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. 2016 Dec 27;61(1):e02056-16. doi: 10.1128/AAC.02056-16

Efficacy of High-Dose Meropenem (Six Grams per Day) in Treatment of Experimental Murine Pneumonia Induced by Meropenem-Resistant Pseudomonas aeruginosa

Kazuhiro Oshima a,b,c, Shigeki Nakamura e,✉, Naoki Iwanaga c, Koji Takemoto f, Taiga Miyazaki b,c, Kastunori Yanagihara d, Yoshitsugu Miyazaki e, Hiroshi Mukae c, Shigeru Kohno c, Koichi Izumikawa b
PMCID: PMC5192154  PMID: 27799201

ABSTRACT

High-dose meropenem (MEPM; 6 g/day) has been approved as a treatment for purulent meningitis; however, little is known regarding its in vivo efficacy in refractory lower respiratory tract infections. The purpose of this study was to evaluate the efficacy of MEPM at 6 g/day in a murine model of severe pneumonia caused by MEPM-resistant Pseudomonas aeruginosa. Experimental pneumonia induced by MEPM-resistant P. aeruginosa was treated with normal-dose MEPM (150 mg/kg of body weight, simulating a 3-g/day regimen in humans) or high-dose MEPM (500 mg/kg, simulating a 6-g/day regimen in humans). Mice treated with high-dose MEPM showed significantly restored survival relative to that of untreated mice and tended to show a survival rate higher than that of mice treated with normal-dose MEPM. The viable bacterial counts (of two clinical isolates) in the lungs decreased significantly in mice treated with high-dose MEPM from those for untreated mice (P < 0.001) or mice treated with normal-dose MEPM (P, <0.01 and <0.05). The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) was also significantly lower in mice treated with high-dose MEPM than in untreated mice. The free MEPM concentration in the epithelial lining fluid (ELF) exceeded 16 μg/ml for 85 min in mice treated with high-dose MEPM, but not for mice treated with normal-dose MEPM. Our results demonstrate that high-dose MEPM (6 g/day) might provide better protection against pneumonia caused by MEPM-resistant strains of P. aeruginosa than the dose normally administered (less than 3 g/day).

KEYWORDS: high dose, meropenem, pharmacokinetics/pharmacodynamics, meropenem-resistant Pseudomonas aeruginosa

INTRODUCTION

Pseudomonas aeruginosa is one of the major causes of hospital-acquired pneumonia (HAP) and opportunistic infections (1). HAP and bacteremia caused by P. aeruginosa can be fatal. A mortality rate of 47% has been reported for P. aeruginosa pneumonia patients. Mortality as a result of P. aeruginosa pneumonia is associated with a delay in initiating effective antimicrobial therapy and with multidrug-resistant P. aeruginosa (MDRP) (2, 3).

Meropenem (MEPM) is an antimicrobial drug that is potent against P. aeruginosa; however, the incidence of MDRP has increased recently (4), and P. aeruginosa is the most common multidrug-resistant bacterial cause of hospital-acquired pneumonia and ventilator-associated pneumonia (1). Approximately 13% of all health care-associated P. aeruginosa infections in the United States are caused by MDRP (5). Although MDRP is responsible for only 0.6% of P. aeruginosa infections detected in Japan, MEPM-resistant P. aeruginosa and imipenem-resistant P. aeruginosa are responsible for 13.8% and 16.3% of P. aeruginosa infections, respectively, indicating an increase in the rate of infection by carbapenem-resistant P. aeruginosa strains (6). Infections with carbapenem-resistant P. aeruginosa generally have a poorer prognosis than infections with carbapenem-sensitive P. aeruginosa (7). In addition, the efficacy of colistin against infections with carbapenem-resistant P. aeruginosa has not been established (8). Appropriate use of antimicrobial drugs is required to control the emergence of drug-resistant bacterial pathogens (9, 10). Pharmacokinetic (PK) parameters, such as the time above the MIC (TAM), the maximum concentration of the drug in serum divided by the MIC (Cmax/MIC), and the area under the concentration-time curve divided by the MIC (AUC/MIC), are used to predict the effectiveness of antimicrobial drugs. However, PK parameters used as efficacy indicators differ among antimicrobial drugs (11, 12). TAM is an efficacy indicator for carbapenems, including MEPM (11). Because the mutant prevention concentration and mutant selection window influence the emergence of drug-resistant bacterial pathogens, high doses of antibiotics can minimize the emergence of drug-resistant bacterial pathogens (13, 14). In addition, high doses of antibiotics can lead to better outcomes than normal doses of antibiotics (15, 16).

