Patent Publication Number: US-8980226-B2

Title: Aerosolized fosfomycin/aminoglycoside combination for the treatment of bacterial respiratory infections

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a continuation of U.S. application Ser. No. 13/085,576, filed Apr. 13, 2011, now U.S. Pat. No. 8,409,549, which is a continuation of U.S. application Ser. No. 11/596,566, filed Nov. 15, 2006, which is the U.S. National Stage of International Application No. PCT/US2005/014690 filed May 2, 2005, now U.S. Pat. No. 7,943,118, which claims priority of U.S. Provisional Applications 60/571,739 filed May 17, 2004 and 60/659,005 filed Mar. 3, 2005, the disclosure of which is incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The current invention concerns a novel, safe, nonirritating and physiologically compatible inhalable fosfomycin plus aminoglycoside combination formulation suitable for treatment of respiratory bacterial infections caused by Gram-negative bacteria, such as  Burkholderia cepacia, Citrobacter  species,  Escherichia coli, Enterobacter  species,  Fusobacterium  species,  Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella oxytoca, Moraxella catarrhalis, Proteus mirabilis, Prevotella  species,  Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans , and Gram-positive bacteria, such as methicillin-resistant  Staphylococcus aureus , methicillin-sensitive  Staphylococcus aureus, Streptococcus pneumoniae , and β-hemolytic  Streptococcus  species. 
     BACKGROUND OF THE INVENTION 
     The most widely accepted therapy for treating respiratory infections caused by Gram-negative bacteria in cystic fibrosis patients involves intravenous administration of a single antibiotic or combinations of antibiotics (Gibson et al., 2003; Ramsey, 1996). This method of treatment has several significant limitations including: (1) narrow spectrum of activity of existing antibiotics, (2) insufficient concentrations of antibiotic reaching the respiratory tract to ensure rapid onset and high rates of bacterial killing, and (3) development of adverse side affects due to high systemic concentrations of drug. 
     Aerosol administration of antibiotics (Conway, 2005; O&#39;Riordan, 2000) addresses several of the limitations of parenteral administration (Flume and Klepser, 2002; Kuhn, 2001). It enables topical delivery of high concentrations of drug to the endobronchial spaces and reduces side effects by lowering systemic exposure to antibiotic. However, cystic fibrosis patients typically receive prolonged and repeated antibiotic therapies over the entire duration of their adult lives (Gibson et al., 2003; Ramsey, 1996). Therefore, cumulative aminoglycoside toxicity and development of resistance remains a significant problem. 
     Fosfomycin 
     Fosfomycin is a broad spectrum phosphonic acid antibiotic (Kahan et. al., 1974; Woodruff et al., 1977) that has bactericidal activity against Gram-negative bacteria including  Citrobacter  spp.,  E. coli, Enterobacter  spp.,  K. pneumoniae, P. aeruginosa, Salmonella  spp.,  Shigella  spp., and  S. marcescens  (Greenwood et. al., 1992; Grimm, 1979; Marchese et. al., 2003; Schulin, 2002), as well as Gram-positive bacteria including vancomycin resistant enterococci, methicillin-resistant  S. aureus  (MRSA), methicillin-sensitive  S. aureus  (MSSA), and  S. pneumoniae  (Greenwood et. al., 1992; Grimm, 1979; Perri et. al., 2002). Fosfomycin has the greatest activity against  E. coli, Proteus  spp.,  Salmonella  spp.,  Shigella  spp., and  S. marcescens  which are generally inhibited at fosfomycin concentrations ≦64 μg/mL (Forsgren and Walder, 1983). Fosfomycin is moderately active against  P. aeruginosa  (Forsgren and Walder, 1983), particularly when compared to tobramycin (Schulin, 2002). 
     Fosfomycin is bactericidal but exhibits time-dependent killing against  E. coli  and  S. aureus  (Grif et al., 2001). The rate and degree of killing depends on the length of time fosfomycin is in contact with the target organism (Craig, 1998; Mueller et al., 2004). Increasing the fosfomycin concentration will not produce a corresponding increase in the rate or degree of killing activity. This feature is significant because it is preferable to treat  P. aeruginosa  infections with antibiotics that exhibit bactericidal, concentration-dependent killing activity (Craig, 1998; Mueller et. al., 2004). 
     Fosfomycin monotherapy is commonly used in the treatment of uncomplicated urinary tract infections caused by  E. coli , and less frequently in treating bacterial respiratory infections including in patients with cystic fibrosis (Kamijyo et al., 2001; Katznelson et al., 1984; Kondo et al., 1996; Reeves, 1994; Bacardi et al., 1977; Bonora et al., 1977; Honorato et al., 1977; Menendez et al., 1977). However, fosfomycin alone has not been widely used to treat infections caused by  P. aeruginosa . Fosfomycin has been administered parenterally in combination with antibiotics of different classes to treat endocarditis (Moreno, et. al., 1986) and cystic fibrosis (Mirakhur et. al., 2003) but has not been delivered directly to the lung environment by aerosol administration. 
     Fosfomycin is available in both oral (fosfomycin calcium and fosfomycin trometamol) and intravenous (fosfomycin disodium) formulations (Woodruff et al., 1977). Fosfomycin trometamol is the preferred formulation for oral administration because it is more readily absorbed into the blood compared to fosfomycin calcium. Following a single intravenous or intramuscular dose of 2 g of fosfomycin, peak serum concentrations range between 25-95 μg/mL within 1-2 hours (Woodruff et al., 1977). By comparison, concentrations reach 1-13 μg/mL in normal lung after parenteral administration of a comparable dose of fosfomycin (Bonora et al., 1977) which is an insufficient to kill most bacterial pathogens, in particular  P. aeruginosa  (Forsgren and Walder, 1983; Schulin, 2002). Patients with cystic fibrosis have altered pharmacokinetics characterized by an increased volume of distribution and rate of clearance (Tan et al., 2003), which would likely further decrease the efficacy of parenterally administered fosfomycin. 
     Fosfomycin is widely distributed in various body tissues and fluids but does not significantly bind to plasma proteins (Mirakhur et al., 2003). Consequently, fosfomycin is available to exert antibacterial effects if it reaches sufficient concentrations at the site of infection. The respiratory tract of cystic fibrosis patients are obstructed with viscous secretions called sputum (Ramsey, 1996). The effectiveness of several classes of antibiotics such as aminoglycosides and β-lactams is reduced due to poor penetration into sputum. Additionally, the activity of these antibiotics is further reduced by binding to sputum components (Hunt et al., 1995; Kuhn, 2001; Ramphal et al., 1988; Mendelman et al., 1985). 
     Development of resistance in bacteria isolated from patients treated with fosfomycin for urinary tract infections occurs very infrequently (Marchese et al., 2003). Cross-resistance with other classes of cell wall inhibiting antibiotics does not occur because fosfomycin acts on the enzyme phosphoenolpyruvate (UDP-N-acetylglucosamine enolpyruval-transferase) which is not targeted by other antibiotics (Kahan et al., 1974; Woodruff et al., 1977). Fosfomycin is actively taken up into bacterial cells by two transport systems; a constitutively functional L-α-glycerophosphate transport and the hexose-phosphate uptake system (Kahan et al., 1974). When fosfomycin resistance occurs, it is typically due to a genetic mutation in one or both of the chromosomally encoded transport systems, and less commonly by modifying enzymes (Area et al., 1997; Nilsson et al., 2003). 
     Tobramycin. 
     Tobramycin is an aminoglycoside antibiotic that is active against Gram-negative aerobic bacilli including  P. aeruginosa, E. coli, Acinetobacter  spp.,  Citrobacter  spp.,  Enterobacter  spp.,  K. pneumoniae, Proteus  spp.,  Salmonella  spp.,  S. marcescens , and  Shigella  spp (Vakulenko and Mobashery, 2003). In particular, tobramycin is highly active against  P. aeruginosa . The tobramycin MICs of susceptible  P. aeruginosa  are typically less than 2 μg/mL (Shawar et al., 1999; Spencker et al., 2002; Van Eldere, 2003). Most Gram-positive bacteria are resistant to tobramycin, with the exception of  S. aureus  and  S. epidermidis  (Vakulenko and Mobashery, 2003). 
     Tobramycin is rapidly bactericidal and exhibits concentration-dependent killing (Vakulenko and Mobashery, 2003). Increasing the tobramycin concentration increases both the rate and extent of bacterial killing. Therefore, to achieve therapeutic success, it is necessary to administer a large enough dose to produce a peak tobramycin level 5-10 times greater than the MIC of the target organism at the site of infection. It is preferable to treat  P. aeruginosa  infections with antibiotics that exhibit bactericidal, concentration-dependent killing activity (Ansorg et al., 1990). 
     Tobramycin is usually administered to treat less serious Gram-negative bacterial infections (Vakulenko and Mobashery, 2003). However, it may be combined with other classes of antibiotics to treat severe infections of the urinary tract and abdomen, as well as endocarditis and bacteremia (Vakulenko and Mobashery, 2003). Parenteral administration of tobramycin in combination with cell-wall inhibiting antibiotics has been used to treat respiratory infections, in particular those caused by  P. aeruginosa  in CF patients (Gibson et al., 2003; Lang et al., 2000; Ramsey et al., 1999; Ramsey et al., 1993; Smith et al., 1999; Spencker et al., 2003). 
     Tobramycin is poorly absorbed orally and must be administered parenterally (Hammett-Stabler and Johns, 1998). Tobramycin is available in both intravenous and aerosol formulations. After parenteral administration, tobramycin is primarily distributed within the extracellular fluid. Tobramycin is rapidly excreted by glomular filtration resulting in a plasma half-life of 1-2 hours (Tan et al., 2003). Penetration of tobramycin into respiratory secretions is very poor and its activity is further reduced by binding to sputum (Kuhn, 2001). Aerosol administration of tobramycin results in significantly higher sputum levels of ≧1000 μg/mL (Geller et al., 2002) compared with parenteral administration, but sputum binding remains a significant problem (Hunt et al., 1995; Mendelman et al., 1985; Ramphal et al., 1988). 
     Nephrotoxicity and ototoxicity are adverse reactions associated with tobramycin therapy (Al-Aloui et al., 2005; Hammett-Stabler and Johns, 1998). Nephrotoxicity results from accumulation of tobramycin within lysosomes of epithelial cells lining the proximal tubules. This causes an alteration of cell function and ultimately cell necrosis (Mingeot-Leclercq and Tulkens, 1999). Clinically, this presents as nonoliguric renal failure. The prevalence of nephrotoxicity in cystic fibrosis patients is estimated to be 31-42% (Al-Aloui et al., 2005). The incidence of ototoxicity, which is characterized by loss of hearing and dizziness, is estimated to be as high as 25% of patients treated with aminoglycosides (Hammett-Stabler and Johns, 1998). Unlike nephrotoxicity, ototoxicity is irreversible. The greatest risk factor for the development of toxicity is cumulative exposure to large doses of tobramycin (Hammett-Stabler and Johns, 1998; Mingeot-Leclercq and Tulkens, 1999). Cystic fibrosis patients are treated with prolonged and repeated high-dosages of tobramycin over their entire lifetime (Tan et al., 2003) and are at increased risk of developing cumulative renal failure (Al-Aloui et al., 2005). 
     Bacterial resistance to tobramycin has become increasingly prevalent and is due to repeated and prolonged antibiotic monotherapy (Conway et al., 2003; Van Eldere, 2003; Mirakhur et al., 2003; Pitt et al., 2005; Schulin, 2002). For example, Cystic fibrosis patients are colonized with  P. aeruginosa  strains which are largely resistant to tobramycin gentamicin, ceftazidime, piperacillin, and ciprofloxacin (Eldere, 2003; Pitt et al., 2005; Pitt et al., 2003; Weiss and Lapointe, 1995). Thus, existing antibiotic therapies are becoming ineffective for treating  P. aeruginosa  infections because of drug resistance. 
     It is clear that there is a continued need for an improved method of treatment for acute and chronic respiratory infections caused by Gram-negative and Gram-positive bacteria, particularly multidrug resistant  P. aeruginosa . This is particularly evident in cystic fibrosis patients where current therapies are limited by problems with development of resistance and toxicity. Such method of treatment would preferably comprise inhalation of an aerosolized antibiotic combination of fosfomycin and an aminoglycoside such as tobramycin that delivers a therapeutically effective amount of the drugs directly to the endobronchial space of the airways or to the nasal passages. Such treatment would be efficacious, reduce the frequency of drug resistance, and improve safety. 
     It would be highly advantageous to provide a formulation and system for delivery of a sufficient dose of fosfomycin plus an aminoglycoside such as tobramycin in a concentrated form, containing the smallest possible volume of solution or weight of dry powder which can be aerosolized and delivered predominantly to the endobronchial space. 
     Thus, it is an objective of this invention to provide a concentrated liquid or dry powder formulation of fosfomycin plus aminoglycoside which contains sufficient but not excessive amounts of fosfomycin and aminoglycoside which can be efficiently aerosolized by nebulization into aerosol particles sizes predominantly within a range of 1 to 5 um and having salinity that is adjusted to permit generation of a fosfomycin plus aminoglycoside aerosol well tolerated by patients, and which has an adequate shelf live. 
     SUMMARY OF THE INVENTION 
     One aspect of this invention is a method for treatment of upper respiratory tract infections like bacterial sinusitis and lower respiratory tract (pulmonary) infections like cystic fibrosis, chronic pulmonary  Pseudomonas  infection in cystic fibrosis patients,  Pseudomonas  infections after first infection, bronchiectasis, hospital and community acquired pneumonia, and ventilator associated pneumonia (VAP) caused by Gram-negative and Gram-positive bacteria comprising the step of administering an effective amount of an aerosol formulation comprising fosfomycin and tobramycin into the endobronchial or nasal space of patients in need of such treatment. 
     Another aspect of the current invention is a concentrated formulation either liquid or dry powder, suitable for delivery of a fosfomycin tobramycin combination into the endobronchial or nasal space of patients to treat lower and upper respiratory bacterial infections. 
     Still another aspect of the current invention is a formulation, either liquid or dry powder, suitable for delivery of aminoglycoside and fosfomycin into the endobronchial and nasal space of patients that reduces the development of antibiotic resistance compared to either drug used alone. 
     Still another aspect of the current invention is a formulation, either liquid or dry powder, suitable for delivery of an aminoglycoside and fosfomycin into the endobronchial or nasal space of patients that increases the post antibiotic affect (PAE) compared to the PAE of either the drug used alone. 
     Still another aspect of the current invention is a formulation comprising from 1-300 mg of fosfomycin and from 1 to 300 mg aminoglycoside in 0.5 to 7 mL of water with a chloride concentration &gt;30 mM wherein said formulation has a pH between 4.5 and 8.0 and is delivered by aerosolization. The aerosol contains particles that have a mass median aerodynamic diameter (MMAD) predominantly between 1 to 5μ, and is administered using a nebulizer able to atomize the particles of the required size. 
     Still another aspect of the current invention is a dry powder formulation comprising from about 1 to about 300 mg of fosfomycin, from about 1 to about 300 mg aminoglycoside and at least one pharmaceutically acceptable excipient in a micronized dry powder form that delivers particles with a MMAD of 1 and 5μ upon aerosolization. 
     The invention concerns an inhalable formulation comprising fosfomycin plus an aminoglycoside suitable for treatment of acute and chronic pulmonary bacterial infections, particularly those caused by the multidrug resistant Gram-negative bacteria  P. aeruginosa  which are resistant to treatment with β-lactams and aminoglycosides. The aerosol formulation has a small volume yet delivers a therapeutically efficacious dose of fosfomycin plus aminoglycoside to the site of infection in amounts sufficient to treat bacterial respiratory infections. The dry powder and reconstituted aminoglycoside plus fosfomycin formulations have a long shelf-life and storage stability. 
     The formulations of the present invention preferably comprise from 5 to 9 parts by weight fosfomycin and 1 to 5 parts by weight aminoglycoside, preferably from about 7 to 9 parts by weight fosfomycin and 1 to 3 parts by weight aminoglycoside and more preferably about 8 parts by weight fosfomycin and about 2 parts by weight aminoglycoside. The most preferred aminoglycoside is tobramycin. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1 and 2  show time-kill curves for a 9:1 fosfomycin:tobramycin combination and demonstrate rapid bactericidal killing of  P. aeruginosa  ATCC 27853 compared to bacteriostatic killing of fosfomycin and tobramycin alone. 
         FIGS. 3 and 4  show time-kill curves for 8:2 fosfomycin:tobramycin combinations and demonstrate rapid bactericidal killing of  P. aeruginosa  ATCC 27853. 
         FIGS. 5 ,  6  and  7  show time-kill curves for 7:3 fosfomycin:tobramycin combinations and demonstrate rapid bactericidal killing of  P. aeruginosa  ATCC 27853. 
         FIGS. 8 and 9  compare kill curves for 9:1, 8:2, and 7:3 fosfomycin:tobramycin combinations and tobramycin alone against  P. aeruginosa  ATCC 27853. 
         FIGS. 10 ,  11  and  12  demonstrate concentration-dependent killing of 9:1, 8:2, and 7:3 fosfomycin:tobramycin combinations against  P. aeruginosa  ATCC 27853. 
         FIGS. 13 ,  14 , and  15  show the killing of  E. coli  in the rat lung after aerosol administration of a 31.1 mg/mL solution of a 9:1, 8:2, and 7:3 fosfomycin:tobramycin combinations, respectively. 
         FIGS. 16 and 17  demonstrate that aerosol administration of a 31.1 mg/mL solution of a 9:1 fosfomycin:tobramycin combination resulted in &gt;5 log 10 killing of  E. coli  in the rat lung, compared to less than 1 log 10 killing fosfomycin and tobramycin alone. 
         FIG. 18  demonstrates that aerosol administration of a 31.1 mg/mL solution of a 8:2 fosfomycin:tobramycin combination resulted in &gt;5 log 10 killing of  E. coli  in the rat lung, compared to 2 log 10 killing by aerosol administration of a 40 mg/mL solution of tobramycin alone. 
         FIGS. 19 and 20  demonstrate that aerosol administration of a 31.1 mg/mL solution of a 8:2 fosfomycin:tobramycin combination resulted in greater killing of  P. aeruginosa  in the rat lung, compared to fosfomycin and tobramycin alone. 
         FIG. 21  demonstrates that aerosol administration of a 60 and 90 mg/mL solution of a 8:2 fosfomycin:tobramycin combination resulted in significant killing of  P. aeruginosa  in the rat lung. 
     
