided herein, the duration of a therapy, e.g., an aerosolized antibiotic therapy can include at least about 1 day /month, at least about 2 days /month, at least about 3 days /month, at least about 4 days /month, at least about 5 days /month, at least about 6 days /month, at least about 7 days /month, at least about 8 days /month, at least about 9 days /month, at least about 10 days /month, at least about 11 days /month, at least about 12 days /month, at least about 13 days /month, at least about 14 days /month, at least about 15 days /month, at least about 16 days /month, at least about 17 days /month, at least about 18 days /month, at least about 19 days /month, at least about 20 days /month, at least about 21 days /month, at least about 22 days /month, at least about 23 days /month, at least about 24 days /month, at least about 25 days /month, at least about 26 days /month, at least about 27 days /month, at least about 28 days /month, at least about 29 days /month, at least about 30 days /month, and at least about 31 days /month.

[0110] Compositions described herein can be administered with a frequency of about 1, 2, 3, 4, or more times daily, 1, 2, 3, 4, 5, 6, 7 or more times weekly, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times monthly. In particular embodiments, the compositions are administered twice daily.

Aerosol delivery [0111] For pulmonary administration, the upper airways are avoided in favor of the middle and lower airways. Pulmonary drug delivery may be accomplished by inhalation of an aerosol through the mouth and throat. Particles having a mass median aerodynamic diameter (MMAD) of greater than about 5 microns generally do not reach the lung; instead, they tend to impact the back of the throat and are swallowed and possibly orally absorbed. Particles having diameters of about 2 to about 5 microns are small enough to reach the upper- to mid-pulmonary region (conducting airways), but are too large to reach the alveoli. Smaller particles, i.e., about 0.5 to about 2 microns, are capable of reaching the alveolar region. Particles having diameters smaller than about 0.5 -22- microns can also be deposited in the alveolar region by sedimentation, although very small particles may be exhaled. 2015275224 22 Dec 2015 (0112] In one embodiment, a nebulizer is selected on the basis of allowing the formation of an aerosol of a fluoroquinolone antimicrobial agent disclosed herein having an MMAD predominantly between about 2 to about 5 microns. In one embodiment, the delivered amount of fluoroquinolone antimicrobial agent provides a therapeutic effect for respiratory infections. The nebulizer can deliver an aerosol comprising a mass median aerodynamic diameter from about 2 microns to about 5 microns with a geometric standard deviation less than or equal to about 2.5 microns, a mass median aerodynamic diameter from about 2.5 microns to about 4.5 microns with a geometric standard deviation less than or equal to about 1.8 microns, and a mass median aerodynamic diameter from about 2.8 microns to about 4.3 microns with a geometric standard deviation less than or equal to about 2 microns. In some embodiments, the aerosol can be produced using a vibrating mesh nebulizer. An example of a vibrating mesh nebulizer includes the PARI E-FLOW® nebulizer. More examples of nebulizers are provided in U.S. Patent Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911; 4,510,929; 4,624,251; 5,164,740; 5,586,550; 5,758,637; 6,644,304; 6,338,443; 5,906,202; 5,934,272; 5,960,792; 5,971,951; 6,070,575; 6,192,876; 6,230,706; 6,349,719; 6,367,470; 6,543,442; 6,584,971; 6,601,581; 4,263,907; 5,709,202; 5,823,179; 6,192,876; 6,644,304; 5,549,102; 6,083,922; 6,161,536; 6,264,922; 6,557,549; and 6,612,303 all of which are hereby incorporated by reference in their entireties, More commercial examples of nebulizers that can be used with the formulations described herein include Respirgard II®, Aeroneb®, Aeroneb® Pro, and Aeroneb® Go produced by Aerogen; AERx® and AERx Essence™ produced by Aradigm; Porta-Neb®, Freeway Freedom™, Sidestream,, Ventstream and I-neb produced by Respironics, Inc.; and PARI LC-Plus®, PARI LC-Star®, produced by PARI, GmbH. By further non-limiting example, U.S. Patent No. 6,196,219, is hereby incorporated by reference in its entirety.

[0113] The amount of levofloxacin or ofloxacin that can be administered (as a respirable dose, nebulizer loaded dose, and/or deposited dose) can include at least about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, -23- about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, and about 800 mg. 2015275224 22 Dec 2015 [0114] The aerosol can be administered to the lungs in less than about 10 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, and about 1 minute.

Indications [0115] Methods and compositions described herein can be used to treat pulmonary infections and disorders, for example, cystic fibrosis. Some embodiments include treating an infection comprising one or more bacteria that can include Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas sp,, eg., Stenotrophomonas maltophilia, Aeromonas hydrophilia, Escherichia coli, Citrohacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteuretta haemolytica, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholera, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeas, Neisseria meningitidis, Burkholderia sp., e.g., Burkholderia cepacia, Francisella tularensis, Kingella, and Moraxella. In some embodiments, the pulmonary -24- infection, can include a gram-negative anaerobic bacteria. In more embodiments, the pulmonary infection can include one or more of the bacteria selected from the group consisting of Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, and Bacteroides splanchnicus. In some embodiments, the pulmonary infection can include a gram-positive bacteria. In some embodiments, the pulmonary infection can include one or more of the bacteria selected from the group consisting of Corynebacterium diphtheriae, Corynebacterium ulcerans. Streptococcus pneumoniae. Streptococcus agalactiae. Streptococcus pyogenes, Streptococcus milleri; Streptococcus (Group G); Streptococcus (Group C/F); Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus saccharolyticus. In some embodiments, the pulmonary infection can include a gram-positive anaerobic bacteria. In some embodiments, the pulmonary infection can include one or more bacteria selected from the group consisting of Clostridium difficile, Clostridium perfringens, Clostridium tetini, and Clostridium botulinum. In some embodiments, the pulmonary infection can include an acid-fast bacteria. In some embodiments, the pulmonary infection can include one or more bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium leprae. In some embodiments, the pulmonary infection can include an atypical bacteria. In some embodiments, the pulmonary infection can include one or more bacteria selected from the group consisting of Chlamydia pneumoniae and Mycoplasma pneumoniae. In some embodiments, the pulmonary infection can comprise a non-fermenting gram-negative bacteria (NFGNB). Examples of NFGNB can include Burkholeria spp., stenotrophomonas spp., acinetobacter spp., pseudomonas spp., and Achromobacter spp. More example of bacteria useful in the methods and compositions provided herein can be found in ‘Bergey's Manual of Systematic Bacteriology,’ Editor-in-chief: Garrity, George M. Boone, David R.; Castenholz, Richard W. (Eds.) Originally published by Williams & Wilkins, 1984, 2nd ed., (2001), incorporated by reference in its entirety. 2015275224 22 Dec 2015 [0116] Some methods and compositions provided here can be especially appropriate for the treatment of pulmonary infections and disorders that include microbial -25- strains that can be difficult to treat using an antimicrobial agent delivered parenterally due to the need for high parenteral dose levels, which can cause undesirable side effects, or due to lack of any clinically effective antimicrobial agents. For example, administration of an aerosol fluoroquinolone antimicrobial directly to the site of infection may reduce systemic exposure and can maximize the amount of antimicrobial agent to the site of microbial infection. Such methods can be appropriate for treating infections involving microbes that are susceptible to fluoroquinolone antimicrobials as a way of reducing the frequency of selection of resistant microbes. 2015275224 22 Dec 2015 [0117] In some embodiments, the aerosol fluoroquinolone antimicrobial agent formulated with divalent or trivalent cations is administered at a level sufficient to overcome the emergence resistance in bacteria or increase killing efficiency such that resistance does not have the opportunity to develop.

[0118] Some embodiments of the methods and compositions described herein include achieving a reduction in a pulmonary infection. A reduction in a pulmonary infection can be measured using a variety of different methods. For example, in a pulmonary infection comprising one or more organisms, a reduction in the density of the organism can be measured. In some embodiments, treatment can achieve a reduction in the density of an organism by at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%. In some embodiments, treatment can achieve a reduction in the density of an organism by at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 100%.

[0119] The density of an organism can be measured in a sample taken from a subject, for example, bronchial alveolar lavage, sputum, and serum. In some embodiments the density of an organism can be reduced by at least about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.8, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5 logjo CFU/g sputum, or more.

[0120] Some embodiments of the methods and compositions described herein can include achieving an improvement in a pulmonary function parameter. Examples of -26- such parameters can include FEV (forced expiratory volume), FEVi (forced expiratory volume in 1 second), and FEF 25-75 (forced expiratory flow 25-75%). In some embodiments, the FEV] of a subject can be increased using the methods and compositions described herein, by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, and more. In some embodiments, the FEV] of a subject can be increased using the methods and compositions described herein, by at least about 0.01 L, 0.02 L, 0.03 L, 0.04 L, and 0.05 L, and by at least about 0.1 L, 0.2 L, 0,3 L, 0.4 L, 0.5 L, 0.6 L, 0.7 L, 0.8 L, 0.9L, 1.0 L, and more. 2015275224 22 Dec 2015 [0121] In some embodiments, the FEF 25-75 of a subject can be increased using the methods and compositions described herein, by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%. In some embodiments, the FEF 25-75 of a subject can be increased using the methods and compositions described herein, by at least about 0.01 L, 0.02 L, 0.03 L, 0.04 L, and 0.05 L, and by at least about 0.1 L, 0.2 L, 0.3 L, 0.4 L, 0.5 L, 0.6 L, 0.7 L, 0.8 L, 0.9L, 1.0 L, and more.

[0122] Some embodiments of the methods and compositions described herein can include reducing the need for a subject to need other inhaled or systemic antibiotics, such as anti-pseudomonal antimicrobials. Such a reduction can be measured by a variety of methods, for example, by the increase in time to need other inhaled or systemic antibiotics. A reduction in such a need can be measured by a variety of statistical means. For example, hazard ratios may be used in a survival analysis. In some embodiments, the hazard ratio is less than about 1.0, 0.9, 0.8, 0.7, 0.6,0.5, 0.4, 0.3, 0.2,0.1, and less.

[0123] Some embodiments of the methods and compositions described herein can include decreasing the frequency of exacerbations, the severity of exacerbations, the duration of exacerbations, or the likelihood that an exacerbation will occur. An exacerbation can be defined by any of several methods and criteria provided by such methods. In some embodiments, a patient can concurrently meet at least 4 symptoms/signs of the Fuchs definition of an exacerbation (Fuchs HJ, et al. Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. N Engl J Med 1994;331:637-642, incorporated by reference in its entirety). The symptoms/signs defined by the Fuchs criteria include: change in sputum; new or increased hemoptysis; increased cough; -27- increased dyspnea; malaise, fatigue or lethargy; temperature above 38°C; anorexia or weight loss; sinus pain or tenderness; change in sinus discharge; change in physical examination of the chest; decrease in pulmonary function by 10 % or more from a previously recorded value; and radiographic changes indicative of pulmonary infection 2015275224 22 Dec 2015 [0124] In some embodiments, a patient with an improved exacerbation profile can have at least 1, at least 2, at least 3, and at least 4 of the following signs/symptoms, where changes can be relative to a patient’s typical experience, for example daily experience, and weekly experience. (1) Change in sputum, e.g., for sputum production: patients have no change, a little less or much less amounts of sputum when coughing up, or for change in sputum appearance: for sputum thickness, patients have a little thinner or much thinner sputum; for sputum color, patients have a better color of sputum (better increases from brown -> green -> yellow -> clear). (2) Hemoptysis, e.g., patients have a little decrease or a large decrease in the amount of blood coughed up. (3) Cough, e.g., for intensity of cough, patients have a little lighter, or much lighter coughs; for frequency of cough, patients cough a little less often or much less often. (4) Dyspnea, e.g., for dyspnea with exertion, patients breathe a little easier or much easier when performing daily activities. (5) Malaise, fatigue or lethargy, e.g., patients have a little more energy or much more energy, and/or patients perform daily activities, e.g., climbing stairs, a little easier, or much easier, (6) Temperature, e.g., patients have normal healthy temperature e.g., about 37°C, or patients have no recent history of fever. (7) Anorexia or weight loss, e.g., patients have no change in weight, or a little weight gain, and/or patients have a little increase in appetite (8) Sinus pain or tenderness, e.g., patient has no sinus pain or tenderness, or less sinus pain or tenderness. (9) Change in sinus discharge, e.g., patients have better sinus discharge (a decrease in thickness and/or better color). (10) Change in physical examination of the chest, e.g., patients have improved signs on examination of the chest and may report for example, a little decrease chest congestion, or a large decrease in chest congestion. -28- (11) Pulmonary function by 10 % or more from a previously recorded value, e.g., patients have improved pulmonary function in pulmonary function tests. 2015275224 22 Dec 2015 (12) Radiographic changes indicative of pulmonary infection, e.g. patients show improved radiographic changes indicating reduced pulmonary infection.

[0125] In some embodiments, exercise tolerance and/or absenteeism from scheduled events, e.g., school or work can be measured as signs/symptoms of exacerbations.