Several reports have shown the efficacy of high-dose antibacterial drugs against severe pneumonia. The efficacy of 3 g/day sulbactam-ampicillin for 4 days against intermediate to severe community-acquired pneumonia has been reported (17). High-dose tigecycline and colistin are effective against pneumonia caused by carbapenem-resistant Klebsiella pneumoniae in liver transplant patients (18). While the indications of high-dose MEPM (6 g/day) are currently limited to purulent meningitis and cystic fibrosis (CF), high-dose MEPM (6 g/day) is used for acute exacerbation of CF. In addition, high-dose MEPM (6 g/day) reduces the bacterial burden in sputum and improves clinical status (19). High-dose MEPM (6 g/day) might be effective against pneumonia caused by MEPM-resistant strains, because high drug concentrations reach the lungs.

This study was designed to evaluate the efficacy of high-dose MEPM (6 g/day) in comparison to that of normal-dose MEPM as a treatment for severe pneumonia caused by MEPM-resistant and low-susceptibility P. aeruginosa strains in mice.

RESULTS

High-dose MEPM treatment protects mice from pneumonia induced by MEPM-resistant P. aeruginosa.

The survival of the mice was observed for 7 days after infection. As shown in Fig. 1, the survival of mice treated with high-dose MEPM was significantly restored relative to that of untreated mice in the model of pneumonia and bacteremia induced by MEPM-resistant strains. In addition, the survival of mice treated with high-dose MEPM was higher than that of mice treated with normal-dose MEPM; however, no significant difference was observed.

FIG 1.

FIG 1

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Survival of mice infected with 3 × 107 (clinical isolate 1) or 1 × 108 (clinical isolate 2) CFU of MEPM-resistant P. aeruginosa and treated four times per day either with MEPM at 500 or 150 mg/kg or with PBS (untreated group) (n, 9 or 10). Statistical differences from the untreated group were determined by the Kaplan-Meier log rank test. Asterisks indicate significant differences from the untreated group (*, P < 0.05; **, P < 0.01).

Higher bactericidal activity of high-dose MEPM than of normal-dose MEPM in the blood and lungs.

Viable bacterial counts in blood were evaluated 4 h after infection (1 h after the first dose of MEPM) in the model of pneumonia and bacteremia induced by MEPM-resistant strains (Fig. 2A and C). The viable bacterial counts in the blood of the group treated four times a day with 500 mg of MEPM/kg of body weight were significantly lower than those in both the untreated group and the group receiving 150 mg/kg MEPM four times a day. In the comparison between the group receiving 500 mg/kg MEPM four times a day and the untreated group, viable bacterial counts in blood were 1.72 ± 0.12 log CFU/ml versus 4.37 ± 0.17 log CFU/ml, respectively (P < 0.001), with clinical isolate 1 and 1.97 ± 0.23 log CFU/ml versus 4.23 ± 0.14 log CFU/ml, respectively (P < 0.001), with clinical isolate 2. In the comparison between the two groups treated with MEPM four times a day (the 500-mg/kg and 150-mg/kg groups), viable bacterial counts in blood were 1.72 ± 0.12 log CFU/ml versus 3.27 ± 0.32 log CFU/ml, respectively (P < 0.05), with clinical isolate 1. However, there was no significant difference between the untreated group and the group receiving 150 mg/kg MEPM four times a day.