    
    
     DEFINITION OF TERMS 
     As used herein: 
     “Quarter normal saline” or “¼ NS” means normal saline diluted to its quarter strength containing 0.225% (w/v) NaCl. 
     “9:1 fosfomycin:tobramycin” means a water solution or dry powder formulation containing a 9:1 ratio by weight of fosfomycin acid to tobramycin base. 
     “8:2 fosfomycin:tobramycin” means a water solution or dry powder formulation containing a 8:2 ratio by weight of fosfomycin acid to tobramycin base such that the amount of fosfomycin is four times the amount of tobramycin. 
     “7:3 fosfomycin:tobramycin” means a water solution or dry powder formulation containing a 7:3 ratio by weight of fosfomycin acid to tobramycin base. 
     “5:5 fosfomycin:tobramycin” means a water solution or dry powder formulation containing a 5:5 ratio by weight of fosfomycin acid to tobramycin base. 
     “Minimal inhibitory concentration (MIC)” means the lowest concentration of antibiotic (s) that prevents visible growth after incubation for 18-20 hours at 35° C. 
     “Minimal bactericidal concentration (MBC)” means the lowest concentration of antibiotic that results in ≧3 Log 10  of bacterial killing. 
     “Time-dependent killing” means higher drug concentrations do not kill bacteria any faster or to a greater extent. 
     Concentration-dependent killing” means the higher the drug concentration, the greater the rate and extent of bacterial killing. 
     “Bacteriostatic” means the antibiotic acts by inhibiting bacterial growth. 
     “Bactericidal” means the antibiotics acts by killing bacteria. 
     “Synergy” means the combined effect of the antibiotics being examined is significantly greater than either drug alone. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The current invention concerns a concentrated fosfomycin plus tobramycin formulation suitable for efficacious delivery by aerosolization into the endobronchial or nasal space. The invention is preferably suitable for formulation of concentrated fosfomycin plus tobramycin for aerosolization by jet, ultrasonic, or equivalent nebulizers to produce aerosol particles between 1 and 5μ necessary for the efficacious delivery of fosfomycin plus tobramycin into the endobronchial or nasal space to treat bacterial infections, particularly those caused by multidrug-resistant  Pseudomonas aeruginosa . The formulation contains a minimal yet efficacious amount of fosfomycin plus tobramycin formulated in the smallest possible volume of physiologically acceptable solution having a salinity adjusted to permit generation of a fosfomycin and tobramycin aerosol well tolerated by patients but minimizing the development of secondary undesirable side effects such as bronchospasm and cough. 
     Primary requirements for any aerosolized formulation are its safety and efficacy. Additional advantages are lower cost, practicality of use, long shelf-life, storage and manipulation of nebulizer. 
     Aerosolized fosfomycin plus tobramycin is formulated for efficacious delivery of fosfomycin plus tobramycin to the lung endobronchial or nasal space. A nebulizer is selected to allow the formation of a fosfomycin plus tobramycin aerosol having a mass median aerodynamic diameter predominantly between 1 to 5μ. The formulated and delivered amount of fosfomycin plus tobramycin is efficacious for the treatment of bacterial pulmonary infections, particularly those caused by multi-drug resistant  Pseudomonas aeruginosa . The formulation has salinity, osmotic strength, and pH adjusted to permit generation of a fosfomycin plus tobramycin aerosol that is well tolerated by patients. The formulation has the smallest possible aerosolizable volume able to deliver an effective dose of fosfomycin plus tobramycin to the site of the infection. Additionally, the aerosolized formulation does not impair the functionality of the airways or nasal passages and minimizes undesirable side effects. 
     I. Evaluation of the Antibiotic Combinations 
     Fosfomycin:aminoglycosides combinations were formulated as described in Example 1. Fosfomycin comprised the major component of the combination because of it&#39;s inherent safety and the need to reduce the toxicity of the aminoglycoside. The fosfomycin: aminoglycoside combinations, in particular fosfomycin:tobramycin were evaluated for (a.) in vitro potency, (b.) killing rates, (c.) frequency of resistance, and (d.) animal efficacy. 
     a. In Vitro Potency 
     The in vitro potency of fosfomycin and tobramycin alone and in combination against a panel of Gram-negative and Gram-positive bacteria representative of species that cause respiratory tract infections in cystic fibrosis, bronchiectasis, sinusitis and ventilator-associated pneumoniae is shown in Tables 1 and 2. The data show that the fosfomycin:tobramycin combinations have antibacterial activity against a broad spectrum of Gram-positive and Gram-negative bacteria. The MICs of the fosfomycin:tobramycin combinations did not improve over fosfomycin or tobramycin alone. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 MIC values of fosfomycin and tobramycin alone and in  
               
               
                 combination against Gram-negative bacteria. 
               