[0126] Table 1 summarizes a variety of methods useful to measure exacerbations. TABLE 1

Sign/ Symptom Method / Protocol Fuchs Ramsey CFF Rosenfeld (Seattle)* Kraynack6 Rabin Biummer Cough X X X X X* X X Sputum X X X X X X X Hemoptysis X X X Dyspnea X X X Exercise Tolerance X X X X Absenteeism X X X X Fatigue X X X Fever X X X X Decreased/Loss Appetite X X X X X X X Sinus pain X X Sinus discharge X Chest exam X X X X X X Tachypnea X X X -29- 2015275224 22 Dec 2015

Sign/ Symptom Method / Protocol Fuchs Ramsey CFF Rosenfeid (Seattle}* Kraynack0 Rabin Blummer Lung function X X X (X) X X X CXR X X X X Sa02 X X Neutrophiia X Criteria 4/12 plus antibiotics 2/5 plus 1/3 3/11 Score Score 3/4 3/4 A: Rosenfeid M. ef a/,, Defining a pulmonary exacerbation in cystic fibrosis. J. of Pediatrics 139:359-365(2001), incorporated by reference in its entirety. B: Kraynack N.C. et a!., Improving care at cystic fibrosis centers through quality improvement. Semin Respir Crit Care Med. 2009 Oct;30(5):547-58), incorporated by reference in its entirety.

[0127] Some embodiments of any of the above methods include administering levofloxacin or ofloxacin in combination with a divalent or trivalent cation in a dosage amount, administration schedule, and/or method of administration sufficient to achieve the above recited outcomes.

Pediatric patient populations [0128] Some embodiments provided herein relate to the use of the compositions provided herein to treating cystic fibrosis in a human. In some embodiments, the human is a pediatric patient. In some embodiments, the human has an age less than about 18 years, less than about 17 years, less than about 16 years, less than about 15 years, less than about 14 years, less than about 13 years, less than about 12 years, less than about 11 years, less than about 10 years, less than about 9 years, less than about 8 years, less than about 7 years, less than about 6 years, less than about 5 years, less than about 4 years, less than about 3 years, less than about 2 years, and less than about 1 year.

[0129] Dosages of aerosol therapeutic agents for pediatric subjects can be less than the dosage for an adult subject. In some embodiments, dosage can be determined, in part, due to the weight of a subject. For example, a subject having a weight from about 14 kg to about 21 kg can receive a dose of about 120 mg, a subject having a weight from -30-about 22 kg to about 30 kg can receive a dose of about 180 mg dose, and a subject having a weight more than about 30 kg can receive a dose of about 240 mg. In some embodiments, the aerosol therapeutic agent can be administered daily, or twice daily. In some embodiments, the aerosol therapeutic agent can be administered for a period of at least about 1 day, 3 days, 5 days, 10 days, 15 days, 20 days, and 30 days. In some embodiments, the aerosol therapeutic agent can be administered for about 14 days. In particular embodiments the aerosol therapeutic agent is administered daily for 14 days. 2015275224 22 Dec 2015 [0130] In some embodiments, the human has a body weight less than about 70 kg, less than about 60 kg, less than about 50 kg, less than about 40 kg, less than about 30 kg, less than about 20 kg, and less than about 10 kg.

[0131] In some embodiments, the human has a body surface area less than about 1.8 m2, less than about 1.6 m2, less than about 1.4 m2, less than about 1.2 m2, less than about 1.0 m2, less than about 0.8 m2, less than about 0.6 m2, and less than about 0.4 m .

[0132] In some embodiments, treating one of the above humans comprises achieving a dose-normalized serum AUC at least about 5 (ng.h/L)/mg Dose, at least about 10 (ng.h/L)/mg Dose, at least about 20 (ng.b/L)/mg Dose, at least about 40 (ng.h/L)/mg Dose, at least about 60 (ng.h/L)/mg Dose, at least about 80 (ng.h/L)/mg Dose, and at least about 100 (ng.h/L)/mg Dose.

[0133] In some embodiments, treating one of the above humans comprises achieving a dose-normalized serum Cmax greater than about 1 pg/L/mg administered dose, greater than about 2 pg/L/mg administered dose, greater than about 3 pg/L/mg administered dose, greater than about 4 pg/L/mg administered dose, greater than about 5 pg/L/mg administered dose, greater than about 6 pg/L/mg administered dose, greater than about 7 pg/L/mg administered dose, greater than about 8 pg/L/mg administered dose, greater than about 9 pg/L/mg administered dose, greater than about 10 pg/L/mg administered dose, greater than about 11 pg/L/mg administered dose, greater than about 12 pg/L/mg administered dose, greater than about 13 pg/L/mg administered dose, greater than about 14 pg/L/mg administered dose, greater than about 15 pg/L/mg administered dose, and greater than about 16 pg/L/mg administered dose.

[0134] Some embodiments of any of the above methods for treating the recited humans include administering levofloxacin or ofloxacin in combination with a divalent or -31-trivalent cation in a dosage amount, administration schedule, and/or method of administration sufficient to achieve the recited outcomes. 2015275224 22 Dec 2015

EXAMPLES

Example 1—Phase lb clinical study with levofloxacin [0135] A Phase lb single-blind, placebo-control, dose-escalating multicenter study was carried out to evaluate the safety, tolerability, and pharmacokinetic (PK) profile of aerosolized levofloxacin administered to stable CF patients. All patients had had P. aeruginosa cultured from sputum within the previous 24 months and at the screening visit Study drug was administered twice daily for up to 14 days at three doses by aerosol using a PARI eFlow device. Respirable delivered doses (RDD) were approximately 40 mg, 80 mg, and 120 mg per treatment, corresponding to loaded doses of 78 mg, 175 mg, and 260 mg, respectively. Thus, the estimated total daily RDDs were 80 mg, 160 mg, and 240 mg. Study drugs were administered at 30 mg/ml (for 40 mg dose) or 50 mg/ml (for 80 mg and 120 mg doses). Table 2 shows the formulations of the study drugs. TABLE2 30 mg/ml Levofloxacin solution 50 mg/ml Levofloxacin solution Placebo Levofloxacin, mg/ml (mM) 30 (81.6) 50 (136) 0 Magnesium, mg/ml (mM) 1.5 (60) 2.4 (100) 2.4(100) Chloride, mg/ml (mM) 4.3 (120) 7.1 (200) 7.1 (200) Lactose, mg/ml (mM) 51.4 (150) 51.4(150) 51.4 (150) pH 6.3 6.3 6.5 Osmolality, mOsm/kg 314 400 424 [0136] All patients used at least 1 concomitant medication during the study. Concomitant medications are those medications taken after the first dose of Study Drug regardless of medication start date. Concomitant medications used by at least 20% of patients included Salbutamol, Domase alfa, Azithromycin, Seretide, Pancrelipase, Adeks, Hypertonic solutions, and Nortase. -32-

Efficacy results 2015275224 22 Dec 2015 {0137] Table 3 summarizes the results for pulmonary function tests for FEVi measurements. The greatest relative improvement in FEV] after 7 and 14 days of dosing was observed in the 120 mg RDD (260 mg loaded) levofloxacin cohort, with a mean improvement of 14.79% and 17.58%, respectively. A mean improvement of 8.90% persisted over the 2 weeks to the follow-up visit. In addition, there appeared to be a dose response in patients receiving levofloxacin with patients in the 80 mg and 120 mg dose groups having an improvement in the FEVi, while those in the 40~mg dose group did not. All 9 patients in the 120 mg dose group had a relative increase in FEVj, with 4 of 9 patients having at least a 20% increase. TABLE3

Median FEVi change (%) for each Treatment Group Time point Placebo 40 mg RDD Levofloxacin 80 mg RDD Levofloxacin 120 mg RDD Levofloxacin Day 7 n=10 n=1Q n=10 n=9 5.75 -0.75 6.15 10.20 Day 14 n=8 1.20 n=10 -1.55 n=10 2.95 n=9 16.90 [0138] Table 4 show the relative changes from baseline across visits (Day 1,2, 7, 14, and 21) in pulmonary function tests for FEVi. In Table 3, relative change from baseline (CBG) was calculated as ‘Result5 FEVi minus ‘Baseline’ FEVi divided by ‘Baseline’ FEV]. TABLE4 FEVi (L) for each Treatment Group Time point Placebo N=10 40 mg RDD Levofloxacin N=10 80 mg RDD Levofloxacin N=10 120 mg RDD Levofloxacin N=1Q Day 1 Predose (Baseline) n 10 10 10 10 Mean (SD) 2.62(1.150) 2.26 (0.534) 2.06 (0.649) 2.56(1.121) Median 2.46 2.30 1.95 2.23 Minimum, maximum 1.31,4.90 1.58, 3.50 1.17, 3.19 1.43, 4.71 Day 2 Predose Result n 10 10 10 10 -33- 2015275224 22 Dec 2015 FEVi (L) for each Treatment Group Time point Placebo N=10 40 mg RDD Levofloxacin N=10 80 mg RDD Levofloxacin N=10 120 mg RDD Levofloxacin N=1Q Mean (SD) 2.75(1.310) 2.31 (0.594) 2.10(0.709) 2.73(1.132) Median 2.43 2.32 2.00 2.38 Minimum, maximum 1.33, 5.50 1.57, 3.60 1.13, 3.46 1.40, 4.72 Day 2 Relative CFB (%) n 10 10 10 10 Mean (SD) 3.85 (6.627) 1.92 ¢8.315) 1,57 (5.149) 7.29(10.081) Median 2.00 -0.30 1.90 6.60 Minimum, maximum -7.10, 12.20 -6.30, 21,70 -7.60, 8.90 -4.70, 23.90 Day 7 Result n 10 10 10 9 Mean (SD) 2.82(1.361) 2.30 (0.544) 2.26 (0.831) 3.07(1.220) Median 2.35 2.30 2.08 2.40 Minimum, maximum 1.39, 5.20 1.49, 3.48 1.20, 3.79 1.79, 4.99 Day 7 Relative CFB (%) n 10 10 10 9 Mean (SD) 5.86(10.196) 1.60 (6.985) 9.42(18.911) 14.79 (12.865) Median 5.75 -0.75 6.15 10.20 Minimum, maximum -6.50, 29.40 -5.70, 17.90 -7.40, 59.90 3.40, 37.70 Day 14 Predose Result n 8 10 10 9 Mean (SD) 2.99(1.272) 2.26 (0.524) 2.09 (0.616) 3.12(1.173) Median 3.11 2.30 2.10 2.57 Minimum, maximum 1.47, 5.20 1.55, 3.38 1.19, 3.21 1.74, 4.78 Day 14 Relative CFB {%) n 8 10 10 9 Mean (SD) 2.00 (5.529) 0.21 (8.440) 2.69(10.125) 17.58(15.089) Median 1.20 -1.55 2.95 16.90 Minimum, maximum -6.70, 8.80 -8.70, 23.10 -19.40, 19.10 1.50, 4150 Day 21/ET Result n 10 10 10 10 -34- 2015275224 22 Dec 2015 FEV-t (L) for each Treatment Group Time point Placebo N=10 40 mg RDD Levofloxacin N=10 80 mg RDD Levofloxacin N=10 120 mg RDD Levofloxacin N=10 Mean (SD) 2.75(1.327) 2.16 (0.517) 2.03 (0.632) 2.75(1.155) Median 2.28 2.07 2.04 2.48 Minimum, maximum 1.17, 5.10 1.46, 3.42 1.06, 2.82 1.49, 4.87 Day 21/ET Relative CFB (%) n 10 10 10 10 Mean (SD) 3.20 (10.157) -4.25 (7.092) -1.03 (8.880) 8.90(15.789) Median 1.75 -5.80 -3.10 10.10 Minimum, maximum -10.70, 20.60 -12.70, 11.80 -11.60, 12.90 -15.90, 38.70 ET = early termination.