FIG 2.

FIG 2

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Dose-dependent bactericidal effects of MEPM with clinical isolate 1 (A and B) and clinical isolate 2 (C and D). (A and C) Numbers of viable bacteria in blood. Mice were inoculated with 3 × 107 (clinical isolate 1) or 1 × 108 (clinical isolate 2) CFU of MEPM-resistant P. aeruginosa. Mice in the different treatment groups (MEPM at 500 mg/kg, MEPM at 150 mg/kg, or no MEPM) were compared at 4 h after infection (1 h after the first dose of MEPM) (A) or 5 h after infection (2 h after the first dose of MEPM) (C). The number of viable bacteria in blood was significantly lower in the 500-mg/kg treatment group. Nine mice per group were used. ***, P < 0.001; *, P < 0.05. (B and D) Numbers of viable bacteria in the lungs. Mice were inoculated with 3 × 106 (clinical isolate 1) or 1 × 107 (clinical isolate 2) CFU of MEPM-resistant P. aeruginosa. At 38 h after infection (24 h after the first dose of MEPM), mice treated four times a day with MEPM at 500 mg/kg or 150 mg/kg and untreated mice were compared. The number of viable bacteria in the lungs was significantly lower in the 500-mg/kg treatment group. Thirteen to 15 (B) or 10 or 11 (D) mice per group were used. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Viable bacterial counts in the lungs were evaluated 36 h after infection (24 h after the first dose of MEPM) in the pneumonia model (Fig. 2B and D). The viable bacterial counts in the lungs of the group treated four times a day with 500 mg/kg MEPM were significantly lower than those in both the untreated group and the group receiving 150 mg/kg MEPM four times a day. In the comparison between the group receiving 500 mg/kg MEPM four times a day and the untreated group, viable bacterial counts in the lungs were 2.61 ± 0.33 log CFU/ml versus 5.11 ± 0.30 log CFU/ml, respectively (P < 0.001), with clinical isolate 1 and 3.56 ± 0.15 log CFU/ml versus 5.28 ± 0.19 log CFU/ml, respectively (P < 0.001), with clinical isolate 2. In the comparison between the two groups treated with MEPM four times a day (the 500-mg/kg and 150-mg/kg groups), viable bacterial counts in the lungs were 2.61 ± 0.33 log CFU/ml versus 4.28 ± 0.31 log CFU/ml, respectively (P < 0.01), with clinical isolate 1 and 3.56 ± 0.15 log CFU/ml versus 4.41 ± 0.15 log CFU/ml, respectively (P < 0.05), with clinical isolate 2. However, there was no significant difference between the untreated group and the group receiving 150 mg/kg MEPM four times a day.

High-dose MEPM treatment inhibits the pulmonary inflammation induced by MEPM-resistant P. aeruginosa.

Quantitative lung cultures and granulocyte counts in bronchoalveolar lavage fluid (BALF) were evaluated for the control group (Fig. 3A and B) over time. The number of bacteria in the lungs and the number of granulocytes in BALF at the evaluation point (38 h after infection) were higher than the corresponding numbers at the previous two time points (6 and 14 h after infection). The number of inflammatory cells in BALF (Fig. 3C) was evaluated 38 h after infection for all groups in the pneumonia model. The group receiving 500 mg/kg MEPM four times a day showed significantly lower numbers of inflammatory cells in BALF than the untreated group (5.44 ± 0.11 log cells/ml versus 6.13 ± 0.13 log cells/ml, respectively [P < 0.01]). However, there was no difference between the group receiving 150 mg/kg MEPM four times a day and the untreated group.

FIG 3.