            
           
           
               
               
            
               
                   
                 MIC (μg/mL) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Bacteria 
                 Fosfomycin 
                 Tobramycin 
                 F9:T1 
                 F7:T3 
                 F5:T5 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 
                   P. 
                   aeruginosa 
                 
                   
                   
                   
                   
                   
               
               
                 COR-003 
                 16 
                 0.5 
                 8 
                 4 
                 1 
               
               
                 COR-009 
                 1024 
                 2048 
                 1024 
                 512 
                 2048 
               
               
                 COR-021 
                 8 
                 64 
                 8 
                 8 
                 8 
               
               
                 COR-027 
                 128 
                 0.5 
                 8 
                 2 
                 1 
               
               
                 
                   E. 
                   coli 
                 
                   
                   
                   
                   
                   
               
               
                 COR-032 
                 1 
                 0.5 
                 1 
                 1 
                 1 
               
               
                 COR-039 
                 64 
                 0.25 
                 2 
                 0.5 
                 0.5 
               
               
                 COR-040 
                 1 
                 32 
                 1 
                 1 
                 2 
               
               
                 
                   H. 
                   influenzae 
                 
                   
                   
                   
                   
                   
               
               
                 COR-042 
                 8 
                 0.5 
                 4 
                 2 
                 1 
               
               
                 COR-049 
                 128 
                 0.5 
                 4 
                 1 
                 1 
               
               
                 
                   B. 
                   cepacia 
                 
                   
                   
                   
                   
                   
               
               
                 COR-090 
                 ≧2048 
                 128 
                 1024 
                 ND 
                 ND 
               
               
                 COR-098 
                 ≧2048 
                 64 
                 1024 
                 ND 
                 ND 
               
               
                 
                   S. 
                   maltophilia 
                 
                   
                   
                   
                   
                   
               
               
                 COR-082 
                 128 
                 4 
                 4 
                 4 
                 8 
               
               
                 COR-083 
                 64 
                 512 
                 256 
                 128 
                 128 
               
               
                 COR-087 
                 ≧512 
                 128 
                 128 
                 128 
                 128 
               
               
                 
                   K. 
                   pneumoniae 
                 
                   
                   
                   
                   
                   
               
               
                 COR-042 
                 8 
                 0.5 
                 4 
                 2 
                 1 
               
               
                 COR-049 
                 128 
                 0.5 
                 4 
                 1 
                 1 
               
               
                 
                   M. 
                   catarrhalis 
                 
                   
                   
                   
                   
                   
               
               
                 COR-109 
                 4 
                 8 
                 4 
                 ND 
                 ND 
               
               
                 COR-113 
                 4 
                 8 
                 4 
                 ND 
                 ND 
               
               
                   
               
               
                 ND = Not determined. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 MIC values of fosfomycin and tobramycin alone and  
               
               
                 in combination against Gram-positive bacteria. 
               
            
           
           
               
               
            
               
                   
                 MIC (μg/mL) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Bacteria 
                 Fosfomycin 
                 Tobramycin 
                 F9:T1 
                 F7:T3 
                 F5:T5 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 
                   S. 
                   aureus 
                 
                   
                   
                   
                   
                   
               
               
                 COR-051 
                 1 
                 0.25 
                 2 
                 1 
                 0.5 
               
               
                 (MSSA) 
                   
                   
                   
                   
                   
               
               
                 COR-055 
                 4 
                 0.25 
                 4 
                 1 
                 0.5 
               
               
                 (MRSA) 
                   
                   
                   
                   
                   
               
               
                 COR-059 
                 2 
                 128 
                 2 
                 4 
                 4 
               
               
                 COR-060 
                 2 
                 64 
                 1 
                 4 
                 8 
               
               
                 (GISA) 
                   
                   
                   
                   
                   
               
               
                 
                   S. 
                   pneumoniae 
                 
                   
                   
                   
                   
                   
               
               
                 COR-061 
                 32 
                 64 
                 16 
                 32 
                 32 
               
               
                 COR-068 
                 8 
                 16 
                 16 
                 16 
                 16 
               
               
                 
                   S. 
                   pyogenes 
                 
                   
                   
                   
                   
                   
               
               
                 COR-104 
                 64 
                 32 
                 ND 
                 ND 
                 ND 
               
               
                 COR-105 
                 16 
                 64 
                 ND 
                 ND 
                 ND 
               
               
                 
                   E. 
                   faecalis 
                 
                   
                   
                   
                   
                   
               
               
                 COR-099 
                 32 
                 512 
                 ND 
                 ND 
                 ND 
               
               
                 COR-103 
                 32 
                 8 
                 ND 
                 ND 
                 ND 
               
               
                   
               
               
                 ND = Not determined 
               
            
           
         
       
     
     Table 3 shows the MIC 50  and MIC 90  values of fosfomycin and tobramycin alone and in combination for 100  P. aeruginosa  strains isolated from lung sputum samples from cystic fibrosis patients. This study demonstrated that in the absence of mucin, tobramycin was the most active antibiotic. Combining fosfomycin and tobramycin resulted in a significant improvement in the MIC 50  and MIC 90  values compared to fosfomycin alone. In the CF mucin binding model (+mucin), the fosfomycin:tobramycin combinations had MIC 50  and MIC 90  values to comparable tobramycin alone. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 MIC values of fosfomycin and tobramycin alone  
               
               
                 and in combination for 100  P.   aeruginosa  strains. 
               
            
           
           
               
               
               
            
               
                   
                 MIC 50  (μg/mL) 
                 MIC 90  (μg/mL) 
               
            
           
           
               
               
               
               
               
            
               
                 Antibiotic 
                 Mucin (−) 
                 Mucin (+) 
                 Mucin (−) 
                 Mucin (+) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Fosfomycin 
                 64 
                 64 
                 ≧512 
                 128 
               
               
                 Tobramycin 
                 2 
                 16 
                 16 
                 64 
               
               
                 F9:T1 
                 16 
                 32 
                 64 
                 128 
               
               
                 F8:T2 
                 8 
                 32 
                 64 
                 128 
               
               
                 F7:T3 
                 8 
                 16 
                 64 
                 64 
               
               
                   
               
            
           
         
       
     
     Table 4 shows the results of checkerboard synergy studies between fosfomycin and nine aminoglycosides. The interactions between the nine different fosfomycin:aminoglycoside combinations were all classified as indifferent. None of the combinations had synergistic activity against  P. aeruginosa  ATCC 27853. This data demonstrates it is not obvious that an improvement in efficacy would be achieved by combining fosfomycin and aminoglycosides. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Checkerboard studies between fosfomycin and  
               
               
                 various aminoglycosides for  P.   aeruginosa  ATCC 27853. 
               
            
           
           
               
               
               
            
               
                   
                 Antibiotic 
                   
               
               
                   
                 Combination 
                 Interaction 
               
               
                   
                   
               
               
                   
                 Fosfomycin:Tobramycin 
                 Indifferent 
               
               
                   
                 Fosfomycin:Gentamicin 
                 Indifferent 
               
               
                   
                 Fosfomycin:Arbekacin 
                 Indifferent 
               
               
                   
                 Fosfomycin:Dibekacin 
                 Indifferent 
               
               
                   
                 Fosfomycin:Kanamycin B 
                 Indifferent 
               
               
                   
                 Fosfomycin:Streptomycin 
                 Indifferent 
               
               
                   
                 Fosfomycin:Amikacin 
                 Indifferent 
               
               
                   
                 Fosfomycin:Neomycin 
                 Indifferent 
               
               
                   
                 Fosfomycin:Netilmicin 
                 Indifferent 
               
               
                   
                   
               
            
           
         
       
     
     Table 5 shows the results of checkerboard synergy studies between fosfomycin and tobramycin against clinical strains of  P. aeruginosa, E. coli, H. influenzae , and  S. aureus . Interactions between fosfomycin and tobramycin were classified as indifferent for the vast majority of isolates examined. The interaction between fosfomycin and tobramycin combination was synergistic against only one strain of  P. aeruginosa  and one strain of  E. coli . This data further suggests it was not obvious that an improvement in efficacy would be achieved by combining fosfomycin and tobramycin. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Checkerboard studies between fosfomycin and tobramycin for  
               
               
                   P.   aeruginosa ,  E.   coli ,  H.   influenzae , and  S.   aureus  strains. 
               
            
           
           
               
               
               
            
               
                 Bacterial 
                   
                 Antibiotic Interaction 
               
            
           
           
               
               
               
               
               
               
            
               
                 Strains 
                 N = 
                 Synergism 
                 Addition 
                 Indifference 
                 Antagonism 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 
                   P. 
                   aeruginosa 
                 
                 17 
                 1 
                 0 
                 16 
                 0 
               
               
                 
                   E. 
                   coli 
                 
                 5 
                 1 
                 0 
                 4 
                 0 
               
               
                 
                   H 
                   influenzae 
                 
                 1 
                 0 
                 0 
                 1 
                 0 
               
               
                 
                   S. 
                   aureus 
                 
                 4 
                 0 
                 0 
                 4 
                 0 
               
               
                   
               
            
           
         
       
     
     Table 6 shows the MBC/MIC values of fosfomycin and tobramycin alone and 9:1, 8:2, and 7:3 fosfomycin:tobramycin combinations for  P. aeruginosa  ATCC 27853. Antibiotics with MBC/MIC values ≦1 are preferable because they inhibit bacteria by killing rather slowing growth. This study unexpectedly demonstrated the MBC/MIC values of the 9:1, 8:2, and 7:3 fosfomycin:tobramycin combinations were identical to tobramycin alone. This finding was unexpected since the fosfomycin alone MBC/MIC ratio was ≧8. The study also showed that a similar finding was not observed with any of the other fosfomycin: aminoglycoside combinations. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 MBC/MIC values of aminoglycosides alone and 9:1,  
               
               
                 8:2, and 7:3 combinations of fosfomycin and  
               
               
                 aminoglycoside for  P.   aeruginosa  ATCC 27853. 
               