[0139] Colony-forming units of P aeruginosa at Day 1 were compared with colony-forming units at Day 7 and 14. Table 5 summarizes the percentage change in sputum P, aeruginosa (log CFU/g) in treatment groups. Declines in sputum P. aeruginosa were observed over all days of dosing. TABLE 5

Time point Median Change in sputum P. aeruginosa (log CFU/g) for Treatment Group Placebo Levofloxacin solution Day 7 0.04 (n=9) -0.50 (n=21) Day 14 0.04 (n=9) -1.23 (n=21)

Safety results [0140] No study drug-related serious adverse events were reported. Most adverse events were mild or moderate in severity and self-limiting. The majority of adverse events were mild, with taste complaints, cough and headache as the most commonly observed adverse events. No patients receiving levofloxacin solution for inhalation met drug intolerability criteria. Adverse events reported in more than 1 patient receiving levofloxacin included abdominal pain, cough, disease progression (acute exacerbation), dysgeusia (bad taste), haemoptysis, headache, nasal congestion, nasopharyngeal pain (sore throat), respiratory tract congestion, and wheezing. Table 6 -35-summarizes adverse events observed in more than one CF patient receiving levofloxacin. Accordingly, these results demonstrated the safety and tolerability of levofloxacin with multiple doses over 14 days. TABLE 6

Adverse event Treatment group 40 mg Levofloxacin (N=10) SO mg Levofloxacin (N=10) 120 mg Levofloxacin (N=10) Placebo (N=10) Abdominal pain 1 (10%) — 1 (10%) — Alopecia — 1 (10%) — — Blood blister -- — 1 (10%) — Breath sounds abnormal — - 1 (10%) — Chest discomfort ~ 1 (10%) — 1 (10%) Cough — 4 (40%) 4 (40%) 1 (10%) Diarrhoea 1 (10%) — -- — Disease progression — - 2 (20%) 1 (10%) Drooling 1 (10%) Dysguesia 3 (30%) 6 (60%) 5 (50%) 1 (10%) Dyspnoea 1 (10%) ~ .. — Fatigue — 1 (10%) .. — Flank pain 1 (10%) — - — Forced expired volume decreased — 1 (10%) — 1 (10%) Hemoptysis — 1 (10%) 1 (10%) - Headache — 2 (20%) 2 (20%) -- Migraine - 1 (10%) — — Nasal congestion - 1 (10%) 1 (10%) — Non-cardiac chest pain -- 1 (10%) — — Oral candidiasis — 1 (10%) — — Paraethesia oral - — 1 (10%) — 2015275224 22 Dec 2015 -36- 2015275224 22 Dec 2015

Adverse event Treatment group 40 mg Levofloxacin (N=10) 80 mg Levofloxacin (N=10) 120 mg Levofloxacin (N=10) Placebo (N=10) Pharyngolaryngeal pain — -- 2 (20%) - Pulmonary function test decreased — ~ 1 (10%) - Pyrexia — - 1 (10%) - Rash erythematous — 1 (10%) - Respiratory tract congestion — — 2 (20%) — Retching — 1 (10%) — - Rhinorrhoea 1 (10%) — — Thirst — 1 (10%) — - Upper respiratory tract infection 1 (10%) — — 1 (10%) Wheezing 1 (10%) — I (10%) —

Example 2—Phase 2 clinical study with levofloxacin [0141] A phase 2, multi-center, randomized, double-blind, placebo-controlled study was carried out to evaluate the safety, tolerability and efficacy of three dosage regimens of levofloxacin formulated with MgCh administered for 28 days to stable CF patients. The following dosage regimens (as nebulizer loaded doses) were evaluated: 120 mg QD (daily); 240 mg QD (daily); and 240 mg BID (twice daily). Based on aerosol characterization studies of the 100 mg/ml formulation, these loaded doses of 120 and 240 mg correspond estimated respirable delivered doses (RDDs) using a PARI eFlow nebulizer of about 65 and 130 mg, respectively. The plasma levofloxacin CmiK and AUC with all dosage regimens chosen for this study should provide concentrations in pulmonary tissues well in excess of those associated with bactericidal activity against CF pathogens (data not shown). -37- [0142] The formulation of levofloxacin solution and placebo is shown in Table 7. Study drug was administered by aerosol using the PARI eFIow® device with a mesh to deliver a particle size smaller than approximately 3,5 pm - 4.0 pm. TABLE 7

Levofloxacin Placebo Levofloxacin, mg/ml (mM) 100(272) 0 Mg, mg/ml (mM) 5.0 (200) 0 Cl, mg/ml (mM) 14.4 (400) 0 pH 5-7 6-8 Osmolality, mOsm/kg 350 - 500 300-500 Saline - 0.9 % 2015275224 22 Dec 2015 [0143] Patient inclusion criteria included: (1) at least 16 years of age; (2) clinically diagnosed with CF; (3) able to elicit an FEV1 > 25% but < 85% predicted value at Screening; (4) received at least 3 courses of inhaled antimicrobials over the preceding 12 months and had received at least 1 course of inhaled tobramycin/(TOBl®)/colistin in the 2 months prior to Visit 1 (Day 1), but none in the 28 days prior to Visit 1 (Day 1); (5) had a sputum specimen at Screening positive for P. aeruginosa and a history of at least 1 positive sputum culture positive for P. aeruginosa within the last 18 months; and (6) clinically stable with no significant changes in health status within the last 30 days.

[0144] Patient exclusion criteria included: (1) use of an investigational agent within 30 days prior to Visit 1 (Day 1); (2) use of any nebulized or systemic antibiotics active against P. aeruginosa within 28 days prior to Visit 1 (Day 1), other than maintenance oral azithromycin, which must have been initiated at least 30 days prior to Visit 1 (Day 1); (3) hypersensitivity to fluoroquinolones or excipients of levofloxacin formulated with MgCh; (4) intolerance to bronchodilators or unwilling to use a bronchodilator during the study; (5) use of oral corticosteroids in doses exceeding the equivalent of 10 mg prednisone/day or 20 mg prednisone every other day; (6) changes in physiotherapy technique or schedule within 14 days prior to Visit 1 (Day 1); (7) changes in medical regimen for treatment of CF (e.g., introduction, dose escalation, or elimination of therapies such as dornase alfa, nonsteroidal anti-inflammatory agents, azithromycin, hypertonic saline, or inhaled corticosteroids) within 30 days of Visit 1 (Day l); (8) history -38-of lung transplantation; (9) evidence of acute upper respiratory tract infection within 10 days or lower respiratory tract infection within 30 days prior to Visit 1 (Day 1); (10) pregnancy, breastfeeding, or unwilling to practice birth control or abstinence during participation in the study (women only); (11) history of seizures or low seizure threshold (e.g., epilepsy); (12) renal dysfunction (calculated creatinine clearance [CrCl] < 50 mL/min) at Screening; (13) aspartate aminotransferase (AST), alanine aminotransferase (ALT), or total bilirubin > 3 x upper limit of normal (ULN) at Screening or evidence of severe liver disease (e.g,, cirrhosis, portal hypertension); (14) history of human immunodeficiency virus (HIV), hepatitis B, or hepatitis C infection/seropositivity; (15) history of hemoptysis > 30 mL over any 24-hour period during the 30 days prior to Visit 1; (16) oxygen saturation < 90% on room air at Screening or Visit 1 (Day 1); and (17) > 15% relative decline in FEV| (L) from Screening to Visit 1 (Day 1). 2015275224 22 Dec 2015 [0145] Patients were assessed on Day 1, 7, 14, and 28, and then up to 28 days after completion of study drug, namely, Days 42 and 56. The formulation of study drug, i.e., levofloxacin, is shown in Table 7. The end of the study was defined as the last visit of the last patient. Study populations included: (1) safety/modified intent to treat (MITT) population which included all patients enrolled in the study that receive at least one dose of study drug; (2) efficacy evaluable (EE) population which included all patients enrolled in the study without major protocol violations that receive at least 80% of study drug doses during the 28 days of study drug therapy; and (3) pharmacokinetic (PK) population which included all patients that receive at least one dose of study drug and have at least one PK blood sample collected.

[0146] Approximately 32 patients per treatment arm provided 80% power to detect a difference between treatment arms using a 2-sided analysis of covariance (ANCOVA), with a 5% alpha, assuming a standard deviation (SD) of 1,5 and a mean log CFU change in P. aeruginosa of 0.75 decrease, 0.75 decrease, no change and 0,25 increase for the levofloxacin 240 mg BID, levofloxacin 240 mg QD, levofloxacin 120 mg QD, and placebo treatment aims, respectively. Sample size calculations assumed a 10% discontinuation rate from the study; therefore patients who discontinue the study were not replaced.

[0147] Efficacy was evaluated by microbiologic assessment of sputum samples, time to need for anti-pseudomonal antimicrobials, the Cystic Fibrosis Questionaire-Revised (CFQ-R), and pulmonary function tests. In addition, a primary -39-efficacy comparison tested Ho: the average decline in log CFUs of P. aeruginosa from the start of levofloxacin or placebo administration (Day 1) to four weeks later is equal for all four groups, versus Hp the average decline in log CFUs is different for at least one of the four groups using a repeated-measures mixed effects model adjusting for baseline levofloxacin minimum inhibitory concentration (MIC) as a continuous variable (log base 2 transformed), baseline lung function, and geographical region (U.S. versus ex-U.S,). Pair-wise comparisons between all the treatment arms were conducted as secondary analyses. 2015275224 22 Dec 2015 [0148] Efficacy endpoints including clinical endpoints, pulmonary function tests, and additional microbiological parameters were assessed as change from Day 1 to subsequent visits where the endpoint data was collected. Time to need for intravenous/oral/inhaled anti-pseudomonal antimicrobials were assessed from Day 1 until final visit using survival analysis. The primary population for efficacy analysis was the EE population, but efficacy endpoints were also analyzed using the MITT population.

[0149] Patients enrolled in the study had a median of 5 courses of aerosolized antibiotics over the previous 12 months. The baseline FEV1 (as percent predicted) was 53% across the entire study and not different among groups. The median baseline MICs of all isolates of Pseudomonas aeruginosa recovered from all patients (n=592) to levofloxacin was 4 mg/L and the MIC90 was 16, also similar across all groups. These MICs are indicative of baseline non-susceptibility/resistance to levofloxacin, as defined by CLSI reference methods and the US FDA.

[0150] Concomitant medication used by patients during the study included Domase alfa, Azithromycin, Salbutamol, Pancrelipase, hypertonic sodium chloride, Seretide, and ADEK, and are summarized for the EE population in Table 8. Table 9 summarizes patient disposition in the study. The results of the study showed were statistically significant advantages of aerosol levofloxacin compared to the placebo in several clinical and microbiological measures, despite the use of concomitant medications, resistance to levofloxacin, and previous use of other aerosolized antibiotics, including tobramycin and colistin. -40- 2015275224 22 Dec 2015 TABLE 8 Medication Placebo (N= 37} Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Total (N=151) Dornase alpha 31 (83.8%) 27 (71.1%) 33 (89.2%) 29 (74.4%) 120 (79.5%) Azithromycin 25 (67.6%) 29 (76.3%) 26 (70.3%) 32 (82.1%) 112 (74.2%) Salbutamol 25 (67.6%) 29 (76.3%) 25 (67.6%) 23 (59.0%) 102 (67.5%) Pancrelipase 19 (51.4%) 21 (55.3%) 22 (59.5%) 21 (53.8%) 83 (55.0%) Sodium chloride 22 (59.5%) 13(34.2%) 13(35.1%) 22 (56.4%) 70 (46.4%) Seretide 19 (51.4%) 20 (52.6%) 16(43.2%) 15(38.5%) 70 (46.4%) ADEK 10 (27.0%) 13(34.2%) 10 (27.0%) 13(33.3%) 46 (30.5%) TABLE9 Disposition Placebo Levofloxacin 120 mg QD Levofloxacin 240 mg QD Levofloxacin 240 mg BID Total Patients Enrolled 37 38 37 39 151 Safety/MITT population 37 (100.0%) 38 (100.0%) 37 (100.0%) 39 (100.0%) 151 (100,0%) Efficacy Evaluable population 32 (86.5%) 35 (92.1%) 35 (94.6%) 34 (87.2%) 136 (90.1%) PK population 37 (100.0%) 37 (97.4%) 37 (100.0%) 39(100.0%) 150 (99.3%) Completed Study 35 (94.6%) 37 (97.4%) 35 (94.6%) 36 (92.3%) 143 (94.7%) Discontinued from Study 2 (5.4%) 1 (2.6%) 2 (5.4%) 3 (7.7%) 8 (5.3%) Primary Reason for Discontinuation from Study3 Adverse Event 2 (5.4%) 1 (2.6%) 1 (2.7%) 2 (5.1%) 6 (4.0%) Withdrawal of consent 0 (0.0%) 0 (0.0%) 1 (2.7%) 0 (0.0%) 1 (0.7%) Other 0 (0.0%) 0 (0.0%) 0 (0.0%) 1 (2.6%) 1 (0.7%) Primary Reason for Study Drug Discontinuation3 5 (13.5%) 3 (7.9%) 1 (2.7%) 2(5.1%) 11 (7.3%) Adverse Event 4 (10.8%) 3 (7.9%) 1 (2.7%) 2(5.1%) 10 (6.6%) -41-

Disposition Placebo Levofloxacin 120 mg QD Levofloxacin 240 mg QD Levofloxacin 240 mg BID Total Other 1 (2.7%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 1 (0.7%) a: Patients could be included in both discontinuation from study and Study Drug categories.