FIG 3

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Numbers of viable bacteria in lungs and of inflammatory cells in BALF. Mice were inoculated with 3 × 106 CFU of MEPM-resistant P. aeruginosa. At 38 h after infection (24 h after the first dose of MEPM), mice treated four times a day with MEPM at 500 mg/kg or 150 mg/kg and untreated mice were compared. (A and B) Shown are numbers of viable bacteria in the lungs (A) and BALF granulocyte counts (B) in the control group over time. The numbers of bacteria in the lungs and of BALF granulocytes at the evaluation point (38 h after infection) were higher than the corresponding numbers at the previous two time points (6 and 14 h after infection). (C) The number of inflammatory cells in BALF was significantly lower for the group treated four times a day with 500 mg/kg. Seven mice per group were used. **, P < 0.01.

Histopathological examination.

As shown in Fig. 4, histopathological analysis of lungs stained with hematoxylin and eosin (HE) at 38 h after infection revealed that treatment with 500 mg/kg MEPM four times a day was more effective than treatment with 150 mg/kg MEPM four times a day.

FIG 4.

FIG 4

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Histopathological analysis of the lungs of mice inoculated with 3 × 106 CFU of MEPM-resistant P. aeruginosa and treated with high-dose MEPM. At 38 h after infection (24 h after the first dose of MEPM), mice treated four times a day with MEPM at 500 or 150 mg/kg and mice with no treatment were compared. HE-stained tissue sections were observed at magnifications of ×40 and ×200. (A) Untreated group; (B) group treated four times a day with 150 mg/kg MEPM; (C) group treated four times a day with 500 mg/kg MEPM. The inflammation of lungs decreased in a dose-dependent manner. Accumulation of inflammatory cells, hemorrhaging in the lungs, and destruction of alveoli were limited in mice treated four times a day with MEPM at 500 mg/kg.

Kinetics of free drug concentrations in the ELF of mice administered high-dose MEPM.

Free MEPM concentrations in epithelial lining fluid (ELF) were evaluated for both infected and uninfected mice and were found to not exceed 16 μg/ml even when a dose of 500 mg/kg was administered to uninfected mice (Fig. 5A). Conversely, in infected mice, free drug concentrations exceeded 16 μg/ml for 85 min after the administration of MEPM at 500 mg/kg; however, they never exceeded 16 μg/ml after the administration of MEPM at 150 mg/kg. The percentage of the time that free drug concentrations remained above the MIC (fTAM) in infected mice receiving 500 or 150 mg/kg MEPM four times a day was 23.6% or 0%, respectively (Fig. 5B). These data suggest that increased penetration of the airway by high-dose MEPM might underlie the protection against pulmonary infection with MEPM-resistant P. aeruginosa.

FIG 5.

FIG 5

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Free MEPM concentrations in ELF at 5, 15, 30, and 60 min after the administration of four doses of MEPM to noninfected mice (A) and to mice infected with 3 × 106 CFU of MEPM-resistant P. aeruginosa (B). For noninfected mice, free MEPM concentrations in the ELF did not reach 16 μg/ml even when MEPM was administered at 500 mg/kg. For infected mice, free MEPM concentrations in the ELF were higher than 16 μg/ml for 85 min in the group treated with 500 mg/kg of MEPM; however, they never exceeded 16 μg/ml in the group treated with 150 mg/kg of MEPM.

DISCUSSION

The efficacy of β-lactam drugs, including MEPM, is generally predicted by comparison between the MIC for the causative bacterium and the concentration of unbound drug in the extracellular fluid. The efficacy of MEPM can be discussed by considering the unbound drug concentration in the extracellular fluid to be equivalent to the total concentration of the drug in plasma, because the plasma protein binding rate of MEPM in humans is as low as 2%. In contrast, the plasma protein binding rate of MEPM is as high as 10% in mice; thus, the efficacy of MEPM should be discussed by taking this difference into consideration. In addition, a number of reports have suggested that the antibacterial drug concentration at the topical infected site reflects the efficacy of the drug. Although the topical infected site is indicative of intracellular substances in the lungs, in pneumonia, because of the presence of extracellular respiratory tract pathogens, including P. aeruginosa, bacteria can exist on the alveolar surface as well. Thus, the drug concentration in ELF is also an important factor that needs to be considered in discussing therapeutic efficacy (20–22). Hence, we measured the changes in MEPM concentrations in ELF over time in order to evaluate their association with therapeutic efficacy.