            
           
           
               
               
            
               
                 Antibiotic 
                 MBC/MIC 
               
            
           
           
               
               
               
               
               
            
               
                 Combination 
                 Aminoglycoside 
                 F9:A1 
                 F8:A2 
                 F7:A3 
               
               
                   
               
               
                 Fosfomycin:Tobramycin 
                 1 
                 1 
                 1 
                 1 
               
               
                 Fosfomycin:Gentamicin 
                 2 
                 4 
                 2 
                 8 
               
               
                 Fosfomycin:Amikacin 
                 2 
                 4 
                 4 
                 2 
               
               
                 Fosfomycin:Netilimicin 
                 1 
                 2 
                 4 
                 2 
               
               
                 Fosfomycin:Arbekacin 
                 1 
                 4 
                 4 
                 2 
               
               
                 Fosfomycin:Streptomycin 
                 4 
                 2 
                 2 
                 2 
               
               
                 Fosfomycin:Neomycin 
                 2 
                 2 
                 2 
                 4 
               
               
                 Fosfomycin:Kanamycin B 
                 ND 
                 4 
                 2 
                 4 
               
               
                 Fosfomycin:Dibekacin 
                 1 
                 4 
                 2 
                 1 
               
               
                   
               
               
                 ND = Not determined. 
               
               
                 “F” = Fosfomycin and “A” = aminoglycoside 
               
            
           
         
       
     
     Table 7 shows the MBC/MIC values of fosfomycin and tobramycin alone and 9:1, 8:2, and 7:3 combinations for  P. aeruginosa  ATCC 27853 , E. coli  ATCC 25922, and  S. aureus  ATCC 29213. For  P. aeruginosa , the MBC/MIC values of the 9:1, 8:2, and 7:3 combinations were identical to tobramycin alone. This finding was unexpected since it was not observed with  E. coli  or  S. aureus . 
                     TABLE 7                  MBC/MIC values of fosfomycin and tobramycin        alone and 9:1, 8:2, and 7:3 combinations        of fosfomycin and tobramycin.                                 MBC/MIC                                     Antibiotic     P.   aeruginosa       E.   coli       S.   aureus                                                   Fosfomycin   ≧8   8   1           Tobramycin   1   1   8           F9:T1   1   1   2           F8:T2   1   2   4           F7:T3   1   2   4                        
b. Killing Rates
 
     Table 8 shows the results of time-kill studies of 9:1, 8:2, and 7:3 fosfomycin:aminoglycoside combinations against  P. aeruginosa  ATCC 27853. Examination of bacterial killing over time revealed synergy between fosfomycin:tobramycin, fosfomycin: gentamicin, and fosfomycin:arbekacin combinations. The remaining fosfomycin:aminoglycoside combinations were not synergistic. This data shows that it was not obvious which combination of fosfomycin:aminoglycoside, nor which ratio of fosfomycin:amino glycoside would be synergistic. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Time-kill studies with 9:1, 8:2, and 7:3 fosfomycin:aminoglycoside  
               
               
                 combinations against  P.   aeruginosa  ATCC 27853. Antibiotics  
               
               
                 were evaluated at a concentration of 32 μg/mL. 
               
            
           
           
               
               
            
               
                 Antibiotic 
                 Synergy 
               
            
           
           
               
               
               
               
            
               
                 Combination 
                 F9:A1 
                 F8:A2 
                 F7:A3 
               
               
                   
               
               
                 Fosfomycin:Tobramycin 
                 Yes 
                 No 
                 No 
               
               
                 Fosfomycin:Gentamicin 
                 Yes 
                 No 
                 No 
               
               
                 Fosfomycin:Amikacin 
                 No 
                 No 
                 No 
               
               
                 Fosfomycin:Netilimicin 
                 No 
                 No 
                 No 
               
               
                 Fosfomycin:Arbekacin 
                 No 
                 No 
                 Yes 
               
               
                 Fosfomycin:Streptomycin 
                 No 
                 No 
                 No 
               
               
                 Fosfomycin:Neomycin 
                 No 
                 No 
                 No 
               
               
                 Fosfomycin:Kanamycin B 
                 No 
                 No 
                 No 
               
               
                 Fosfomycin:Dibekacin 
                 No 
                 No 
                 No 
               
               
                   
               
               
                 “F” = Fosfomycin and “A” = aminoglycoside 
               
            
           
         
       
     
     Table 9 shows the time to achieve bactericidal killing of  P. aeruginosa  ATCC 27853 by various fosfomycin:aminoglycoside combinations. Antibiotics were evaluated at a concentration of 32 μg/mL. This study demonstrated that the 9:1, 8:2, and 7:3 fosfomycin:tobramycin combinations achieved bactericidal killing faster than any other fosfomycin:aminoglycoside combination. The data also showed that three of the fosfomycin:aminoglycoside combinations did not exhibit bactericidal killing. These results were unexpected because tobramycin alone reached bactericidal killing at 1 hour, and fosfomycin alone was not bactericidal. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Time to reach bactericidal killing of  P.   aeruginosa   
               
               
                 ATCC 27853 by 9:1, 8:2, and 7:3  
               
               
                 fosfomycin:aminoglycoside combinations. 
               
            
           
           
               
               
               
            
               
                   
                 Antibiotic 
                 Time (h) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Combination 
                 F9:A1 
                 F8:A2 
                 F7:A3 
               
               
                   
                   
               
               
                   
                 Fosfomycin:Tobramycin 
                 2 
                 1 
                 1 
               
               
                   
                 Fosfomycin:Gentamicin 
                 6 
                 4 
                 2 
               
               
                   
                 Fosfomycin:Amikacin 
                 4 
                 4 
                 2 
               
               
                   
                 Fosfomycin:Netilimicin 
                 NC 
                 NC 
                 6 
               
               
                   
                 Fosfomycin:Arbekacin 
                 NC 
                 4 
                 2 
               
               
                   
                 Fosfomycin:Streptomycin 
                 NC 
                 NC 
                 NC 
               
               
                   
                 Fosfomycin:Neomycin 
                 NC 
                 NC 
                 NC 
               
               
                   
                 Fosfomycin:Dibekacin 
                 NC 
                 4 
                 4 
               
               
                   
                   
               
               
                   
                 “NC” refers to not cidal 
               
               
                   
                 “F” = Fosfomycin and “A” = aminoglycoside 
               
            
           
         
       
     
       FIGS. 1 and 2  show time-kill curves for a 9:1 fosfomycin:tobramycin combination and demonstrate rapid bactericidal killing of  P. aeruginosa  ATCC 27853 compared to bacteriostatic killing of fosfomycin and tobramycin alone. This was unexpected since checkerboard analysis did not demonstrate synergy between fosfomycin and tobramycin. 
       FIGS. 3 and 4  show time-kill curves for 8:2 fosfomycin:tobramycin combinations and demonstrate rapid bactericidal killing of  P. aeruginosa  ATCC 27853. At a concentration of 32 μg/mL, the killing activity of the combination was significantly superior to fosfomycin and slightly superior to tobramycin. At 16 μg/mL, the combination was superior to tobramycin and fosfomycin alone. 
       FIGS. 5 ,  6  and  7  show time-kill curves for 7:3 fosfomycin:tobramycin combinations and demonstrate rapid bactericidal killing of  P. aeruginosa  ATCC 27853. At concentrations of 16 μg/mL and 32 μg/mL, the killing activity of the combination was significantly superior to fosfomycin and slightly superior than tobramycin alone. At 8 μg/mL, the combination was superior to both tobramycin and fosfomycin alone. 
       FIGS. 8 and 9  compare kill curves for 9:1, 8:2, and 7:3 fosfomycin:tobramycin combinations and tobramycin alone against  P. aeruginosa  ATCC 27853. At a concentration of 32 ug/mL, all the antibiotics had comparable rates and degrees of killing. At 16 ug/mL, tobramycin was the most active antibiotic followed by F7:A3, F8:A2, and F9:A1. 
       FIGS. 10 ,  11  and  12  demonstrate concentration-dependent killing of 9:1, 8:2, and 7:3 fosfomycin:tobramycin combinations against  P. aeruginosa  ATCC 27853. This was unexpected because the major component of the combination, fosfomycin, exhibits time-dependent killing. Tobramycin alone exhibits concentration-dependent killing. 
     c. Frequency of Resistance 
     Table 10 shows the frequency of development of resistance to fosfomycin and tobramycin alone, and a 9:1 fosfomycin:tobramycin combination for five  P. aeruginosa  strains. This study demonstrated the frequency of development of resistance to the 9:1 fosfomycin:tobramycin combination was 1000-100,000 times less than fosfomycin alone and 10-1000 times less than tobramycin alone. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Frequency of development of resistance to fosfomycin,  
               
               
                 tobramycin and a 9:1 fosfomycin:tobramycin combination. 
               
            
           
           
               
               
               
            
               
                   
                 
                   P. 
                   aeruginosa 
                 
                 Frequency of Resistance 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Strains 
                 Fosfomycin 
                 Tobramycin 
                 Fos + Tob 
               
               
                   
                   
               
               
                   
                 COR-002 
                 1.4 × 10 −3   
                 6.7 × 10 −6   
                 &lt;2.9 × 10 −8   
               
               
                   
                 COR-003 
                 5.5 × 10 −4   
                 3.2 × 10 −6   
                 &lt;1.2 × 10 −9   
               
               
                   
                 COR-013 
                 6.4 × 10 −3   
                 2.6 × 10 −6   
                  2.0 × 10 −7   
               
               
                   
                 COR-014 
                 1.5 × 10 −5   
                 5.0 × 10 −6   
                 &lt;1.4 × 10 −9   
               
               
                   
                 ATCC 27853 
                 4.1 × 10 −5   
                 3.4 × 10 −6   
                 &lt;2.5 × 10 −9   
               
               
                   
                   
               
            
           
         
       
     
     Table 11 shows the fold increase in the MIC of  P. aeruginosa  mutants isolated after a single exposure to fosfomycin, tobramycin, or a 9:1 combination of fosfomycin:tobramycin. This study demonstrated that the 9:1 mutants did not have a significant increase in MIC (1-2 fold), compared to fosfomycin (128→512 fold) or tobramycin (2-16 fold) mutants. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Fold increase in MIC after a single exposure to antibiotic. 
               