Microbiological evaluations 2015275224 22 Dec 2015 [0151] Changes in P. aeruginosa density (logio CFU/g sputum) for sputum samples taken from the EE and MITT populations are summarized in Tables 10 and 11, respectively (SD - standard deviation). Changes in MIC for P. aeruginosa density (logio CFU/g sputum) to levofloxacin (all organisms) for EE and MITT populations are shown in Tables 12 and 13, respectively. In addition, changes in the highest MIC values for P. aeruginosa isolates to levofloxacin were determined for EE and MITT populations, and are summarized in Tables 14 and 15, respectively. Tables 16 and 17 summarize the percentage of EE and MITT populations, respectively, with categorical changes in highest MIC values for P. aeruginosa isolates to levofloxacin. Tables 18 and 19 summarize changes in non-fermenting gram negative bacilli density (NFGNB), excluding P. aeruginosa, with only patients with positive NFGNB counts at baseline, for EE and MITT populations, respectively. Tables 20 and 21 summarize changes for presumptive S. aureus density (logio CFU/g sputum) for only patients with positive S. aureus counts at baseline, for EE and MITT populations, respectively, TABLE 10 EE population - P. aeruginosa density (log10 CFU/g sputum) Placebo (N= 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N« 34) Combined Levofloxacin 240mg QD and BID (N= 69) Day 1 Mean (SD) 7.49 (1.674) 7.43(1.622) 7.16(1.597) 7.26(1.433) 7.21 (1.508) Median 8.11 8.17 7.97 7.84 7.90 Day 7 Mean (SD) 7.37 (1.746) 6.64(1.818) 6.94 (1.783) 6.23(1.850) 6.59 (1.838) Median 8.14 6.86 7.15 6.52 6.77 Change from Baseline Mean (SD) -0.12 (1.422) -0.76(1.263) -0.25(1.789) -1.03(1.921) -0.64 (1.883) Median 0.09 -0.58 -0.18 -0.88 -0.37 -42- 2015275224 22 Dec 2015 EE population - P. aeruginosa density (log10 CFU/g sputum} Placebo (N* 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) to Day 7 P-value 0.0277 0.5892 0.0051 0.0533 Day 14 Mean (SD) 7.48 (1.616) 7.32(1.643) 6.69(1.628) 6.43(1.725) 6.56 (1.669) Median 7.85 8.00 6.46 6.81 6.78 Change from Baseline to Day 14 Mean (SD) 0.03 (1.355) -0.10(1.240) -0.47 (1.905) -0.83(1.901) -0.65(1.898) Median -0.01 0.00 -0.11 -0.91 -0.46 P-value 0.3929 0.0998 0.0047 0.0105 Day 28 Mean (SD) 7.85 (1.050) 7.25(1.431) 6.78(1.896) 6.65 (1.440) 6.72(1.683) Median 8.28 7.78 7.38 6.40 6.90 Change from Baseline to Day 28 Mean (SD) 0.36 (1.329) -0.31 (1.050) -0.38 (1.780) -0.74 (1.488) -0.55(1.645) Median 0.00 -0.20 -0.52 -0.68 -0.58 P-value 0.0093 0.0075 0.0002 0.0002 TABLE 11 MITT population - P. aeruginosa density (log10 CFU/g sputum) Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Day 1 Mean (SD) 7.45 (1.656) 7.43(1.571) 7.08(1.742) 7.17 (1.726) 7.12(1.723) Median 8.10 8.16 7.97 7.90 7.91 Day 7 Mean (SD) 7.21 (1.750) 6.68 (1.778) 6.79(1.854) 6.16 (1.969) 6.47 (1.927) Median 7.97 6.98 6.95 6.53 6.69 Change from Baseline to Day 7 Mean (SD) -0.24 (1.429) -0.70(1.257) -0.31 (1.823) -0.97 (1.846) -0.65 (1.852) Median 0.00 -0.53 -0.18 -0.76 -0.35 P- value 0.1091 0.6986 0.0124 0.0956 -43- 2015275224 22 Dec 2015 MITT population - P. aeruginosa density (log™ CFU/g sputum) Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Day 14 Mean (SD) 7.33 (1.651) 7.33(1.602) 6.63(1.744) 6.32(1.875) 6.48(1.806) Median 7.85 8.00 6.46 6.70 6.61 Change from Baseline to Day 14 Mean (SD) -0.07 (1.373) -0.07(1.213) -0.44(1.855) -0.81 (1.838) -0.63 (1.843) Median -0.02 0.00 -0.10 -0.79 -0.40 P- value 0.7941 0.1972 0.0091 0.0251 Day 28 Mean (SD) 7.66 (1.236) 7.26(1.394) 6.82(1.869) 6.58(1.704) 6.70(1.781) Median 8,26 7.78 7.38 6.48 6.90 Change from Baseiine to Day 28 Mean (SD) 0.21 (1.377) -0.27(1.035) -0.25(1.857) -0.66 (1.422) -0.46 (1.658) Median 0.00 -0.20 -0.39 -0.56 -0.52 P- value 0.0728 0.0698 0.0014 0.0039 TABLE 12 EE population - MIC of P. aeruginosa to levofloxacin (all organisms) (pg/ml) Placebo Levofloxacin 120mg QD Levofloxacin 240mg QD Levofloxacin 240mg BID Combined Levofloxacin 240mg QD and BID N 124 140 140 136 276 Day 1 Mean (SD) 6.0 (5.00) 11.5 (21.98) 6.3 (8.85) 6.8 (8.80) 6.5 (8.82) MIC*, 4 4 4 4 4 MICgo 16 32 16 16 16 Day N 124 136 132 132 264 7 Mean (SD) 6,6 (6.73) 9.9 (17.28) 6.8(16.13) 8.8 (17.28) 7.8(16.71) -44- 2015275224 22 Dec 2015 EE population - MIC of P. aeruginosa to ievofloxacin (all organisms) (pg/ml) Placebo Levofloxacin 120mg QD Levofloxacin 240mg QD Levofloxacin 240mg BID Combined Levofloxacin 240mg QD and BID Min, Max 0.25, 32 0.13, 128 0.13, 128 0.13, 128 0.13, 128 MICso 4 4 2 4 4 MIC90 8 16 16 16 16 N 120 140 136 132 268 Day 14 Mean (SD) 5.6 (6.79) 13.0(29.34) 6.6 (9.44) 8.4 (14.47) 7.5(12.19) MICm 4 4 4 4 4 MIC90 8 32 16 16 16 N 128 132 136 124 260 Day 28 Mean (SD) 5.0 (3.97) 8.9 (13.47) 6.0 (7.57) 10.1 (16.32) 8.0 (12.67) MICso 4 4 4 4 4 MICso 8 16 16 32 16 TABLE 13 r MITT population - MIC of P. aeruginosa to levofloxacin (all organisms) (pg/mi) Placebo Levofloxacin 120mg QD Levofloxacin 240mg QD Levofloxacin 240mg BID Combined Levofloxacin 240mg QD and BID Day 1 N 140 152 148 152 300 Mean (SD) 6.1 (5.40) 11.0(21.17) 6.1 (8.64) 6.8 (8.47) 6.4 (8.55) MICso 4 4 4 4 4 MICso 16 32 16 16 16 Day 7 N 136 144 140 140 280 Mean (SD) 6.8 (7.33) 9.6 ¢16.83) 6.7 (15.69) 8.6 (16.82) 7.6 (16.27) MICso 4 4 2 4 4 MICso 16 16 16 16 16 Day I ! 128 144 144 140 284 -45- 2015275224 22 Dec 2015 MITT population - MiC of P. aeruginosa to levofloxacin (ail organisms) (pg/ml) r Placebo Levofloxacin 120mg QD Levofloxacin 240mg QD Levofloxacin 240mg BID Combined Levofloxacin 240mg QD and BID 14 Mean (SD) 5.8 (7.09) 12.9 (28.95) 6.5 (9.23) 8.2 (14.11) 7.3 (11.90) MICso 4 6 4 4 4 MlCgo 16 32 16 16 16 N 132 136 140 132 272 Day 28 Mean (SD) 4.9 (3.97) 8.8(13.32) 5.9 (7.50) 9,9(15.98) 7.9(12.51) MIC» 4 4 4 4 4 MIC® 8 16 16 32 16 TABLE 14 EE population - Highest MIC of P. aeruginosa to levofloxacin (pg/ml) Placebo (N= 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) Day 1 Mean (SD) 8.79 (6.661) 14.98 (22.383) 9.35(12.991) 11.58(13.399) 10.45 (13.144) MICS0 8 8 4 6 4 MIC® 16 32 32 32 32 Day 7 Mean (SD) 8.77 (7.455) 15.71 (22.523) 10.86 (22.222) 13.05 (22.838) 11.95(22.385) MiCSo 8 8 4 4 4 MICso 16 32 16 32 32 Day 14 Mean (SD) 8.42 (11.437) 21.16(43.639) 9.56 (12.409) 15.18(25.541) 12.33 (20.032) MICso 6 8 8 4 8 MICso 16 32 16 32 32 Day 28 Mean (SD) 7.31 (4.961) 12.91 (15.669) 9.42(11.658) 17.97 (26.008) 13.56 (20.252) MIC50 8 8 8 8 8 MlCgo 16 32 16 32 32 -46- 2015275224 22 Dec 2015 TABLE 15 MITT population - Highest MIC of P. aeruginosa to levofloxacin (pg/ml) Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD <N- 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Day 1 Mean (SD) 9.04 ¢7.593) 14.32 (21.583) 9.06(12.685) 11.41 (12.816) 10.25(12.720) MIC» 8 8 4 6 4 mic90 16 32 32 32 32 Day 7 Mean (SD) 9.06 (8.203) 15.17(21.991) 10.53 (21.615) 12.96 (21.902) 11.76 (21,639) MICso 8 8 4 6 4 M!C90 16 32 16 32 16 Day 14 Mean (SD) 8.78 (11.554) 20.34 (42.555) 9.28 (12.136) 14.86 (24.522) 12.07(19.415) MICso 6 8 8 6 8 MlCgo 16 32 16 32 32 Day 28 Mean (SD) 7.78 (6.334) 12.74(15.286) 9.17(11.389) 17.91 (25.067) 13.48(19.734) MICso 8 8 8 8 8 MICso 16 32 16 32 32 TABLE 16 EE population - Categorical change in highest MIC of P. aeruginosa to levofloxacin (% population) Placebo (N= 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) Change from Baseline to Day 7 >= 4 fold increase 6.5% 2.9% 12.1% 9.1% 10.6% < 4 fold increase 93.5% 97.1% 87.9% 90.9% 89.4% Change from Baseline to Day 14 >= 4 fold increase 10.0% 11.4% 5.9% 18.2% 11.9% < 4 fold increase 90.0% 88.6% 94,1% 81.8% 88.1% -47- 2015275224 22 Dec 2015 EE population - Categorical change in highest MIC of P. aeruginosa to levofloxacin {% population) Placebo (N= 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240m g QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) Change from Baseline to Day 28 >= 4 fold increase 9.4% 9.1% 8.8% 15.6% 10.6% < 4 fold increase 90.6% 90.9% 91.2% 84.4% 87.9% Change from Baseline to Day 56/Early Term >= 4 fold increase 22.6% 6.2% 5.7% 15.2% 10.3% < 4 fold increase 77.4% 93.8% 94.3% 84.8% 89.7% TABLE 17 MITT population - Categorical change in highest MIC of P. aeruginosa to levofloxacin {% population) Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Change from Baseline to Day 7 >=4 fold increase 5.7% 2.8% 11.4% 8.3% 9.9% < 4 fold increase 94.3% 97.2% 88.6% 91.7% 90.1% Change from Baseline to Day 14 >-4 fold increase 8.8% 10.8% 5.6% 16.7% 11.1% < 4 fold increase 91.2% 89.2% 94.4% 83.3% 88.9% Change from Baseline to Day 28 >= 4 fold increase 8.3% 8.6% 8.3% 14.3% 11.3% < 4 fold increase 91.7% 91.4% 91.7% 85.7% 88.7% -48- 2015275224 22 Dec 2015 MITT population - Categorical change in highest MIC of P. aeruginosa to levofloxacin (% population) Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Change from Baseline to Day 56/Early Term >=4 fold increase 20.0% 5.9% 5.4% 13.9% 9.6%) < 4 fold increase 80.0% 94.1% 94.6% 86.1% 90,4% TABLE 18 EE population - Change in non-fermenting gram negative bacilli density (iog10 CFU/g sputum) Placebo (N= 5) Levofloxacin 120mg QD (N=5) Levofloxacin 240mg QD (N=5) Levofloxacin 240mg BID (N= 4) Combined Levofloxacin 240mg QD and BID (N= 9) Combined Levofloxacin (N= 14) Day 1 Mean (SD) 6.60 (1.113) 5.69 (2.674) 4.65 (2.271) 5.52 (2.291) 5.03 (2.182) 5.27 (2.288) Day 7 Mean (SD) 6.26 ¢3.018) 4.54(2.361) 2.18(1.614} 5.01 (3.068) 3.44 (2.657) 3.83 (2.522) Change from Baseline to Day 7 Mean (SD) -0.34 (2.610) -1.15(1.607) -2.47 (3.346) -0.51 (3.184) -1.60 (3.235) -1.44 (2.699) P- value 0.1206 0.0476 0.7552 0.1755 0.1178 Day 14 Mean (SD) 5.94 (3.002) 3.15(3.122) 2.04(2.315) 6.02(1.894) 3.80 (2.903) 3.57 (2.880) Change from Baseline to Day 14 Mean (SD) -0.66 (2.315) -2.54 (3.057) -2.61 (3.222) 0.50 (1.638) -1.23 (2.980) -1.70 (2.961) P- value 0.0894 0.1571 0.5983 0.5932 0.2824 Day 28 Mean (SD) 5.60 (3.012) 5.10(2.938) 1.36 (0.808) 5.01 (2.758) 2.98 (2.621) 3.63 (2.787) Change from Baseline to Day 28 Mean (SD) -1.00 (3.262) -1.44 (4.739) -3.29 (2.401) -0.51 (1.438) -2.05 (2.407) -1.87 (3.092) P- value 0.2997 0.0479 0.9881 0.2237 0.1956 -49- 2015275224 22 Dec 2015 TABLE 19 MITT population - Change in non-fermenting gram negative bacilli density (log™ CFU/g sputum) Placebo <N=6) Levofloxacin 120mg QD (N=5) Levofloxacin 240mg QD (N=7) Levofloxacin 240mg BID (N- 5) Combined Levofloxacin 240mg QD and BID (N= 12) Combined Levofloxacin (N= 17) Day 1 Mean (SD) 5.88 (2.018) 5.69 (2.674) 5.26 (2.203) 6.05 (2.317) 5.59 (2.184) 5.62 ¢2.251) Day 7 Mean (SD) 5.44 (3.375) 4.54 (2.361) 2.52 (1.997) 5.63 (3.004) 3.82 (2.832) 4.03 (2.650) Change from Baseline to Day 7 Mean (SD) -0.45 (2.350) -1.15(1.607) -2.73 (2.882) -0.42 (2.765) -1.77 (2.955) -1.59 (2.594) P- value 0.2680 0.0485 0.6562 0.3749 0.2748 Day 14 Mean (SD) 5.17 (3.286) 3.15(3.122) 3.08 (2.599) 6.50 (1.964) 4.50 (2.862) 4.11 (2.911) Change from Baseline to Day 14 Mean (SD) -0.72 (2.075) -2.54 (3.057) -2.18(2.784) 0.45(1.423) -1.08 (2.605) -1.51 (2.733) P- value 0.1020 0.1924 0.2832 0.9333 0.4410 Day 28 Mean (SD) 4.88 (3.215) 5.10 (2.938) 2.77 (2.505) 5.63 (2.762) 3.96 (2.892) 4.24 (2.850) Change from Baseline to Day 28 Mean (SD) -1.00 (2.917) -1.44 (4.739) -2.49 (2.538) -0.43 (1.260) -1.63 (2.284) -1.58 (2.885) P- value 0.5733 0.1606 0.4532 0.7286 0.6211 TABLE 20 EE population - Changes for presumptive S. aureus density (log™ CFU/g sputum) Placebo (N= 16) Levofloxacin 120mg QD (N= 16) Levofloxacin 240mg QD (N- 19) Levofloxacin 240mg BID (N= 13) Combined Levofloxacin 240mg QD and BID (N= 32) Combined Levofloxacin (N= 48) Day 1 Mean (SD) 5.67 (2.125) 5.72 (1.871) 5.64 (1.985) 5.68 (2.196) 5.65 (2.038) 5.68(1.964) Day 7 Mean (SD) 5.53 (2.152) 4.53 (2.272) 5.34 (2.321) 4.96 (2.992) 5.18 (2.574) 4.97 (2.473) -50- 2015275224 22 Dec 2015 EE population - Changes for presumptive 5. aureus density (log10 CFU/g sputum) Placebo (N= 16) Levofloxacin 120mg QD (N= 16) Levofloxacin 240mg QD (N= 19) Levofloxacin 240mg BID (N= 13) Combined Levofloxacin 240mg QD and BID (N= 32) Combined Levofloxacin (N= 48) Change from Baseline to Day 7 Mean (SD) -0.14 (0.968) -1.19(1.722) -0.30(1.579) -0.72(1.159) -0.47 (1.419) -0.71 (1.546) P- value 0.0347 0.5969 0.2039 0.2891 0.1059 Day 14 Mean (SD) 5.35 (2.469) 5.30 (2.543) 5.14(2.594) 4.70 (2.829) 4.96 (2.656) 5.08 (2.597) Change from Baseline to Day 14 Mean (SD) -0.32 (1.259) -0.42 (1.273) -0.50 (1.766) -0.98 (0.998) -0.69(1.502) -0.60(1,422) P- value 0.6684 0.5883 0.1593 0.2497 0.3259 Day 28 Mean (SD) 5.46 (2.647) 5.31 (2.482) 4.56 (2.724) 5.42 (2.973) 4.89 (2.806) 5.04 (2.680) Change from Baseline to Day 28 Mean (SD) -0.21 (1.137) -0.41 (1.146) -1.08 (2.622) -0.38(1.144) -0.81 (2.173) -0.67 (1.882) P- value 0.6124 0.1186 0.6403 0.2589 0.3159 TABLE 21 MITT population - Changes for presumptive S. aureus density (log10 CFU/g sputum) Placebo (N= 19) Levofloxacin 120mg QD (N= 17) Levofloxacin 240mg QD (N= 21) Levofloxacin 240mg BID (N= 16) Combined Levofloxacin 240mg QD and BID (N-37) Combined Levofloxacin (N= 54) Day 1 Mean (SD) 6.06 (2.152) 5.89(1.941) 5.63 (1.894) 5.51 (2.318) 5.58 (2.058) 5.68 (2.009) Day 7 Mean (SD) 5.78 (2.071) 4.75 (2.382) 5.23 (2.249) 4.65 (2.954) 4.99 (2.543) 4.91 (2.472) Change from Baseline to Day 7 Mean (SD) -0.28 ¢0.985) -1.14(1.681) -0.40(1.615) -0.66 (1.243) -0.51 (1.458) -0.71 (1.545) P- vaiue 0.0563 0.6856 0.3058 0.3995 0.1653 Day 14 Mean (SD) 5.64 (2.387) 5.48 (2.568) 4.97 (2.579) 4.32 (2.820) 4.70 (2.662) 4.95 (2.633) -51- 2015275224 22 Dec 2015 MITT population - Changes for presumptive S. aureus density (log™ CFU/g sputum) Placebo (N= 19) Levofloxacin 120mg QD (N= 17) Levofloxacin 240mg QD (N= 21) Levofloxacin 240mg BID (N= 16) Combined Levofloxacin 240mg QD and BID (N= 37) Combined Levofloxacin <N= 54) Change from Baseline to Day 14 Mean (SD) -0.42 (1.216) -0.41 (1.233) -0.66 (1.907) -0.99 (0.981) -0.80 (1.578) -0.68 (1.475) P- value 0.8238 0.5691 0.2231 0.2939 0.4058 Day 28 Mean (SD) 5.65 (2.511) 5.49 (2.510) 4.48 (2.633) 4.95 (3.018) 4.66 (2.760) 4.93 (2.684) Change from Baseline to Day 28 Mean (SD) -0.41 (1.240) -0.41 (1.110) -1.15(2.578) -0.44(1.150) -0.87 (2.131) -0.72 (1.860) P- value 0.8341 0.1623 0.7846 0.3528 0.4572 [0152] P. aeruginosa densities were reduced from baseline values on (Day 1) over the course of the study in all three levofloxacin treatment groups in both the EE and MITT populations. Figures 3 and 4 show mean changes in P. aeruginosa densities over time for each treatment group.