A noninfection mouse model and a mouse model of P. aeruginosa infection were used to measure MEPM concentrations in ELF. Because the migration of MEPM into ELF was found to be poor in the noninfection model, the concentration of unbound MEPM in ELF did not exceed 16 μg/ml in either group receiving MEPM four times a day (at 150 or 500 mg/kg per dose). In the P. aeruginosa infection model, however, for the group receiving 150 mg/kg MEPM four times a day, the unbound MEPM concentration in ELF did not exceed 16 μg/ml and the fTAM was 0%, whereas for the group receiving 500 mg/kg MEPM four times a day, the fTAM was 23.6%. Carbapenems, including MEPM, have been reported to exert a bacteriostatic effect at a TAM of 20% to 30% and an antimicrobial effect at a TAM of 40% to 50% (23, 24). The TAM in the group receiving 500 mg/kg MEPM four times a day was 23.6%, supporting the following effects of MEPM in this group: a trend toward an improved survival rate, a significant reduction in viable bacterial counts in the lungs, and improvement of inflammatory cell infiltration in the lungs in comparison with that in BALF and as observed by histopathological images. In this study, a trend toward an improved survival rate and a reduction in viable bacterial counts in the lungs were observed in the group receiving 150 mg/kg MEPM four times a day relative to those for the untreated group. The following factors may have potentially contributed to the reduction in viable bacterial counts in the lungs and the improvement in the survival rate: the plasma MEPM concentration achieved a TAM of 17.2%; a certain antimicrobial effect of MEPM was obtained at the sub-MIC level (25); and MEPM itself enhanced the phagocytosis of bacteria by macrophages (26).

The results of this study indicate that high-dose MEPM (6 g/day) was more effective than normal-dose MEPM against pneumonia caused by MEPM-resistant P. aeruginosa. P. aeruginosa is a typical causative bacterium in HAP and in health care-associated pneumonia with high mortality (1). Because delayed administration of effective antimicrobial therapy and failure of initial therapy result in poor prognosis, clinicians should select antibacterial drugs and initiate therapy without waiting for drug sensitivity test results for patients suspected to have P. aeruginosa pneumonia. Carbapenem antibiotics, including MEPM, are first-line drugs for the treatment of P. aeruginosa pneumonia. In this study, the concentration of the drug at the topical infection site (both the concentration in plasma and that in ELF) of MEPM-resistant P. aeruginosa (for which the MEPM MIC is 16 μg/ml) was unsatisfactory upon administration of normal-dose MEPM (3 g/day), and this may have led to treatment failure. However, high-dose MEPM (6 g/day) achieved a TAM (in both plasma and ELF) that enabled a sufficient treatment effect against MEPM-resistant P. aeruginosa. The MIC90 (MIC at which 90% of the isolates tested are inhibited) of MEPM against P. aeruginosa is ≤16 μg/ml in Japan and other countries (27–34); thus, according to PK-pharmacodynamic (PD) theory, high-dose MEPM (6 g/day) might be able to achieve clinical efficacies of >90% against P. aeruginosa. In clinical practice, high-dose MEPM may be indicated for patients suspected to have severe P. aeruginosa pneumonia, for whom failure of initial therapy is not permissible. Moreover, high-dose (6-g/day) MEPM may be highly effective for patients with MEPM-sensitive P. aeruginosa pneumonia and for immunocompromised patients with neutropenia, lung abscess, cystic fibrosis, and idiopathic pulmonary fibrosis (IPF) that limit drug migration into the topical infected site. Therefore, further validation, including clinical studies, is necessary.

MATERIALS AND METHODS

Bacterial isolates.