            
           
           
               
               
               
            
               
                   
                 
                   P. 
                   aeruginosa 
                 
                 Fold Increase in MIC 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Strain 
                 F9:T1 
                 Fos 
                 Tob 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 COR-002 
                 1 
                 &gt;256 
                 1 
               
               
                   
                 COR-003 
                 2 
                 &gt;256 
                 2 
               
               
                   
                 COR-013 
                 1 
                 &gt;64 
                 8 
               
               
                   
                 COR-014 
                 1 
                 &gt;512 
                 16 
               
               
                   
                 ATCC 27853 
                 2 
                 128 
                 1 
               
               
                   
                   
               
            
           
         
       
     
     Table 12 shows the development of resistance in a clinical  P. aeruginosa  strain after 28 days of continuous exposure to fosfomycin, tobramycin or a 9:1 fosfomycin:tobramycin combination. Exposure to the 9:1 combination caused a 8-fold increase in MIC by day 14, but the MIC did not exceed the fosfomycin resistance breakpoint of 256 μg/mL. Exposure to tobramycin increased the MIC 128-fold which exceeded the tobramycin resistance breakpoint of 16 μg/mL. A rapid and dramatic development of resistance was observed with  P. aeruginosa  exposed to fosfomycin alone. 
                     TABLE 12                  Development of resistance after continuous        exposure to antibiotic.                                 MIC (μg/mL)                                             Antibiotic   0d   7d   14d   21d   28d                                                         F9:T1   8   16   64   64   64           Tobramycin   0.5   0.5   0.5   32   64           Fosfomycin   16   &gt;512   &gt;512   &gt;512   &gt;512                        
d. Animal Efficacy
 