[0153] In the EE population, there was a decrease of P. aeruginosa density in patients administered levofloxacin 240 mg BID from a median value of 7.84 logio CFU/g sputum on Day 1 to a median value of 6.40 logio CFU/g sputum on Day 28, representing a reduction in P. aeruginosa density in sputum of approximately 96%. In patients administered levofloxacin 240 mg QD, there was a decrease in P. aeruginosa density from a median value of 7.97 logio CFU/g sputum on Day 1 to a median value of 7.38 logio CFU/g sputum on Day 28, representing a reduction in P. aeruginosa density in sputum of approximately 74%. In patients administered levofloxacin 120 mg QD, there was a decrease in P. aeruginosa density from a median value of 8.17 logio CFU/g sputum on Day 1 to a median value of 7.78 logio CFU/g sputum on Day 28, representing a reduction in P. aeruginosa density in sputum of approximately 59%. The largest differences for P. aeruginosa density in sputum between patients administered placebo and patients administered levofloxacin were 1.62 logic CFU/g sputum on Day 7, 1.39 logio CFU/g sputum on Day 14, and 1.88 logio CFU/g sputum on Day 28.

[0154] In the MITT population, there was a decrease of P. aeruginosa density in patients administered levofloxacin 240 mg BID from a median value of 7.90 logio -52- CFU/g sputum on Day 1 to a median value of 6.48 logio CFU/g sputum on Day 28, representing a reduction in P. aeruginosa density in sputum of approximately 96%. In patients administered levofloxacin 240 mg QD, there was a decrease in P. aeruginosa density from a median value of 7.97 logic CFU/g sputum on Day 1 to a median value of 7.38 logio CFU/g sputum on Day 28, representing a reduction in P. aeruginosa density in sputum of approximately 74%. In patients administered levofloxacin 120 mg QD, there was a decrease in P. aeruginosa density from a median value of 8.16 logio CFU/g sputum on Day 1 to a median value of 7.78 logic CFU/g sputum on Day 28, representing a reduction in P, aeruginosa density in sputum of approximately 58%. The largest differences for P. aeruginosa density in sputum between patients administered placebo and patients administered levofloxacin were 1.44 logic CFU/g sputum on Day 7, 1.39 logio CFU/g sputum on Day 14, and 1.78 logic CFU/g sputum on Day 28. 2015275224 22 Dec 2015 [0155] For the EE and MITT populations, baseline MIC50 values for P. aeruginosa isolates from patients administered placebo, 120 mg QD, 240 mg QD, and 240 mg BID were 8 pg/ml, 8, 4 pg/ml, and 6 pg/ml, respectively. On Day 28, MICso values for P. aeruginosa isolates from patients administered placebo, 120 mg QD, 240 mg QD, and 240 mg BID were 8 pg/ml, 8 pg/ml, 8 pg/ml, and 8 pg/ml, respectively. Baseline MIC90 values for P. aeruginosa isolates from patients administered placebo, 120 mg QD, 240 mg QD, and 240 mg BID were 16 pg/ml, 32 pg/ml, 32 pg/ml, and 32 pg/ml, respectively. On Day 28, MIC90 values for P. aeruginosa isolates from patients administered placebo, 120 mg QD, 240 mg QD, and 240 mg BID were 16 pg/ml, 32 pg/ml, 16 pg/ml, and 32 pg/ml, respectively.

[0156] The similarity of corresponding MIC50 and MIC90 values between Day 1 and Day 28 for the EE and MITT populations indicates that P. aeruginosa cultures from patients did not develop any significant resistance to levofloxacin.

Clinical evaluations—CFQ-R questionnaire [0157] Patients completed the CFQ-R questionnaire with domains that included: respiratory, body image, digestion, eating, emotion, health perception, physical, role/school, social, treatment burden, vitality, and weight. Tables 22 and 23 summarize results for the respiratory domain of the CFQ-R for EE and MITT populations, respectively. Table 24 summarizes changes in score for various domains of the CFQ-R from baseline to visit 4 in the MITT population. -53- 2015275224 22 Dec 2015 TABLE 22 EE-popuiation - Respiratory domain of CFQ-R Placebo (N= 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) Day 1 Mean (SD) 66.7 (14.39) 63.5(15.84) 60.8(15.83) 60.3 (16.42) 60.5 (16.00) Median 66.7 66.7 61.1 61.1 61.1 Day 14 Mean (SD) 66.7 (15.84) 67.8(13.79) 63.2 (19.34) 68.8(19.15) 66.0 (19.31) Median 63.9 66.7 66.7 72.2 66.7 Change from Baseline to Day 14 Mean (SD) -0.0 (11.20) 4.3 (9.82) 2.1 (16.41) 8.5 (15.55) 5.3(16.19) Median 0.0 5.5 0.0 5.6 5.6 P- value 0.3953 0.9534 0.0374 0.2171 Day 28 Mean (SD) 64.9 (18.53) 65.1 (17.28) 61.7(19.37) 66.0(19.68) 63.8 (19.49) Median 63.9 66.7 66.7 72.2 66.7 Change from Baseline to Day 28 Mean (SD) -1.7 (14.90) 1,6(12.32) 1.0(14.91) 5.2 (17.56) 3.0 (16.27) Median 0.0 0.0 0.0 5.5 0.0 P- value 0.6183 0.8145 0.0924 0.2690 TABLE 23 MITT-population - Respiratory domain of CFQ-R Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Day 1 Mean (SD) 64.9 (14.17) 62.3(15.88) 61.3(15.52) 60.5(16.31) 60.9(15.83) Median 66.7 61.1 61.1 61.1 61.1 Day 14 Mean (SD) 64.6 (15.84) 67.1 (13.70) 63.0 (19.74) 68.4(19.51) 65.8 (19.68) Median 61.1 66.7 66.7 72.2 69.4 -54- 2015275224 22 Dec 2015 MITT-population - Respiratory domain of CFQ-R Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Change from Baseline to Day 14 Mean (SD) -0.3 ¢10.79) 4.5 (9.78) 1.4 (17.54) 7.5 (15.30) 4.5 (16.59) Median 0.0 5.5 0.0 5.6 5.5 P- value 0.2559 0.8964 0.0334 0.1937 Day 28 Mean (SD) 63.1 (18.01) 64.6 (16.94) 61.6 (19.74) 64.4 (20.39) 63.0(19.98) Median 61.1 66.7 66.7 72.2 66.7 Min, Max 22, 94 17, 100 17, 89 22. 94 17, 94 Change from Baseline to Day 28 Mean (SD) -1.8 (14.11) 2.0(12.23) 0.3(16.14) 3.0(19.18) 1.7(17.66) Median 0.0 0.0 0.0 0.0 0.0 P- value 0.5029 0.8365 0.2174 0.4069 TABLE 24 CFQ-R Scale LS Mean Change from Baseline LS Mean Difference (95% Cl) P- value Placebo (N =37) Levofloxacin 240 mg BID (N = 39) Respiratory -0.44 4.06 4.50 (-2.68, 11.67) 0.2174 Body Image 0.59 0.34 -0.25 (-6.00, 5.50) 0.9315 Digestion -1.35 1.11 2.46 (-3.27, 8.18) 0.3975 Eating -3.29 2.41 5.70 (0.78, 10.62) 0.0235 Emotion 2.32 2.22 -0.09 (-4.34, 4.15) 0.9651 Health Perception -2.1 0.52 2.58 (-3.70, 8.86) 0.4177 Physical -2.80 3.14 5.94(0.75, 11.13) 0.0252 Role/School -1.41 -1.94 -0.53 (-5.43, 4.38) 0.8324 Social -1.68 0.69 2.37 (-2.66, 7.40) 0.3530 Treatment Burden -0.99 -0.93 0.06 ¢-5.84, 5.95) 0.9851 Vitality -1.88 1.23 3.11 ¢-2.91, 9.13) 0.3085 -55- LS Mean Change from Baseline LS Mean Difference (95% Cl) CFQ-R Scale Placebo (N Levofloxacln 240 mg BID "37) (N = 39) Weight 4.72 10.06 5.34 (-4.38, 15.06) [0158] For the EE population, mean changes from baseline to Day 28 for respiratory factors measured by the CFQ-R for patient administered placebo, 120 mg QD, 240 mg QD, and 240 mg BID treatment groups were 1.6, 1.0, 5.2, and 3.0 units, respectively. For the MITT population, mean changes from baseline to Day 28 for respiratory factors for patient administered placebo, 120 mg QD, 240 mg QD, and 240 mg BID treatment groups were 2.0, 0.3, 3.0, and 1.7 units, respectively. The 240mg BID group in the EE population demonstrated a statistically significant improvement in respiratory score on Day 14. 2015275224 22 Dec 2015 [0159] The CFQ-R eating score also showed improvement with the difference in the 240mg BID group in the MITT population showing statistically significant improvement at Day 28.