Clinical isolates of MEPM-resistant (MEPM MIC, 16 μg/ml) P. aeruginosa were utilized in this study. The MEPM MIC was determined by the broth microdilution method according to CLSI guidelines (42). Isolates stored in Trypticase soy broth with 10% glycerol stocks maintained at −80°C at Nagasaki University Hospital were spread on LB agar (Sigma, Tokyo, Japan) and were incubated overnight at 37°C under 5% CO2 prior to use in the experiments. The mechanism of MEPM resistance was not investigated in this study; however, we confirmed that these strains did not produce metallo-beta-lactamase.

Laboratory animals.

Pathogen-free ddY mice (7 weeks old, female) weighing about 30 g were purchased from SLC Inc., Shizuoka, Japan. All of the animals were housed in a pathogen-free environment and received sterile food and water at the laboratory of the Animal Center for Biomedical Science at Nagasaki University (Nagasaki, Japan). The Ethics Review Committee for Animal Experimentation at Nagasaki University approved all experimental protocols used in this study.

Pharmacokinetic (PK) studies and determination of dosing regimen.

Plasma MEPM concentrations in mice were measured after intraperitoneal administration of 100 mg/kg MEPM combined with 100 mg/kg cilastatin, and those in humans after intravenous administration of 1 g of MEPM over 30 min were obtained from previous data (35, 36). The PK parameters were calculated using a two-compartment model with the MULTI program (37). The percentage of the time that free drug concentrations remained above the MIC (fTAM) was calculated using the PK parameters, protein binding levels, and MICs. The level of protein binding of MEPM in mice is 10% (38). Because the level of binding of MEPM to protein in human plasma is very low (2%) (39), total-drug concentrations in the plasma of humans were used as free-drug concentrations. Table 1 shows the fTAMs of the MEPM regimens for humans and mice. The fTAM of MEPM for humans was calculated when 1 g or 2 g of MEPM was administered to humans for 30 min, three times a day. Four-dose intraperitoneal administration of MEPM (10 ml/kg/dose) at 2-h intervals was chosen to alleviate the pain of the mice. The dose regimens required for mice infected with the MEPM-resistant strain to achieve fTAMs equivalent to those with human regimens of 3 g/day and 6 g/day were 150 mg/kg four times per day and 500 mg/kg four times per day, respectively (Table 1). The half-life of meropenem-cilastatin in mice is short (12 min), and concentrations of meropenem-cilastatin in plasma at 2 h after administration were less than 1/500 of those at 5 min after administration. Therefore, the PK of the first dose of meropenem was equal to that of other doses of meropenem, and the TAM in the regimen with 2-h intervals was the same as that in the regimen with 6-h intervals.

TABLE 1.

fTAMs of MEPM dose regimens for humans and mice with pneumonia caused by MEPM-resistant P. aeruginosa

Host Dose regimen fTAM (%)a
Human 2 g, 3 times/day 25.1
Mouse 500 mg/kg, 4 times/day 24.8
Human 1 g, 3 times/day 15.4
Mouse 150 mg/kg, 4 times/day 17.4
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a

fTAM, the percentage of the time that free drug concentrations remain above the MIC.

Murine models of pneumonia caused by P. aeruginosa.

P. aeruginosa was cultured on LB agar and was incubated overnight at 37°C under 5% CO2. The organisms were suspended in normal saline. For the study of pneumonia with bacteremia, 20 μl of the suspended MEPM-resistant strain (3 × 107 CFU of clinical isolate 1 and 1 × 108 CFU of clinical isolate 2) was inoculated intranasally with anesthesia. For the pneumonia study, 20 μl of the suspended MEPM-resistant strain (3 × 106 CFU of clinical isolate 1 and 1 × 107 CFU of clinical isolate 2) was inoculated intranasally with anesthesia. MEPM was administered intraperitoneally 3 h after inoculation in the model of pneumonia with bacteremia and 14 h after inoculation in the pneumonia model. MEPM at 150 mg/kg or 500 mg/kg was administered four times a day at 2-h intervals in combination with 100 mg/kg cilastatin to yield a PK profile similar to that in humans (3 g/day or 6 g/day, respectively). The treatment lasted for 2 days in the model of pneumonia with bacteremia and for 1 day in the pneumonia model. The model of pneumonia with bacteremia was used to evaluate the survival rate and the viable bacterial counts in blood, whereas the pneumonia model was used to evaluate the viable bacterial counts in the lungs, the number of inflammatory cells in BALF, and free MEPM concentrations in epithelial lining fluid (ELF), and for histopathological analysis of the lungs.