       FIGS. 13 ,  14 , and  15  show the killing of  E. coli  in the rat lung after aerosol administration of a 31.1 mg/mL solution of a 9:1, 8:2, and 7:3 fosfomycin:tobramycin combinations, respectively. This study demonstrated complete eradication (5-6 Log 10 CFU) of the  E. coli  lung infection after 45-60 minutes of treatment. 
       FIGS. 16 and 17  demonstrate that aerosol administration of a 31.1 mg/mL solution of a 9:1 fosfomycin:tobramycin combination resulted in &gt;5 log 10 killing of  E. coli  in the rat lung, compared to less than 1 log 10 killing fosfomycin and tobramycin alone. 
       FIG. 20  demonstrates that aerosol administration of a 31.1 mg/mL solution of a 8:2 fosfomycin:tobramycin combination resulted in &gt;5 log 10 killing of  E. coli  in the rat lung, compared to 2 log 10 killing by tobramycin alone. This was unexpected because the in vitro potency data did not demonstrate that the combination was superior to tobramycin alone. 
       FIGS. 19 and 20  demonstrate that aerosol administration of a 31.1 mg/mL solution of a 8:2 fosfomycin:tobramycin combination resulted in greater killing of  P. aeruginosa  in the rat lung, compared to fosfomycin and tobramycin alone. 
       FIG. 21  demonstrates that aerosol administration of a 60 and 90 mg/mL solution of a 8:2 fosfomycin:tobramycin combination resulted in significant killing of  P. aeruginosa  in the rat lung. 
     II. Aerosol Fosfomycin/Tobramycin Formulation 
     Aminoglycosides useful in the invention are antibiotics, such as tobramycin, gentamicin, kanamycin B, amikacin, arbekacin, dibekacin, streptomycin, neomycin, and netilmicin. Fosfomycin compounds particularly useful in the invention are antibiotics, such as fosfomycin trometamol, fosfomycin disodium salt, and fosfomycin calcium. 
     The preferred fosfomycin plus tobramycin formulation according to the invention contains 10-500 mg of fosfomycin plus tobramycin per 0.5-7 mL of water with a chloride concentration &gt;30 mM. This corresponds to doses that would be required to prevent colonization or to treat severe upper and lower respiratory tract infections caused by a range of susceptible organisms. 
     Patients can be sensitive to the pH, osmolarity, and ionic content of a nebulized solution. Therefore these parameters should be adjusted to be compatible with fosfomycin plus tobramycin and tolerable to patients. The most preferred solution or suspension of fosfomycin plus tobramycin will contain a chloride concentration &gt;30 mM at pH 4.5-8.0. 
     The formulation of the invention is nebulized predominantly into particle sizes allowing delivery of the drug into the terminal and respiratory bronchioles or nasal passages where the bacteria reside during infection and colonization. For efficacious delivery of fosfomycin plus tobramycin to the lung endobronchial space of airways in an aerosol, the formation of an aerosol having a mass median aerodynamic diameter predominantly between 1 to 5μ is necessary. The formulated and delivered amount of fosfomycin plus tobramycin for the treatment and prophylaxis of endobronchial infections, particularly those caused by the  P. aeruginosa  must effectively target the lung surface. The formulation must have the smallest possible aerosolizable volume able to deliver an effective dose of fosfomycin plus tobramycin to the site of the infection. The formulation must additionally provide conditions which would not adversely affect the functionality of the airways and nasal passages. Consequently, the formulation must contain enough of the drug formulated under the conditions to allow its efficacious delivery, while avoiding undesirable reactions. The new formulation according to the invention meets all these requirements. 
     According to the invention, fosfomycin plus tobramycin is formulated in a liquid dosage form intended for inhalation therapy by patients with, or at risk to acquire, bacterial upper or lower respiratory infection. Since the patients reside throughout the world, it is imperative that the liquid dosage formulation has reasonably long shelf-life. Storage conditions and packaging thus become important. 
     The formulation of fosfomycin plus tobramycin can be aseptically prepared as an aqueous solution in a dual blow-fill ampoule such that each antibiotic is independently formulated in water and pH adjusted. Tobramycin is formulated at acidic pH (1-6) while fosfomycin is formulated at basic pH (8-13). The low pH formulation for tobramycin assures that all of its basic nitrogen atoms are protonated thus protecting the molecule from amine oxidation and degradation (tobramycin solutions turn yellow at room temperature). Thus, the low pH solutions of tobramycin are stable at room temperature for indefinite periods of time. Fosfomycin is most stable at high pH as the reactive epoxide ring is prone to hydrolysis (ring opening) at acidic or low pH. High pH solutions of fosfomycin are also stable at room temperature. Therefore, the dual ampoule blow-fill container allows for the separate and room temperature stable formulation of each antibiotic at high and low pH such that the drug product combination and final pH is created by mixing both solutions in the nebulizer immediately before use. The storage suitability of the formulation allows reliable usage of the formulated fosfomycin plus tobramycin suitable for aerosolization. 
     III. Nebulizers for Pulmonary Delivery of the Antibiotic Combination 
     A device able to nebulize the formulation of the invention into aerosol particles predominantly in the 1 to 5μ size range is utilized to administer the formulations of the present invention. Predominantly in this application means that at least 70% but preferably more than 90% of all generated aerosol particles are within the 1 to 5μ range. Typical devices include jet nebulizers, ultrasonic nebulizers, pressurized aerosol generating nebulizers, and vibrating porous plate nebulizers. Representative suitable nebulizers include the eFlow® nebulizer available from Pari Inovative Manufactures, Midlothian, Va.; the iNeb® nebulizer available from Profile Drug Delivery of West Sussex, United Kingdom; the Omeron MicroAir® nebulizer available from Omeron, Inc. of Chicago, Ill. and the AeroNebGo® nebulizer available from Aerogen Inc. of Mountain View, Calif. 
     A jet nebulizer utilizes air pressure to break a liquid solution into aerosol droplets. An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets. Pressurized systems general force solutions through small pores to generate small particles. A vibrating porous plate device utilizes rapid vibration to shear a stream of liquid into appropriate droplet sizes. However, only some formulations of fosfomycin plus tobramycin can be efficiently nebulized as the devices are sensitive to the physical and chemical properties of the formulation. 
     The invention is a small volume, high concentration formulation of fosfomycin plus tobramycin that can be delivered as an aerosol at efficacious concentrations of the drug to the respiratory tract in people at risk for, or suffering from infection caused by susceptible bacteria. The formulation is safe, well tolerated, and very cost effective. Furthermore, the formulation provides adequate shelf life for commercial distribution. The foregoing may be better understood from the following examples, which are presented for the purposes of illustration and are not intended to limit the scope of the inventive concepts. 
     EXAMPLE 1 
     Preparation of Fosfomycin/Tobramycin Solutions for Aerosolization 
     9:1 Fosfomycin/Tobramycin Solution 
     Fosfomycin disodium (18.057 g, 13.99 g free acid) was dissolved in 250 mL of water and the pH was adjusted to 7.41 by the dropwise addition of 1.53 mL of 4.5 N HCl. To the resulting solution was added 1.56 g of 97.5% tobramycin base. The pH of the solution was adjusted to 7.60 by the addition of 2.45 mL of 4.5 N HCl. The solution was diluted to 500 mL with water and filtered through a 0.2 μm Nalge Nunc 167-0020 membrane filter. The final pH was 7.76, the osmolality was 537 mOsmol/kg, the fosfomycin/tobramycin ratio was calculated to be 9:1, and the chloride concentration was 35.8 mM. 
     8:2 Fosfomycin/Tobramycin Solution 
     A solution of fosfomycin/tobramycin in a 8:2 ratio was prepared. 3.1680 g of fosfomycin disodium (2.4013 g free acid) was dissolved in 50 ml water. 0.6154 g of 97.5% tobramycin base (0.6000 g of pure tobramycin base) was dissolved in the fosfomycin solution. The pH adjusted by adding 0.910 mL of 6 M HCl. The solution was diluted to 100 mL with water. The final pH of the solution was 7.65, osmolality was 477 mOsmol/kg, and the chloride concentration was 54.6 mM. The final fosfomycin/tobramycin ratio was calculated to be 8:2 
     7:3 Fosfomycin/Tobramycin Solution 
     Using the procedure described for the 9:1 solution, above, a solution of fosfomycin/tobramycin in a 7:3 ratio was prepared; 17.466 g of fosfomycin disodium (13.239 g free acid) was dissolved in water; the pH adjusted to 7.43 by adding 1.46 mL of 4.5 N HCl, 5.819 g of 97.5% tobramycin base (5.674 g of pure tobramycin base), and the pH of the combined solution was adjusted by adding 9.20 mL of 4.5 N HCl. The final pH of the solution was 7.68, the osmolality was 560 mOsmol/kg, the fosfomycin/tobramycin ratio was 7:3, and the chloride concentration was 95.9 mM. 
     EXAMPLE 2 
     Preparation of A 8:2 Hyperosmolar Solution of Fosfomycin and Tobramycin 
     7.9165 g of fosfomycin disodium (6.0007 g of fosfomycin free acid) and 1.5382 g of 97.5% tobramycin base (1.4997 g of pure tobramycin base) were dissolved in 50 mL water. The pH was adjusted to 7.62 by the addition of 2.3 mL of 6 M HCl. The combined solution was diluted to 100 mL. The final pH was 7.64, the osmolality was 1215 mOsmol/kg, the final chloride concentration was 138 mM, and the fosfomycin/tobramycin ratio was 8:2. 
     EXAMPLE 3 
     Preparation of Individual Solutions of High pH Fosfomycin and Low pH Tobramycin for Reconstitution 
     A fosfomycin solution was prepared by dissolving 5.891 g of fosfomycin disodium (4.465 g of fosfomycin free acid) in water and diluting to 100 mL. The pH was 9.42, and the osmolality was 795 mOsmol/kg. 
     A tobramycin solution was prepared by dissolving 1.869 g of 97.5% tobramycin base (1.822 g of pure tobramycin base) in 60 mL water, adjusting the pH to 4.90 by adding 18.8 mL of 1 M HCl, and diluting to 100 ml with water. The pH was 4.89, and the osmolality was 148 mOsmol/kg. 
     1 mL of the fosfomycin solution and 1 mL of the tobramycin solution were combined. For the combined drug product solution, the pH was 7.30, the osmolality was 477 mOsmol/kg, the chloride concentration was 94.0 mM, and the fosfomycin/tobramycin ratio was 7:3. 
     EXAMPLE 4 
     Preparation of an 8:2 Fosfomycin/Tobramycin Solution from Commercial Tobramycin for Inhalation Solution (TOBI) and Lyophilized (dry) Fosfomycin Disodium (FOSFO) 
     3.1665 g of fosfomycin disodium (FOSFO, 2.4002 g of fosfomycin free acid) was dissolved in two 5 mL (60 mg/mL) ampoules of Tobramycin Solution for Inhalation (TOBI, 600 mg of tobramycin base) plus 40 mL water. The initial pH of the solution was 7.50. To the solution was added 667 μl of 4.5 M NaCl. The combined solution was diluted to 100 mL with water. The final formulation had the following properties; pH 7.49, osmolality 502 mOsmol/kg, chloride concentration 33.8 mM, and the fosfomycin/tobramycin ratio was 8:2, and drug concentration of 24 mg/mL fosfomycin and 6 mg/mL of tobramycin. 
     Examples 5-10 set forth the procedures that have been used to generate the date presented in the Tables and Figures of this application. 
     EXAMPLE 5 
     Determination of Minimal Inhibitory Concentrations (MIC) 
     The efficacy of antibiotics and antibiotic combinations against Gram-positive and Gram-negative bacteria representative of species that cause respiratory infections in patients with cystic fibrosis, bronchiectasis, sinusitis, and ventilator-associated pneumonia were evaluated in MIC assays.  P. aeruginosa  strains were isolated from lung sputum samples collected from cystic fibrosis patients, blood cultures, respiratory tract infections, and skin or soft tissue infections.  E. coli, H. influenzae, B. cepacia, S. maltophilia, K. pneumoniae, M. catarrhalis, S. aureus, S. pneumoniae, S. pyogenes , and  E. faecalis  were isolated from respiratory tract infections.  E. coli  ATCC 25922 , P. aeruginosa  ATCC 27853, and  S. aureus ATCC  29213 were used as quality control stains. 
     Method A: 
     The MICs of fosfomycin alone, tobramycin alone, or combinations of fosfomycin plus tobramycin were determined by the agar-plate dilution method according to NCCLS guidelines (NCCLS, 2003). Bacterial strains were streaked onto Trypic Soy Agar plates (PML Microbiologicals, Wilsonville, Oreg.) containing 5% defibrinated sheep blood (hereafter referred to as blood agar plates) and incubated overnight at 35° C. Two to three bacterial colonies from the overnight cultures were inoculated into 3 mL of sterile normal saline, vortexed briefly, and adjusted to a 0.5 McFarland standard (NCCLS, 2003). The bacterial suspension was diluted 1:40 in sterile normal saline and served as the inoculum. Mueller-Hinton agar plates (hereafter referred to as MHA) were prepared by combining 16 g of agarose (Becton-Dickinson, Sparks, Md.), 22 g of Mueller-Hinton broth powder (Becton-Dickinson, Sparks, Md.), and adjusted to 1 L with distilled water. The agar was sterilized by autoclaving, cooled to 55° C., and supplemented with 25 μg/mL of glucose-6-phosphate (Sigma-Aldrich, St. Louis, Mo.). Twenty-five mL of cooled agar was aliquoted into 50 mL conical tubes and supplemented with appropriate concentrations of antibiotic to achieve concentrations ranging from 0.06 μg/mL to 512 μg/mL. After gently mixing the agar and antibiotic, the suspension was poured into sterile 100 mm petri dishes and allowed to solidify at room temperature. The antibiotic agar plates were inoculated with approximately 2×10 4  CFU/spot with a 48-point inoculator (Sigma-Aldrich, St. Louis, Mo.). The MIC was defined as the lowest concentration of antibiotic (s) that prevented visible growth after incubation for 18-20 hours at 35° C. The efficacy of a particular antibiotic or antibiotic combination on large populations of  P. aeruginosa  was determined by calculating the MIC 50  and MIC 90  values. The MIC 50  value was defined as the concentration of antibiotic(s) which inhibited 50% of the  P. aeruginosa  strains. The MIC 90  value was defined as the concentration of antibiotic(s) which inhibited 90% of the  P. aeruginosa  strains (Wiedemann and Grimm, 1996). 
     Method B: 