Time to patient need for anti-pseudomonal antimicrobials [0160] Time to administration of intravenous/oral/inhaled anti-pseudomonal antimicrobials from Day 1 until Final visit was measured for patients with at least one of the following: decreased exercise tolerance, increased cough, increased sputum/chest congestion, and decreased appetite. The proportion of patients requiring anti-pseudomonal antimicrobials (inhaled or systemic) over time was analyzed using a Cox proportional hazards model. Table 25 summarizes results for time to need for anti-pseudomonal antimicrobials for EE and MITT populations. -56- 2015275224 22 Dec 2015 TABLE 25 EE and MITT populations - Parameters for time to need anti-pseudomonal antimicrobials Placebo (N= 32) Levofloxacin 120mg QD {N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) EE population Patients Requiring Anti- Pseudomonal Antimicrobials 40.6% 20.0% 25.7% 20.6% 23.2% Hazard Ratio [95% CIJ 0.39 [0.15. 1.01] 0.48 [0.20. 1.17] 0.26 [0.10, 0.69] 0.37 [0.17, 0.81] P-va!ue 0.0522 0.1050 0.0067 0.0123 MITT population Number {%) of Patients Requiring Anti- Pseudomonal Antimicrobials 48.6% 18.4% 27.0% 20.5% 23.7% Hazard Ratio [95% Cl] 0.29 [0.12, 0.71] 0.39 [0.17, 0.87] 0.21 [0.09, 0.52] 0.30 [0.15, 0.60] P-vaiue 0.0069 0.0215 0.0007 0.0007 [0161] The measured need for additional anti-pseudomonai microbials was reduced in all levofloxacin treatment groups. In addition, significant hazard ratios were observed in all levofloxacin treatment groups. Hazard ratios are related to the relative risk that an event may occur. The hazard ratios were 0.29 for the levofloxacin 120 mg QD group, 0.39 for the levofloxacin 240 mg QD group, and 0.21 for the levofloxacin 240 mg BID group in the MITT Population compared to the placebo group, and were statistically significant compared to placebo. Figures 5 and 6 are plots of survival distribution function over time for each treatment group and show that the survival distribution function for patients treated with placebo begins to fall at a shorter time than patients treated with levofloxacin. In sum, at least 240 mg BID showed significant efficacy over placebo at Day 28 in time to need for anti-pseudomonal antimicrobials. -57-

Pulmonary function evaluations 2015275224 22 Dec 2015 [0162] Changes in FEV] (forced expiratory volume in 1 second), FVC (forced vital capacity) and FEF 25-75 (forced expiratory flow 25-75%) from Day 1 to all subsequent visits were determined for patients. Tables 26 and 27 summarize the results for FEVt measurements in EE and MITT populations, respectively. Figures 7 and 8 show graphs of percent change in FEV] (L) and percent change in FEV] (L) vs. placebo, respectively, at Day 28 for the EET population treated with placebo, 120 mg QD, 240 mg QD, or 240 mg BID. Figure 9 shows a graph of the categorical change in percent predicted FEV] at Day 28 for the EET population treated with 120 mg QD, 240 mg QD, or 240 mg BID. Tables 28 and 29 summarize the results for predicted FEV] measurements in EE and MITT populations, respectively. Tables 30 and 31 summarize results for FEF 25-75 measurements in EE and MITT populations, respectively. TABLE 26 EE population - Changes in FEV·, values Placebo (N- 32) Levofloxacin 120mg QD (N= 35} Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) Day 1 Mean (SD) 1.98 (0.61) 1.99 (0.83) 2.09 (0.75) 1.86(0.68) 1.98 (0.72) Median 2.01 1.87 2.02 1.70 1.81 Day 14 Mean (SD) 1.97 (0.63) 2.00 (0.83) 2.16 (0.78) 2.02 (0.76) 2.09 (0.77) Median 1.93 1.82 2.09 1.88 2.03 Percent Change from Baseline to Day 14 Mean (SD) -0.89 (6.86) 1.13(7.48) 3.84(10.23) 8.87 (12.42) 6.32(11.56) Median -0.17 0.56 1.92 7.51 3.94 P-value vs. Placebo 0.4940 0.0737 0.0001 0.0013 Day 28 Mean (SD) 1.95 (0.65) 2.03 (0.89) 2.13(0.76) 1.90 (0.68) 2.02 (0.73) Median 1.91 1.91 2.15 1.79 1.98 Percent Change from Mean (SD) -2.55 (10.37) 1.71 (9.73) 2.51 (13.25) 5.05(11.49) 3.75 (12.40) Median -3.91 1.60 1.28 4.55 2.80 -58- 2015275224 22 Dec 2015 EE population - Changes in FEV-ι values Placebo {N= 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) Baseline to Day 28 P-value vs. Placebo 0.1726 0.1121 0,0102 0,0168 TABLE 27 MITT population - Changes in FEV·] values Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Day 1 Mean (SD) 1.94 (0.61) 1.95 (0.81) 2.05 (0.75) 1.88 (0.68) 1.96 (0.71) Median 1.97 1.83 1.90 1.70 1.80 Day 14 Mean (SD) 1.92 (0.63) 1.97 ¢0.81) 2.12(0.78) 2.04 (0.77) 2.08 (0.77) Median 1.92 1.79 2.04 1.90 1.97 Percent Change from Baseline to Day 14 Mean (SD) -1.21 (6,46) 1.87 (9.41) 3.81 ¢10.32) 9.34 (13.72) 6.61 (12.40) Median -0.90 0.56 1.92 7.51 3.94 P- value 0.2873 0.0497 <.0001 0.0005 Day 28 Mean (SD) 1.89 (0.64) 1.99 (0.88) 2.09 (0.76) 1.93 (0.69) 2.01 (0.73) Median 1.85 1.83 2.10 1.82 1.95 Percent Change from Baseline to Day 28 Mean (SD) -2.96 (9.81) 1.92 (9.95) 2.35 (13.03) 5.93(15.07) 4.14 (14.11) Median -3.92 1.60 1.28 3.55 2.50 P- value 0.1292 0.0831 0.0026 0.0063 -59- 2015275224 22 Dec 2015 TABLE 28 EE population - Changes in % predicted FEVi Placebo (N= 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levoffoxacin 240mg QD and BID (N= 69) Day 1 Mean <SD) 54.4 (12.93) 54.0(17.79) 55.9 (14.60) 48.2 (14.88) 52,1 (15.13) Median 54.5 53.0 56.0 45.5 51.0 Day 14 Mean <SD) 53.9 (13.21) 54.7(17.61) 57.7 (15.19) 51.7(16.93) 54.7 (16.23) Median 54.5 53.0 62.0 49.5 52.0 Relative Percent Change from Baseline to Day 14 Mean (SD) -0.77 (6.85) 1,77 (6.08) 3.57 (10.75) 7.67 (14.70) 5.59 (12.92) Median 0.00 1.15 1.52 7.42 4.00 Day 28 Mean (SD) 53.1 (14.05) 55.0(19.30) 57.3 (15.52) 50.5 (15.36) 54.0(15.71) Median 55.0 52.0 58.0 49.0 54.0 Relative Percent Change from Baseline to Day 28 Mean (SD) -2.47 (10.34) 1.82 (9.81) 2.99 (14.54) 7.39 (17.48) 5.13(16.06) Median -3.74 2.30 0.00 4.88 4.00 P- value 0.2655 0.1411 0.0034 0.0113 TABLE 29 .. MITT population - Changes in % predicted FEV, Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Day 1 Mean (SD) 52.4 (13.42) 52.9(17.68) 55.4 (14,41) 48.8 (15.15) 52.0(15.07) Median 53.0 53.0 56.0 46.0 51.0 Day 14 Mean (SD) 51.7 (13.84) 53.9 (17.42) 57.1 (14.96) 52.1 (17.16) 54.6(16.20) Median 53.0 53.0 61.0 49.5 52.0 -60- 2015275224 22 Dec 2015 MITT population - Changes In % predicted FEV-i Placebo (N= 37) Levofloxacin 120mg QD (N= 38) Levofloxacin 240mg QD (N= 37) Levofloxacin 240mg BID (N= 39) Combined Levofloxacin 240mg QD and BID (N= 76) Mean (SD) -0.68 (3.37) 0.81 (3.24) 1.70 (5.39) 3.66 (7.49) 2.69 (6.57) Median -1.00 1.00 1.00 2.50 2.00 Relative Percent Change from Mean (SD) -1.30 (6.58) 2.46 (8.47) 3.51 (10.83) 8.30 (15.80) 5.93 (13.70) Median -1.89 1.15 1.52 7.42 4.00 Baseline to Day 14 P- value 0.2284 0.0780 0.0005 0.0025 Day 28 Mean (SD) 50.9 (14.46) 54.1 (19.19) 56.7 (15.33) 50.8 (15.40) 53.8 ¢15.54) Median 52.0 50.0 54.0 49.0 53.5 Relative Percent Change from Mean (SD) -3.08 (9.82) 2.00 (10.07) 2.77 (14.30) 7.97 (19.49) 5.37(17.18) Median -3.77 2.30 0.00 4.44 3.85 Baseline to Day 28 P- value 0.1759 0.0888 0.0008 0.0036 TABLE30 EE population - Changes in FEF 25-75 values Placebo (N= 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) Day 1 Mean (SD) 1.02 (0.57) 1.17(0.78) 1.22 (0.84) 0.85 (0.53) 1.04(0.72) Median 0.94 0.98 1.10 0.68 0.83 Day 14 Mean (SD) 1.03 (0.64) 1.18(0.83) 1.33 (0.93) 1.00 (0.78) 1.17(0.87) Median 0.86 0.99 1.11 0.78 0.92 Percent Change from Baseline to Day 14 Mean (SD) 1.95 (19.45) 0.41 (12.28) 9.58 (21.16) 16.99 (32.60) 13.23 (27.45) Median -1.72 0.45 4.41 11.66 8.79 P- value 0.6634 0.2532 0.0072 0.0275 -61- 2015275224 22 Dec 2015 EE population - Changes in FEF 25-75 values Placebo (N= 32) Levofloxacin 120mg QD (N= 35) Levofloxacin 240mg QD (N= 35) Levofloxacin 240mg BID (N= 34) Combined Levofloxacin 240mg QD and BID (N= 69) Day 28 Mean (SD) 1.00 (0.64) 1.23 (0.93) 1.27(0.88) 0.93 (0,59) 1.10(0.77) Median 0.94 1.03 1.15 0.78 0.97 Percent Change from Baseline to Day 28 Mean (SD) -2.73 (16.74) 0.73 (15.76) 4.70(16.11) 13.11 (28.50) 8.78 (23.19) Median -4.61 2.70 5.77 5.71 5.74 P- value 0.6038 0.2223 0.0007 0.0076 TABLE 31 MITT population - Changes in FEF 25-75 values Placebo (N= 37} Levofloxacin solution 120mg QD (N= 38) Levofloxacin solution 240mg QD (N= 37) Levofloxacin solution 240mg BID (N= 39) Combined Levofloxacin solution 240mg QD and BID (N= 76) Day 1 Mean (SD) 0.98 (0.55) 1.12(0.77) 1.18(0.83) 0.87 (0.53) 1.02 (0.71) Median 0.92 0.89 1.09 0.68 0.83 Day 14 Mean (SD) 0.97 (0.63) 1.15(0.82) 1.29 (0.92) 1.01 (0.78) 1.15(0.86) Median 0.81 0.85 1.08 0.78 0.90 Percent Change from Baseline to Day 14 Mean (SD) -1.78 (22.86) 1.73 (13.76) 9.53 (20.70) 16.70 (31.72) 13.16(26.92) Median -4.46 1.35 4.41 11.66 8.79 P- value 0.5910 0.0699 0.0007 0.0027 Day 28 Mean (SD) 0.94 (0.62) 1.20 (0.92) 1.23 (0.87) 0.95 (0.61) 1.09 (0.76) Median 0.91 0.83 1.12 0.82 0.93 Percent Change from Baseline to Day 28 Mean (SD) -6.40 (20.27) 1.94(16.55) 5.02 (15.98) 14.19 (28.56) 9.61 (23.44) Median -8.66 3.38 5.77 7.46 5.88 P- vaiue 0.1241 0.0489 <.0001 0.0003 -62- 2015275224 22 Dec 2015 [0163] In the EE population, FEVi values for patients administered levofloxacin 240 mg BID increased from a median value of 1.70 L at Day I (baseline) to 1.79 L at Day 28, representing an increase in FEVi of approximately 5%. In patients administered levofloxacin 240 mg QD, FEVi values increased from a median value of 2.02 L on Day 1 to a median value of 2.15 L on Day 28, representing an increase in FEVi of approximately 6%. In patients administered levofloxacin 120 mg QD, FEVI values increased from a median value of 1.87 L on Day 1 to a median value of 1.91 L on Day 28, representing an increase in FEVI of approximately 2%. The largest differences for FEVj between patients administered placebo and patients administered levofloxacin were 0.16 L on Day 14, and 0.24 L on Day 28.