Lung preparation for CFU determination and histopathological analysis.

Whole lungs were removed under aseptic conditions and were homogenized in 1.0 ml phosphate-buffered saline (PBS). P. aeruginosa was quantified by placing serial dilutions of the lung homogenates on LB agar plates and incubating them at 37°C under a 5% CO2 atmosphere. For histopathological analysis, lung specimens were fixed in a 10% formalin-buffered solution, and then the lung tissue sections were paraffin embedded and were stained with hematoxylin and eosin (HE) using standard procedures (40).

BALF cell analysis.

BALF analysis was performed with mice different from those used for CFU determination and histopathological analysis in order to assess inflammatory cell accumulation in the airspace. The chest was opened to expose the lungs after the mouse was anesthetized, and a disposable sterile feeding tube (Toray Medical Co., Chiba, Japan) was inserted into the trachea. BAL was performed using 1.0 ml of PBS, and the recovered fluid was pooled for each mouse. Total-cell counts were performed by Turk staining with a hemacytometer (40, 41).

Measurement of MEPM concentrations in ELF.

BALF samples were mixed with 4 volumes of methanol, vortex mixed, and centrifuged at 10,000 × g for 10 min at 4°C. The supernatants were stored at −80°C until the measurement of MEPM concentrations by high-performance liquid chromatography (HPLC). The supernatants (50 μl) were separated on an XTerra MS C18 reverse-phase column (particle size, 3.5 μm; inside diameter, 4.6 mm; length, 20 mm; Nihon Waters K.K., Tokyo, Japan) with methanol–5 mM sodium dihydrogen phosphate (pH 7.0) (3:17) as the mobile phase delivered at 1.0 ml/min. The HPLC system (LC-2010C; Shimadzu Co., Kyoto, Japan) was controlled by a Class VP workstation (Shimadzu), and the wavelength for MEPM detection was 300 nm. Five-point standard curves (0.1 to 10 μg/ml) were linear, with an r2 of >0.98. The lower limit of quantitation was 0.1 μg/ml. The concentration of MEPM in ELF was calculated as follows: (concentration in BALF) × [(urea in serum)/(urea in BALF)]. Calculating the fTAM in ELF is the same as calculating the fTAM in plasma [see the “Pharmacokinetic (PK) studies and determination of dosing regimen” paragraph above]. Serum samples were also collected just before BAL fluid was obtained from the same mice used for the urea assay.

Urea assay.

The rate of decline in NADH levels induced by NH3 in the samples was measured. Urea was hydrolyzed by urease to produce NH3. The NH3 produced reacted with α-ketoisohexanoic acid and NADH by the action of leucine dehydrogenase to form leucine and NAD. The rate of decline in NADH levels at this point was measured optically, and the urea content in the sample was calculated by subtracting the rate of decline resulting from the endogenous ammonia reaction.

Statistical analysis.

All data were analyzed by using Prism 5 (GraphPad Software) and were expressed as means ± standard errors of the means (SEMs). Survival analysis was performed using the log rank test, and the survival rate was calculated by the Kaplan-Meier method. Differences between groups were examined using the Kruskal-Wallis test and Dunn's multiple-comparison test. A P value of <0.05 was considered to indicate a statistically significant difference.

ACKNOWLEDGMENTS

This work was supported by a grant from the Program for Nurturing Global Leaders in Tropical and Emerging Communicable Diseases, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.

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