     The MICs of fosfomycin alone, tobramycin alone, or combinations of fosfomycin plus tobramycin were determined for  P. aeruginosa  strains in the presence of porcine gastric mucin to evaluate the effect of mucin and protein binding on antibiotic activity. Methodologies were identical to that described in Example 5, Method A, with the exception that 2% (weight/volume) porcine gastric mucin (Sigma Chemical Co., St. Louis, Mo.) was added to the MHA prior to autoclaving. 
     Method C: 
     The MICs of amikacin, arbekacin, dibekacin, gentamicin, kanamycin, netilimicin, neomycin, streptomycin, and tobramycin alone were determined for  P. aeruginosa  ATCC 27853 by the broth-microdilution method according to NCCLS standards (NCCLS, 2003).  E. coli  ATCC 25922 and  S. aureus  ATCC 29213 were used as quality control stains. Bacterial strains were streaked onto blood agar plates, and incubated at 35° C. for 18 hours. Two to three bacterial colonies from the overnight cultures were inoculated into 3 mL of sterile normal saline, vortexed briefly, and adjusted to a 0.5 McFarland standard (NCCLS, 2003). The bacterial suspension was diluted 1:100 in cation-adjusted Mueller-Hinton broth (hereafter referred to as CAMHB). Fifty microliters of bacterial inoculum (approximately 2×10 5  CFU/mL) was pipeted into individual wells of 96-well plates containing 50 μl of CAMHB (Remel, Lenexa, Kanas) supplemented with 2-fold dilutions of antibiotics ranging in concentration from 0.125 μg/mL to 128 μg/mL. The MIC was defined as the lowest concentration of antibiotic (s) that prevented visible growth after incubation at 35° C. for 18-24 hours. 
     EXAMPLE 6 
     Checkerboard Synergy 
     Potential interactions between fosfomycin and amikacin, arbekacin, dibekacin, gentamicin, kanamycin B, neomycin, netilimicin, streptomycin, or tobramycin were determined by the checkerboard method (Eliopoulos and Moellering, 1996). Two-fold serial dilutions of fosfomycin and aminoglycosides, which bracketed the expected MIC value for both compounds, were evaluated. Bacterial strains were streaked onto blood agar plates and incubated at 35° C. for 18-24 hours. Two to three bacterial colonies from the overnight cultures were inoculated into 3 mL of sterile normal saline, vortexed briefly, and adjusted to a 0.5 McFarland standard (NCCLS, 2003). Fifty microliters of bacterial inoculum (approximately 2×10 5  CFU/mL) was pipeted into individual wells of 96-well plates containing 50 μl of CAMHB supplemented with 2-fold dilutions of the two antibiotics of interest. The fractional inhibitory concentration (FIC) was calculated as the MIC of compound #1 in combination with a second compound, divided by the MIC of compound #1 alone. A summation of FIC (SFIC) was calculated for each drug combination as the sum of the individual FICs of compound #1 and #2. The FIC was calculated as the lowest concentration of antibiotics that that prevented visible growth after incubation at 18-20 hours at 35° C. Synergy was defined as an SFIC of 0.5, indifference as an SFIC &gt;0.5 and &lt;4, and antagonism as an SFIC &gt;4. The lowest SFIC was used for final interpretation of drug interactions. 
     EXAMPLE 7 
     Determination of Time-Kill Kinetics 
     Time-kill experiments were performed in the presence of 2% porcine gastric mucin to evaluate the effect of mucin and protein binding on antibiotic activity. Two to three bacterial colonies were inoculated into 10 mL CAMHB and incubated at 35° C. in a shaking water bath (250 rpm) for 18-24 hours. A 1:40 dilution of the overnight culture was made in 10 mL of fresh CAMHB and incubated at 35° C. in a shaking water bath (250 rpm) for 1-2 hours. The resulting culture was adjusted to a 0.5 McFarland standard (NCCLS, 2003). To reduce variability in the bacterial inoculum size when comparing multiple antibiotics, one master tube of CAMHB containing 2% (weight/volume) of porcine gastric mucin was inoculated with a 1:200 dilution of bacterial inoculum (approximately 5×10 5  CFU/mL), supplemented with 25 μg/mL of glucose-6-phosphate, and briefly vortexed. Ten milliliter aliquots were then pipeted into 50 mL conical tubes. Fosfomycin alone, tobramycin alone, and combinations of fosfomycin plus amikacin, arbekacin, dibikacin, gentamicin, kanamycin, netilimicin, neomycin, streptomycin, or tobramycin were added to the culture medium at concentrations equal to 1, 2, 4, and 8-fold multiples of the fosfofomycin MIC (4 μg/mL) for  P. aeruginosa  ATCC 27853. The killing activity of fosfomycin plus amikacin, arbekacin, dibikacin, gentamicin, kanamycin, netilimicin, neomycin, streptomycin, or tobramycin were also compared to the killing activity of their individual components alone. For example, 32 μg/mL of a 9:1 fosfomycin:tobramycin combination was compared to killing activity of 28.8 μg/mL of fosfomycin alone and 3.2 μg/mL of tobramycin alone. A no drug control was run in each experiment. Cultures were incubated with antibiotic at 35° C. in a shaking (250 rpm) water bath for 24 hours. Bacterial killing was determined at 0, 1, 2, 4, 6, and 24 hours by making 10-fold serial dilutions of the cultures in sterile normal saline, and spreading 100 μl aliquots on blood agar plates. Culture plates were incubated at 35° C. for 18-24 hours and the number of colonies enumerated manually. The limit of detection for the colony counting method was 1 Log 10 ). Time-kill curves were constructed by plotting the Log 10  number of CFU/mL culture versus time. Antibiotic concentrations that reduced the original inoculum by ≧3 Log 10  CFU/mL were considered bactericidal, and concentrations that reduced the original inoculum by ≦2 Log 10  CFU/mL were defined as bacteriostatic (NCCLS, 1999). Synergism was defined as a reduction of bacterial colony counts of ≧2 Log 10  CFU/mL with the antibiotic combinations compared to the most active single antibiotic (NCCLS, 1999). 
     EXAMPLE 8 
     Determination of Minimal Bactericidal Concentration (MBC) 
     The MBCs of fosfomycin, amikacin, arbekacin, dibikacin, gentamicin, kanamycin B, netilimicin, neomycin, streptomycin, or tobramycin alone for  P. aeruginosa  ATCC 27853 , E. coli  ATCC 25922, and  S. aureus  ATCC 29213 were determined by the broth microdilution method according to NCCLS standards (NCCLS, 1999). Bacterial strains were streaked onto blood agar plates and incubated at 35° C. for 18-24 hours. Two to three bacterial colonies from the overnight cultures were inoculated into 3 mL of sterile normal saline, vortexed briefly, and adjusted to a 0.5 McFarland standard (NCCLS, 2003). Fifty microliters of bacterial inoculum (approximately 2×10 5  CFU/mL) was pipeted into individual wells of 96-well plates containing 50 μl of CAMHB (Remel, Lenexa, Kanas) supplemented with 2-fold dilutions of antibiotics ranging in concentration from 0.125 μg/mL to 128 μg/mL. Plates were incubated at 35° C. for 18-24 hours and MIC determined as described in Example 5, Method C. The contents of wells showing no growth (MIC and above) were mixed with a pipetor and duplicate 10 μl samples spread onto blood agar plates. Culture plates were incubated at 35° C. for 18-24 hours and the number of bacterial colonies on each plate enumerated manually. Rejection values were determined by NCCLS methods which considers the final inoculum size, single or double sampling, pipetting error, and the Poisson distribution of sample responses (NCCLS, 1999). For example, if the final inoculum was 5×10 5  CFU/mL and duplicate samples were evaluated, the lowest dilution having fewer than a total of 25 colonies was considered the MBC. The MBC was defined as the ≧3 Log 10  decrease in CFU/mL of the original inoculum as described by NCCLS standards (NCCLS, 1999). The MBC/MIC ratios were calculated by dividing the MBC by the MIC. 
     EXAMPLE 9 
     Determination of Frequency of Single-Step Resistance 
     The frequency of single-step spontaneous resistance mutation was determined for 5 susceptible  P. aeruginosa  strains. The bacterial inoculum was prepared by inoculating 5 mL of CAMHB with 2-3 bacterial colonies and incubating at 35° C. in a shaking water bath (250 rpm) for 18 h. A 1:20 dilution of the overnight culture was made into 50 mL of fresh CAMHB in a 125 ml erlenmyer flask and incubated at 35° C. in a shaking waterbath (250 rpm) for 8 hours. Cultures were centrifuged at 2,500 rpm, room temperature, for 20 min. The supernatant was decanted off and the cell pellet from the 50 mL of culture resuspended in 500 μl-1000 μl CAMHB. The CFU/mL in the bacterial suspension was determined by making 10-fold serial dilutions in sterile normal saline, and spreading 100 μl aliquots on blood agar plates. Culture plates were incubated at 35° C. for 18-24 hours and the number of colonies enumerated manually. One hundred microliters of bacterial cell suspension (approximately 10 9  CFU) was spread onto MHA plates containing 128 μg/mL of fosfomycin, 8 μg/mL of tobramycin, or 128 μg/mL of fosfomycin plus 8 μg/mL of tobramycin. The culture plates were incubated at 35° C. for 48-72 hours and the number of colonies on each plate was enumerated manually. The frequency of resistance was calculated by dividing the number of bacteria growing at the defined antibiotic concentration by the number of bacteria in the original inoculum. Representative mutants were evaluated for changes in MIC to fosfomycin alone, tobramycin alone, and fosfomycin plus tobramycin combinations as described in Example 5, Method A. 
     EXAMPLE 10 
     Multistep Resistance Analysis 
     Development of resistance during continuous serial passage was evaluated with a clinical  P. aeruginosa  strain susceptible to fosfomycin (MIC-8 μg/mL) and tobramycin (MIC=0.5 μg/mL). A single colony of  P. aeruginosa  COR-014 was inoculated into 5 mL of MHCAB and briefly vortexed. Two-fold serial dilutions of fosfomycin alone covering the range of 1 μg/mL-512 μg/mL, tobramycin alone covering 0.0625 μg/mL-512 μg/mL, and a 9:1 fosfomycin:tobramycin combination covering 0.0625 μg/mL-512 μg/mL were made in CAMHB. Ten microliters of bacterial suspension was pipetted into 3 mL of each antibiotic dilution and incubated statically at 35° C. for 18-24 hours. Tubes with the highest concentration of antibiotic having visible bacterial growth were selected from each antibiotic dilution series and 100 μl of the culture was pipeted into a fresh antibiotic dilution series. Tubes were incubated statically at 35° C. for 18-24 hours. This process was repeated for a total of 28 times. MICs of fosfomycin alone, tobramycin alone, and fosfomycin plus tobramycin were determined for the parent strain and resistant isolates collected after each passage as described in Example 5, Method C. 
     EXAMPLE 11 
     Determination of Animal Efficacy 
     The in vivo efficacy of aerosolized fosfomycin alone, tobramycin alone and combinations of fosfomycin plus tobramycin were evaluated against  E. coli  ATCC 25922 and  P. aeruginosa  ATCC 27853 in rat lungs. The bacterial inoculum used to establish the rat lung infections consisted of a mixture of bacteria and agarose beads.  E. coli  ATCC 25922 or  P. aeruginosa  ATCC 27853 was streaked onto blood agar plates and incubated for 18-24 hours at 35° C. Two to three colonies were inoculated into 10 mL Tryptic Soy Broth (hereafter referred to as TSB) and incubated statically at 35° C. for 1.5 to 2 hours. The culture was adjusted to an optical density of 0.1 (625 nm) in fresh TSB and then diluted 1:5 in TSB. One milliliter of bacterial suspension (approximately 10 7  CFU/mL) was added to 10 mL of a 2% noble agar solution and briefly mixed by inversion. The mixture was added to 150 mL of heavy white mineral oil equilibrated to 55° C. and stirred on ice for 5 minutes. The cooled suspension was poured into a 250 mL centrifuge tube and centrifuged at 3,000 rpm, 4° C., for 10 minutes. The supernatant was decanted off, the pellet resuspended in 25 mL of normal sterile saline, and transferred to a 50 mL conical centrifuge tube. Following centrifugation at 3,000 rpm, 4° C., for 10 minutes, the supernatant was decanted off and the pellet resuspended in 10 mL of saline. The suspension was centrifuged at 3,000 rpm, 4° C., for 10 minutes and the supernatant decanted off. The pellet was resuspended in approximately 10 mL sterile normal saline and then diluted 1:30 in a sterile 2% agarose bead solution to give approximately 10-1000 CFU/mL. 
     Prior to intratracheal installation of the bacterial inoculum, male Sprague-Dawly rats (200-250 g) were anesthetized by exposure to isoflurane for 5 minutes. An intratracheal needle was inserted into the trachea and 80 μA of agarose beads containing approximately 10-100 CFU of  E. coli  ATCC 25922 or  P. aeruginosa  ATCC 27853 were instilled into the lungs with a 1 mL syringe. The rats were placed into individual cages and allowed to recover for approximately 18 hours. 
     Fosfomycin alone, tobramycin alone, and fosfomycin:tobramycin combinations were administered to rats using an aerosol exposure device (In Tox Products, New Mexico). The system consisted of a central chamber having separate aerosol supply and exhaust paths. The central chamber had 24 ports that were directly connected to the aerosol supply system. Rats were placed into individual aerosol exposure tubes and restrained with an adjustable push plate and end cap assembly so they could not turn around or back away from the end of the tube. The restraint tubes containing the rats were loaded onto ports on the central chamber and the air flow adjusted to 1 liter/min. The air (breathable quality air) flow to the PARI LC Star nebulizer was held constant at 6.9 liters per minute. Fosfomycin alone, tobramycin alone, or 9:1, 8:2, or 7:3 fosfomycin:tobramycin combinations were nebulized at 0.2 mL/min and delivered to the rats via the aerosol supply path. Additional air (hereafter referred to as dilution air) was delivered to nebulizer (where) to balance the over pressure of the air used to deliver aerosolized drugs to the rats. Rodents were exposed to aerosolized antibiotics for up to 2 hours, twice daily for 3 consecutive days. Each treatment group consisted of 5-8 animals per group. A non-treatment control was included in each experiment. 
     Bacterial killing was evaluated 18 hours after the last exposure. Rats were anesthetized with isoflurane and euthanized by intraperitoneal administration of 500 μl of phenobarbitol. The lungs were removed aseptically, excess tissue removed and the lung weight determined. Lungs were placed in 10 mL glass vials, and 3 mL of normal sterile saline added per gram tissue. Samples were homogenized with hand-held homogenizer for 30 seconds. Bacterial killing was determined by making 10-fold serial dilutions of the lung homogenate in sterile normal saline, and spreading 100 μl aliquots on blood agar plates. Culture plates were incubated at 35° C. for 18-24 hours and the number of bacterial colonies enumerated manually. Antibiotic efficacy was determined by comparing the CFU&#39;s/lung from the non-treatment control group to the treatment groups. 
     REFERENCES 
     