[0164] In the MITT population, FEVi values for patients administered levofloxacin 240 mg BID increased from a median value of 1.70 L at Day 1 (baseline) to 1.82 L at Day 28, representing an increase in FEV] of approximately 7%. In patients administered levofloxacin 240 mg QD, FEV] values increased from a median value of 1.90 L on Day 1 to a median value of 2.10 L on Day 28, representing an increase in FEVi of approximately 10%. The largest differences for FEVi between patients administered placebo and patients administered levofloxacin were 0.12 L on Day 14, and 0.25 L on Day 28.

[0165] FEF 25-75 values relate to the average flow of air coming out of the lungs during the middle portion of the expiration. In small airway diseases this value can be reduced. In the EE population, FEF 25-75 values for patients administered levofloxacin 240 mg BID increased from a median value of 0.68 at Day I (baseline) to 0.78 at Day 28, representing an increase in FEF 25-75 of approximately 15%. In patients administered levofloxacin 240 mg QD, FEF 25-75 values increased from a median value of 1.10 on Day 1 to a median value of 1.15 on Day 28, representing an increase in FEF 25-75 of approximately 4%. In patients administered levofloxacin 120 mg QD, FEF 25-75 values increased from a median value of 0.98 on Day 1 to a median value of 1.03 on Day 28, representing an increase in FEF 25-75 of approximately 5%. The largest differences for FEF 25-75 between patients administered placebo and patients administered levofloxacin were 0.25 L on Day 14, and 0.21 L on Day 28.

[0166] In the MITT population, FEF 25-75 values for patients administered levofloxacin 240 mg BID increased from a median value of 0.68 at Day 1 (baseline) to -63-0.82 at Day 28, representing an increase in FEF 25-75 of approximately 20%. In patients administered levofloxacin 240 mg QD, FEF 25-75 values increased from a median value of 1.09 on Day 1 to a median value of 1.12 on Day 28, representing an increase in FEF 25-75 of approximately 3%. The largest differences for FEF 25-75 between patients administered placebo and patients administered levofloxacin were 0.27 L on Day 14, and 0.21 L on Day 28. 2015275224 22 Dec 2015

Safety evaluations [0167] Adverse events and drug intolerability from Day 1 through the end of study were evaluated. No significant adverse events were reported.

[0168] Flouroquinolone-induced arthralgia and myalgia have been previously reported in the use of some fluoroquinolones, for example in the treatment of sinusitis (O-Lee T., et al “Fluoroquinolone-induced arthralgia and myalgia in the treatment of sinusitis" Am. J. Rhinol. (2005) 19:395-9, incorporated by reference in its entirety). In this study, Arthralgia was reported in 5.4% of patients administered with placebo. No arthralgia was reported in patients administered any levofloxacin formulations.

Example 3—Phase I Clinical Study in Pediatric CF Patients [0169] A Phase 1 multicenter, open-label study was carried out to evaluate the safety, tolerability and pharmacokinetics of weight-adjusted doses of levofloxacin formulated with MgCh administered once daily for 14 days to stable pediatric CF patients. Patients were divided into 2 groups based on their age: 6-11 years of age and 12-16 years of age. The daily dose administered of levofloxacin formulated with MgCk was divided as follows: patients that weighed 14-21 kg received a 120 mg dose, patients that weighed 22-30 kg received a 180 mg dose, and patients that weighed more than 30 kg received a 240 mg dose. A total of 27 patients were enrolled and all patients completed the study. There were 14 patients in the 6-11 years of age group and 13 patients in the 12-16 years of age group. Seven patients (all in the 6-11 age group) received 180 mg QD levofloxacin formulated with MgCk and the remaining 20 patients received 240 mg QD levofloxacin formulated with MgC^. Figure 10 shows a graph of dose normalized serum AUC in pediatric CF patients vs. patient body weight. Figures 11A-9B show graphs of dose normalized serum AUC in pediatric CF patients vs. patient age, and vs. BSA, -64-respectively. Figures 11C-9E show graphs of dose normalized serum Cmax in pediatric CF patients vs. patient body weight, vs. patient age, and vs. BSA, respectively. 2015275224 22 Dec 2015 [0170] Serum levofloxacin exposures with either the 180 or 240 mg dose of levofloxacin formulated with MgCl2 appear to be in the ranges observed in adults CF patients studied in a related clinical trial (data not shown).

Example 4—Phase III Clinical Study [0171] A Phase 3, multi-center, multinational, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of levofloxacin formulated with MgCb in stable CF patients is performed. Following a 14-day screening period, patients are randomized at Visit 1/Day 1 in a 2;1 ratio to receive levofloxacin formulated with MgC^ or placebo. Randomization is stratified by geographic region (US vs. non-US), age (12-18 years vs. >18 years of age) and by FEVi percent predicted (< 55% vs. > 55%). Patients receive 28 days of either levofloxacin formulated with MgCl2 or placebo followed by 28 days of observation. Patients should remain off anti-pseudomonal antimicrobials, other than Study Drug (levofloxacin formulated with MgCl2 or placebo) and maintenance oral azithromycin (if applicable), for the duration of the study unless they meet the protocol defined definition of an exacerbation or unless determined to be necessary for safety reasons by the Investigators. The end of the study is defined as the last visit of the last patient.

[0172] Levofloxacin formulated with MgCl2 (levofloxacin inhalation solution, Aeroquin™) will be provided in single use ampules ready for administration. Each ampule contains 240 mg of levofloxacin formulated with MgCl2 in 2.4 mL (100 mg/mL). A 240 mg dose (1 ampule) is administered using a PARI investigational eFlow® nebulizer twice daily (BID) approximately 8-12 hours apart. Placebo is riboflavin 5'-phosphate (solubilized form of vitamin B2) in 0.9% saline provided in single use ampules ready for administration. Each ampule contains 9.6 pg of riboflavin 5'-phosphate in a volume of 2.4 mL. A PARI investigational eFIow® nebulizer is optimized and customized for use only with levofloxacin formulated with MgCl2. Both levofloxacin formulated with MgCl2 and the placebo control are administered using this nebulizer system. Analysis populations include: (1) safety/modified intent to treat (MITT) population (all patients enrolled in the study who receive at least one dose of Study Drug (levofloxacin formulated with MgCl2 or placebo); (2) efficacy evaluable Population (all patients -65-enrolled in the study, without major protocol violations, who receive at least 80% of Study Drug (levofloxacin formulated with MgCl2/placebo) doses); and (3) pharmacokinetic population (all patients who receive a least one dose of Study Drug (levofloxacin formulated with MgCl2/placebo) and have at least one pharmacokinetic (PK) blood or sputum sample collected). 2015275224 22 Dec 2015 [0173] Inclusion criteria for study patients included : at least 12 years of age; weigh at least 30 kg or 66 pounds; and have documented CF diagnosis as evidenced by one or more clinical features consistent with the CF phenotype. Exclusion criteria for study patients included: use of an investigational agent within 28 days prior to Visit 1; use of any nebulized or systemic antimicrobials active against P. aeruginosa within 28 days prior to Visit 1, other than maintenance oral azithromycin, which must have been initiated at least 28 days prior to Visit 1; and use of oral corticosteroids in doses exceeding the equivalent of 10 mg prednisone/day or 20 mg prednisone every other day at Screening or Visit 1.

Primary Efficacy Evaluations [0174] The primary endpoint includes the time (in days) to an exacerbation from Baseline (Visitl/Day 1) until Final Visit. To meet the endpoint a patient must concurrently meet 4 of the 12 symptoms/signs that make up the Fuchs definition of an exacerbation (Fuchs HJ, et al. Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. N Engl J Med 1994;331:637-642, incorporated by reference in its entirety). [0190] The 12 symptoms/signs defined by the Fuchs criteria include: change in sputum; new or increased hemoptysis; increased cough; increased dyspnea; malaise, fatigue or lethargy; temperature above 38°C; anorexia or weight loss; sinus pain or tenderness; change in sinus discharge; change in physical examination of the chest; decrease in pulmonary function by 10 % or more from a previously recorded value; and radiographic changes indicative of pulmonary infection [0175] The above symptoms associated with an exacerbation are recorded from each patient using a standardized questionnaire, the Respiratory and Systemic Symptoms Questionnaire (RSSQ) at patient visits. Changes the patient experiences are relative to what the patient considers normal on a day-to-day basis. It is anticipated that patients administered aerosolized levofloxacin formulated with MgCl2 provide the -66-following signs/symptoms that are included in the Fuchs criteria and that are recorded using the RSSQ: 2015275224 22 Dec 2015 (1) Increased sputum production: patients have no change, a little less or much less amounts of sputum when coughing up. (2) Change in sputum appearance: for sputum thickness, patients have a little thinner or much thinner sputum; for sputum color, patients have a better color of sputum (better increases from brown -> green -> yellow -> clear). (3) Increased chest congestion: patients have a little decrease, or a large decrease in chest congestion. (4) New or increased coughing up of blood: patients have a little decrease or a large decrease in the amount of blood coughed up. (5) Increased cough: for intensity of cough, patients have a little lighter, or much lighter coughs; for frequency of cough, patients cough a little less often or much less often. (6) Decreased exercise tolerance: patients perform daily activities, e.g., climbing stairs, a little easier, or much easier. (7) Increased dyspnea with exertion: patients breathe a little easier or much easier when performing daily activities (8) Malaise, fatigue or lethargy: patients have a little more energy or much more energy since last visit. (9) Fever: patients have no fever since last visit (10) Weight loss: patients have no change in weight, or a little weight gain. (11) Sinus pain and tenderness: patient has no pain or tenderness. (12) Change in sinus discharge: patient has better sinus discharge (a decrease in thickness and/or better color). (13) School or work absenteeism (due to illness): patient has no absenteeism from schedules activities. (14) Decreased appetite: patient has a little increase in appetite.

Secondary Efficacy Evaluations (0176] Secondary Endpoints include clinical, pulmonary function, microbiology, and patient reported outcome characteristics. -67-

Clinical characteristics 2015275224 22 Dec 2015 [0177] Clinical characteristics include the time (in days) to administration of systemic (oral or IV) and/or inhaled anti-pseudomonal antimicrobials from Baseline (Visit 1/Day 1) until Final Visit. To meet this endpoint, patients must have at least one of four worsening respiratory symptoms (increased cough, increased sputum/chest congestion, decreased exercise tolerance, decreased appetite) at the time of administration of the anti-pseudomonal antimicrobial agent. Clinical characteristics also include the proportion of patients who miss at least 1 day of school/work secondary to worsening respiratory status.

[0178] It is anticipated that patients administered aerosolized levofloxacin formulated with MgCb have an increased time to administration of systemic (oral or IV) and/or inhaled anti-pseudomonal antimicrobials from Baseline (Visit 1/Day 1) until Final Visit compared to patients administered placebo. In addition, the proportion of patients administered aerosolized levofloxacin formulated with MgC^ who miss at least 1 day of school/work secondary to worsening respiratory status is less than the proportion of patients administered placebo who miss at least 1 day of school/work secondary to worsening respiratory status.

Pulmonary Function characteristics [0179] Pulmonary function characteristics include: percent change in FEY1 (L) from Baseline to Day 28; relative change in FEV1 (percent predicted) from Baseline to Day 28; percent change in FEF25-75 (L/s) from Baseline to Day 28; percent change in FVC (L) from Baseline to Day 28; and categorical assessment of percent change in FEV1 (L) and relative change in percent predicted FEV1 from Baseline to Day 28.

[0180] It is anticipated that patients administered aerosolized levofloxacin formulated with MgCb have more advantageous pulmonary function characteristics compared to patients administered placebo.