         
         Al-Aloui, M., H. Miller, S. Alapati, P. A. Stockton, M. J. Ledson, and M. J. Walshaw. 2005. Renal impairment in cystic fibrosis patients due to repeated intravenous aminoglycoside use. Ped. Pulmon. 39:15-20. 
         Ansorg, R., K.-D. Muller, and W. Wiora. 1990. Comparison of inhibitory and bactericidal activity of antipseudomonal antiotics against  Pseudomonas aeruginosa  isolates from cystic fibrosis patients. Chemother. 36:222-229. 
         Arca, P., G. Reguera, and C. Hardisson. 1997. Plasmid-encoded fosfomycin resistance in bacteria isolated from the urinary tract in a multicentre survey. J. Antimicrob. Chemother. 40:393-399. 
         Bacardi, R., J. Tomos, I. Moga, N. Marti, P. Alomar, L. Gaztelurrutia, and C. Villalonga. 1977. Treatment of respiratory infections with fosfomycin. Chemother. 23 (Suppl. 1):343-347. 
         Bonora, V., C. Lozano, M. Santos, M. Paz, J. Baguena, and M. Gobernado. 1977. Fosfomycin in treatment of respiratory bacterial infections. Chemother 23 (Suppl. 1):337-341. 
         Conway, S. P. 2005. Nebulized antibiotic therapy: the evidence. Chronic Respir. Dis. 2:35-41. 
         Conway, S P., K G Brownlee, M Denton, and D G Peckham. 2003. Antibiotic treatment of multidrug-resistant organisms in cystic fibrosis. Am J Respir Med 2(4):321-332. 
         Craig, W. A. 1998. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin. Infect. Dis. 26 (1):1-12. 
         Forsgren, A. and M. Walder. 1983. Antimicrobial activity of fosfomycin in vitro. J. Antimicrob. Chemother. 11(5):467-471. 
         Flume, P. and M. E. Klepser. 2002. The rationale for aerosolized antibiotics. Pharmacotherapy 22 (3 Pt 2):71S-79S. 
         Geller, D. E., W. H. Pitlick, P. A. Nardella, W. G. Tracewell, and B. W. Ramsey. 2002. Pharmacokinetics and bioavailability of aerosolized tobramycin in cystic fibrosis. Chest. 122:219-226. 
         Gibson, R L., J L Burns, and B W Ramsey. 2003. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am. J. Respir. Crit. Care. Med. 168(8):918-951. 
         Greenwood, D., J. Brown, and R. Edwards. 1992. The influence of anaerobiosis on the activity fosfomycin tromtamol. Infection. 20(4):S305-S309. 
         Grif, K., M. P. Dierich, K. Pfaller, P. A. Miglioli, and F. Allerberger. 2001. In vitro activity of fosfomycin in combination with various antistaphylococcal substances. J Antimicrob Chemother 48:209-217. 
         Grimm, H. 1979. In vitro investigations with fosfomycin on Mueller-hinton agar with and without glucose-6-phosphate. Infect. 7(4):256-259. 
         Hammett-Stabler, C. A. and T. Johns. 1998. Laboratory guidelines for monitoring of antimicrobial drugs. Clin. Chem. 44(5):1129-1140. 
         Honorato, J., R. R. P. Ortola, R. Masso, and J. M. Reparaz. 1977. fosfomycin in acute bronchopneumonopathies. Chemother 23 (Suppl. 1):331-336. 
         Hunt, B. E., A. Weber, A. Berger, B. Ramsey, and A. L. Smith. 1995. Macromolecular mechanisms of sputum inhibition of tobramycin activity. Antimicrob. Agents. Chemother. 39(1):34-39. 
         Kahan, F. M., J. S. Kahan, P. J. Cassidy, and H. Kropp. 1974. The mechanism of action of fosfomycin (phoshonomycin). Ann N Y Acad Sci 253:364-386. 
         Katznelson, D., Y. Yahev, and E. 1984. Rubinstein. Fosfomycin in the treatment of cystic fibrosis. Eur J Clin Microbiol 3(3):213. 
         Kamijyo, A., Z. Matsuzaki, K. Kikushima, J. Ogino, I. Nozawa, T. Matsuoka, S. Endo, and Y. Okamoto. 2001. Fosfomycin nebulizer therapy to chronic sinusitis. Auris. Nasus. Larynx. 28(3):227-232. 
         Kondo, H., K. Suzuki, I. Takagi, N. Miyamoto, S. Baba, T. kobayashi, A. Yokota, I. Tanaka, and K. Sugiyama. 1996. Transitional concentration of antibacterial agent to the maxillary sinus via a nebulizer. Acta Otalaryngol (Stockh) Suppl 525:64-67. 
         Mirakhur, A., M. J. Gallagher, M. J. Ledson, C. A. Hart, and M. J. Walshaw. 2003. Fosfomycin therapy for multiresistant  Pseudomonas aeruginosa  in cystic fibrosis. J Cystic Fibrosis 2:19-24. 
         Kuhn, R. J. 2001. Formulation of aerosolized therapeutics. Chest. 120:94 S-98S. 
         Lang, B. J., S. D. Aaron, W. Ferris, P. C. Herbert, and N. E. MacDonald. 2000. Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with multiresistant strains of  Pseudomonas aeruginosa . Am. J. Respir. Crit. Care. Med. 162:2241-2245. 
         Marchese, A., L. Gualco, E. A. Debbia, G. C. Schito, and A. M. Schito. 2003. In vitro activity of fosfomycin against gram-negative urinary pathogens and the biological cost of fosfomycin resistance. Int. J. Antimicrob. Agents. 22(2):53-59. 
         Mendelman, P. M., A. L. Smith, J. Levy, A. Weber, B. Ramsey, and R. L. Davis. 1985. Aminoglycoside penetration, inactivation, and efficacy in cystic fibrosis sputum. Am. Rev. Respir. Dis. 132(4):761-765. 
         Menendez, A., A. Tutor, and A. S. Sousa. 1977. Treatment of respiratory infections with fosfomycin. Chemother 23 (Suppl. 1):348-357. 
         Mingeot-Leclercq, M-P. and P. M. Tulkens. 1999. Aminoglycosides: Nephrotoxity. Antimicrob. Agents. Chemother. 43(5):1003-1012. 
         Mirakhur, A., M. J. Gallagher, M. J. Ledson, C. A. Hart, and M. J. Walshaw. 2003. Fosfomycin therapy for multiresistant  Pseudomonas aeruginosa  in cystic fibrosis. J. Cyst fibros. 2(1):19-24. 
         Moreno, S., C. Ezpeleta, F. Parras, C. Banos, J. Martinez Beltran, and L. Buzon. 1986. Cure of a case of  Haemophilus aphrophilus  endocarditis with a combination of fosfomycin and gentamicin. J. Antimicrob. Chemother. 18(6): 771-772. 
         Mueller, M., A. de la Pena, and H. Derendorf. 2004. Issues in pharmacokinetics and pharmacodynamics of anti-infective agents: kill curves versus MIC. Antimicrob. Agents. Chemother. 48(2):369-377. 
         National Committee for Clinical Laboratory Standards (NCCLS). 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grows aerobically; approved standard. M7-A6, Vol. 23 No. 2. National Committee for clinical Laboratory Standards, Wayne, Pa. 
         National Committee for Clinical Laboratory Standards (NCCLS). 1999. Methods for determining bactericidal activity of antimicrobial agents; approved guideline. M26-A, Vol. 19 No. 18. National Committee for clinical Laboratory Standards, Wayne, Pa. 
         Nilsson, A. I., O. G. Berg, O. Aspevall, G. Kahlmeter, and D. I. Andersson. 2003. Biological costs and mechanisms of fosfomycin resistance in  Escherichia coli . Antimicrob. Agents. Chemother. 47(9):2850-2858. 
         O&#39;Riordan, T. G. 2000. Inhaled antimicrobial therapy: from cystic fibrosis to the flu. Respir. Care. 45(7):836-845. 
         Perri, M. B., E. Hershberger, M. Ionescu, C. Lauter, and M. J. Zervos. 2002. In vitro susceptibility of vancomycin-resistant enterococci (VRE) to fosfomycin. Diagn. Microbiol. Infect. Dis. 42:269-271. 
         Pitt, T L., M Sparrow, M Warner, and M Stefanidou. 2005. Survey of resistance of  Pseudomonas aeruginosa  from UK patients with cystic fibrosis to six commonly prescribed antimicrobial agents. Thorax 58(9):794-796. 
         Ramphal, R., M. Lhermitte, M. Filliat, and P. Roussel. 1988. The binding of antipseudomonal antibiotics to macromolecules from cystic fibrosis sputum. J. Antimicrob. Chemother. 22:483-490. 
         Ramsey, B W., M S Pepe, J M Quan, K L Otto, A B Montgomery, J Williams-Warren, K M Vasiljev, D Borowitz, C M Bowman, B C Marshall, S Marshall, and A L Smith. 1999. Intermittent administration of inhaled tobramycin in patients with cystic fibrosis. Cystic fibrosis inhaled tobramycin study group. N Engl J Med 340(1):23-30. 
         Ramsey B W. 1996. Management of pulmonary disease in patients with cystic fibrosis. N Engl J Med 335(3):179-188. 
         Ramsey, B W., H L Dorkin, J D Eisenberg, R L Gibson, I R Harwood, R M Kravitz, D V Schidlow, R W Wilmott, S J Astley, M A McBuurnie 1993. Efficacy of aerosolized tobramycin in patients with cystic fibrosis. N Engl J Med 328(24):1740-1746. 
         Reeves, D. S. 1994. Fosfomycin trometamol. J Antimicrob Chemother 34:853-858. 
         Schulin, T. 2002. In vitro activity of the aerosolized agents colistin and tobramycin and five intravenous agents against  Pseudomonas aeruginosa  isolated from cystic fibrosis patients in southwestern Germany. J Antimicrob Chemother 49:403-406. 
         Shawar, R M., D L MacLeod, R L Garber, J L Burns, J R Stapp, C R Clausen, and S K Tanaka. 1999. Activities of tobramycin and six other antibiotics against  Pseudomonas aeruginosa  isolates from patients with cystic fibrosis. Antimicrob Agents Chemother. 43(12):2877-2880. 
         Smith, A. L., C. Doersbuk, D. Goldman, E. Gore, B. Hilman, M. Marks, R. Moss, B. Ramsey, G. Redding, T. Rubio, J. Williams-Warren, R. Wilmott, H. D. Wilson, and R. Yogev. 1999. Comparison of a β-lactam alone versus β-lactam and an aminoglycoside for pulmonary exacerbations in cystic fibrosis. J. Pediatr. 134:413-421. 
         Spencker, F. B., L. Staber, T. Lietz, R. Schille, and A. C. Rodloff. 2002. Development of resistance in  Pseudomonas aeruginosa  obtained from patients with cystic fibrosis at different times. Clin. Microbiol. Infect. 9:370-379. 
         Tan, K. V., M. Mulheran, A. J. Knox, and A. R. Smyth. 2003. Aminoglycoside prescribing and surveillance in cystic fibrosis. Am. J. Respir. Crit Care med. 167(6):819-823. 
         Van Eldere, J. 2003. Multicenter surveillance of  Pseudomonas aeruginosa  susceptibility patterns in nosocomial infections. J. Antimicrob. Chemother. 51:347-352. 
         Vakulenko, S. B. and S. Mobashery. 2003. Versatility of aminoglycosides and prospects for their future. Clin. Microbiol. Rev. 16(3):430-450. 
         Weiss, K., and J. R. Lapointe. 1995. Routine susceptibility testing of four antibiotic combinations for improvement of laboratory guide to therapy of cystic fibrosis infections caused by  Pseudomonas aeruginosa . Antimicrob. Agents. Chemother. 39(11):2411-2414. 
         Woodruff, H. B., J. M. Mata, S. Hernandez, S. Mochales, A. Rodriguez, E. O. Stapley, H. Wallick, A. K. Miller and D. Hendlin. 1977. Fosfomycin: Laboratory studies. Chemother. 23(1):1-22)