Microbiology characteristics [0181] Microbiology characteristics include: change in P. aeruginosa density (loglO colony-forming units [CFU] per gram sputum) from Baseline to Day 28; categorical assessment of change in P. aeruginosa density (loglO colony-forming units [CFU] per gram sputum) from Baseline to Day 28; and change in Stenotrophomonas sp., Achromobacter sp., Burkhalderia sp. and S. aureus density (loglO colony-forming units [CFU] per gram sputum) from Baseline to Day 28. -68- [0182] It is anticipated that patients administered aerosolized levofloxacin formulated with MgCl2 have more advantageous microbiology characteristics, e.g., decreased P. aeruginosa sputum density, decreased Stenotrophomonas sp., Achromobacter sp., Burkholderia sp. and S. aureus sputum density, compared to patients administered placebo. 2015275224 22 Dec 2015

Patient reported outcome characteristics [0183] Patient reported outcome characteristics include: change in the respiratory domain of the CFQ-R from Baseline to Day 28; and categorical assessment of change in the respiratory domain of the CFQ-R from Baseline to Day 28.

[0184] It is anticipated that patients administered aerosolized levofloxacin formulated with MgCl2 have more advantageous patient reported outcome characteristics than patients administered placebo.

Example 5—In vivo antibacterial activity of levofloxacin inhalation solution against Burkholderia cepacia [0185] Burkholderia cepacia is an opportunistic pathogen capable of causing pulmonary infection in CF patients. Infections with certain strains of B. cepacia cause “cepacia syndrome” which is characterized by progressive and invasive necrotizing pneumonia and septicemia. Most B. cepacia have high MICs to many antibiotics. Aerosolized levofloxacin formulated with MgCl2 enables aerosol delivery of high drug concentrations to the lung.

[0186] Five B. cepacia strains with levofloxacin MICs ranging between 0.25 -8 mg/L were tested in a mouse model of pulmonary infection. Female BALB/c mice were injected with 150 mg/kg of cyclophosphamide 3 days prior to infection. On day 4, the mice were infected with 50 μΐ of bacterial suspension (~106 CFU/ml) using a curved bead-tipped oral gavage syringe under isoflurane anesthesia. Treatment with levofloxacin formulated with MgCl2 (60 mg/kg BID) or saline only was initiated 72 hours postinfection and continued twice daily for four days using a microspray aerosol device. Sixteen hours after the last treatment, mice were sacrificed, lungs halves ted, homogenized, and plated to determine colony counts (CFU).

[0187] As part of a Phase 2b trial of aerosolized levofloxacin formulated with MgCl2 in CF patients, a 16 year old male patient infected with B. cepacia (levofloxacin MIC = >128 mg/L) received levofloxacin formulated with MgCl2 240 mg QD for 28 days. -69- [0188] Aerosolized levofloxacin formulated with MgCl2 produced at least one log CFU of bacterial killing for all strains in the mouse infection model (Table 32). In the CF patient, a 1.7 log CFU decrease in bacterial counts was observed over 28 days. TABLE 32

Genomovar il Genomovar III Strains BC1012 | BC1013 BC1014 BC1020 BC1021 MIC (mg/L) 4 | 0.25 1 8 8 Mean Change in Log CFU/Lungs -1.03 -2.08 -1.47 -1.11 -1.35 2015275224 22 Dec 2015 [0189] An increase in the CF patient’s FEV1 was observed. On day 1 of the study, the patient’s FEV1 was 1.21L, this increased to 1.30L on day 28, a 7% improvement. From a population model, Cmax and AUC values were calculated to be approximately 12,900 mg/L and 4,400 mg*h/L, respectively.

[0190] Aerosol administration of levofloxacin formulated with MgCl2 produced significant bacterial killing in strains with a wide range of MICs. The non-clinical and clinical data support the future clinical evaluation of levofloxacin formulated with MgCl2 in the management of chronic pulmonary infections due to B. cepacia.

[0191] To the extent publications and patents or patent applications incorporated by reference herein contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

[0192] Unless otherwise defined, all terms (including technical and scientific terms) are to be given their ordinary and customary meaning to a person of ordinary skill in the art, and are not to be limited to a special or customized meaning unless expressly so defined herein.

[0193] Terms and phrases used in this application, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation’ or the like; the term ‘comprising’ as used herein is synonymous with ‘including,’ ‘containing,’ or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term ‘example’ is used to provide exemplary instances of the item in discussion, not an -70- exhaustive or limiting list thereof; adjectives such as ‘known’, ‘normal’, ‘standard’, and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass known, normal, or standard technologies that may be available or known now or at any time in the future; and use of terms like ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important to the structure or function of the invention, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the invention. Likewise, a group of items linked with the conjunction ‘and’ should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as ‘and/or’ unless expressly stated otherwise. Similarly, a group of items linked with the conjunction ‘or’ should not be read as requiring mutual exclusivity among that group, but rather should be read as ‘and/or’ unless expressly stated otherwise. In addition, as used in this application, the articles ‘a’ and ‘an’ should be construed as referring to one or more than one (/. e., to at least one) of the grammatical objects of the article. By way of example, ‘an element’ means one element or more than one element. 2015275224 22 Dec 2015 [0194] The presence in some instances of broadening words and phrases such as ‘one or more’, ‘at least’, ‘but not limited to’, or other like phrases shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent.

[0195] All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term ‘about.’ Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0196] Furthermore, although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, it is apparent to those skilled in the art that certain changes and modifications may be -71-practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention to the specific embodiments and examples described herein, but rather to also cover all modification and alternatives coming with the true scope and spirit of the invention. 2015275224 22 Dec 2015 -72-

Claims (34)

1. WHAT IS CLAIMED IS:

   1. The use of an aerosol of a solution for the manufacture of a medicament for the treatment of a pulmonary Pseudomonas aeruginosa infection in a human with cystic fibrosis; wherein the solution comprises levofloxacin at a concentration between about 75 mg/ml to about 150 mg/ml, a magnesium cation concentration between about 150 mM to about 250 mM, a pH between about 5 to about 7; and an osmolality of between about 300 mOsmol/kg to about 500 mOsmol/kg; and wherein the solution comprises about 240 mg levofloxacin and the aerosol is administered to the human twice daily for about 28 days.
2. 2. The use of an aerosol of a solution for the manufacture of a medicament in accordance with claim 1 wherein the solution comprises levofloxacin at a concentration from about 90 mg/ml to about 110 mg/ml, and a magnesium cation at a concentration from about 175 mM to about 225 mM wherein the solution has a pH from about 5 to about 7, and an osmolality from about 300 mOsmol/kg to about 500 mOsmol/kg.
3. 3. The use of claim 1 or claim 2, wherein the magnesium cation is in the form of magnesium chloride.
4. 4. The use of claim 3, wherein the solution comprises a levofloxacin concentration between about 90 mg/ml to about 110 mg/ml, a magnesium chloride concentration between about 175 mM to about 225 mM, a pH between about 5 to about 7; and an osmolality of between about 300 mOsmol/kg to about 500 mOsmol/kg.
5. 5. The use of claim 3 or 4, wherein the solution comprises a levofloxacin concentration of about 100 mg/ml, and a magnesium chloride concentration of about 200 mM.
6. 6. The use of any one of claims 3 to 5, wherein the solution comprises a levofloxacin concentration of 100 mg/ml, a magnesium concentration of 200 mM, a chloride concentration of 400 mM, a pH of from 5 to 7 and an osmolality of from 350 to 500 mOsmol/kg.
7. 7. The use of any one of claims 3 to 5, wherein the solution comprises a levofloxacin concentration of about 100 mg/ml, a magnesium chloride concentration of about 200 mM, a pH between about 6.0 to about 6.5, and an osmolality of between about 300 mOsmol/kg to about 500 mOsmol/kg.
8. 8. The use of any of claims 1 to 7, wherein the aerosol is administered to the human twice daily with an interval between doses of 8 to 12 hours.
9. 9. The use of any of claims 1 to 8, wherein the aerosol is produced using a vibrating mesh nebulizer.
10. 10. The use of any of claims 1 to 9, wherein the aerosol is administered to the human in less than about 10 minutes.
11. 11. The use of any one of claims 1 to 10, wherein the aerosol of the solution comprises a mass median aerodynamic diameter from about 2 microns to about 5 microns with a geometric standard deviation less than or equal to 2.5 microns.
12. 12. The use of an aerosol of a solution for the manufacture of a medicament for the treatment of a pulmonary Pseudomonas aeruginosa infection in a human with cystic fibrosis; of any one of claims claim 1 to 11, wherein the treatment further comprises administering an antibiotic, a bronchodilator, an anticholinergic agent, a glucocorticoid, an eicosanoid inhibitor, or a combination of two or more thereof.
13. 13. The use of any of claims 1 to 12, wherein the human is treated concomitantly with a medication selected from dornase alfa, azithromycin, salbutamol, pancrelipase, hypertonic sodium chloride, seretide, and ADEK.
14. 14. The use of any one of claims 1 to 12, further comprising administering by inhalation dornase alfa, azithromycin, salbutamol, pancrelipase, sodium chloride, seretide, vitamin A, vitamin D, vitamin E, vitamin K, or a combination of two or more thereof.
15. 15. The use of any one of claims 1 to 14, further comprising the step of discontinuing administration of the levofloxacin for 28 days.
16. 16. The use of any one of claims 1 to 15, wherein the pulmonary infection further comprises one or more bacteria selected from the group consisting of Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholera, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Burkholderia cepacia, Francisella tularensis, Kingella, and Moraxella.
17. 17. The use of any one of claim 1 to 16, wherein the human is a pediatric patient.
18. 18. A method for treating a pulmonary infection in a human having cystic fibrosis, wherein the pulmonary infection comprises Pseudomonas aeruginosa, the method comprising administering via inhalation 240 mg of levofloxacin twice daily for 28 days to the human having cystic fibrosis to treat the Pseudomonas aeruginosa pulmonary infection; wherein the levofloxacin is in an aerosol of a solution comprising levofloxacin at a concentration between about 75 mg/ml to about 150 mg/ml, a magnesium cation concentration between about 150 mM to about 250 mM, a pH between about 5 to about 7; and an osmolality of between about 300 mOsmol/kg to about 500 mOsmol/kg; and wherein the solution comprises about 240 mg levofloxacin and the aerosol is administered to the human twice daily for about 28 days.
19. 19. The method of claim 18 wherein the solution comprises levofloxacin at a concentration from about 90 mg/ml to about 110 mg/ml, a magnesium cation at a concentration from about 175 mM to about 225 mM, wherein the solution has a pH from about 5 to about 7, and an osmolality from about 300 mOsmol/kg to about 500 mOsmol/kg.
20. 20. The method of claim 18 or 19, wherein the magnesium cation is in the form of magnesium chloride.
21. 21. The method of claim 20, wherein the solution comprises a levofloxacin concentration between about 90 mg/ml to about 110 mg/ml, a magnesium chloride concentration between about 175 mM to about 225 mM, a pH between about 5 to about 7; and an osmolality of between about 300 mOsmol/kg to about 500 mOsmol/kg.
22. 22. The method of claim 20 or claim 21, wherein the solution comprises a levofloxacin concentration of about 100 mg/ml, and a magnesium chloride concentration of about 200 mM.
23. 23. The method of any one of claims 20 to 22, wherein the solution comprises a levofloxacin concentration of 100 mg/ml, a magnesium concentration of 200 mM, a chloride concentration of 400 mM, a pH of from 5 to 7 and an osmolality of from 350 to 500 mOsmol/kg.
24. 24. The method of any one of claims 20 to 22, wherein the solution comprises a levofloxacin concentration of about 100 mg/ml, a magnesium chloride concentration of about 200 mM, a pH between about 6.0 to about 6.5, and an osmolarity of between about 300 mOsmol/kg to about 500 mOsmol/kg.
25. 25. The method of any of claims 18 to 24, wherein the aerosol is administered to the human twice daily with an interval between doses of 8 to 12 hours.
26. 26. The use of any of claims 18 to 25, wherein the aerosol is produced using a vibrating mesh nebulizer.
27. 27. The method of any of claims 18 to 26, wherein the aerosol is administered to the human in less than about 10 minutes.
28. 28. The method of any one of claims 18 to 27, wherein the aerosol of the solution comprises a mass median aerodynamic diameter from about 2 microns to about 5 microns with a geometric standard deviation less than or equal to 2.5 microns.
29. 29. The method of any one of claims 18 to 28, further comprising administering an antibiotic, a bronchodilator, an anticholinergic agent, a glucocorticoid, an eicosanoid inhibitor, or a combination of two or more thereof.
30. 30. The method of any of claims 18 to 29, wherein the human is treated concomitantly with a medication selected from domase alfa, azithromycin, salbutamol, pancrelipase, hypertonic sodium chloride, seretide, and ADEK.
31. 31. The method of any one of claims 18 to 29, further comprising administering by inhalation dornase alfa, azithromycin, salbutamol, pancrelipase, sodium chloride, seretide, vitamin A, vitamin D, vitamin E, vitamin K, or a combination of two or more thereof.
32. 32. The method of any one of claims 18 to 31, further comprising the step of discontinuing administration of the levofloxacin for 28 days.
33. 33. The method of any one of claims 18 to 32, wherein the pulmonary infection further comprises one or more bacteria selected from the group consisting of Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholera, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Burkholderia cepacia, Francisella tularensis, Kingella, and Moraxella.
34. 34. The method of any one of claim 18 to 33, wherein the human is a pediatric patient.