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We determined the concentration-time profiles of ciprofloxacin and amikacin in serum and alveolar epithelial lining fluid (ELF) of rats with or without pulmonary fibrosis and investigated the effect of pulmonary fibrosis on the capacity for penetration of antimicrobials into the ELF of rats. Pulmonary fibrosis was induced in rats with a single intratracheal instillation of bleomycin. After intravenous injection of ciprofloxacin or amikacin, blood and bronchoalveolar lavage fluid samples were collected. Urea concentrations in serum and lavage fluid were determined using an enzymatic assay. Ciprofloxacin and amikacin concentrations were determined by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry, respectively. The mean ratio of ELF to plasma concentrations of ciprofloxacin at each time point in the normal group did not significantly differ from that in the pulmonary fibrosis group. However, the ratio of the ciprofloxacin area under the concentration-time curve from 0 to 24 h (AUC0–24) in ELF to the AUC0–24 in plasma was 1.02 in the normal group and 0.76 in the pulmonary fibrosis group. The mean ELF-to-plasma concentration ratios of amikacin at each time point in the normal group were higher than those in the pulmonary fibrosis group, reaching a statistically significant difference at 1, 2, and 4 h. The ratio of the AUC0–24 in ELF to the AUC0–24 in plasma was 0.49 in the normal group and 0.27 in the pulmonary fibrosis group. In conclusion, pulmonary fibrosis can influence the penetration of antimicrobials into the ELF of rats and may have a marked effect on the penetration of amikacin than that of ciprofloxacin.
Though the concentration-time data antimicrobials in plasma can allow us to gain valuable insight into the link between the drug doses and the outcomes of infections, the antimicrobial concentration at the site of infection is a direct factor affecting therapeutic effectiveness (1). In pneumonia, the alveolar epithelial lining fluid (ELF) and alveolar cells are known as the intrapulmonary sites where the extracellular and intracellular “drug-pathogen” interactions occur (1, 2). Except for a few intracellular pathogens, most cases of community- or hospital-acquired pneumonia are caused by extracellular pathogens, such as Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Enterobacteriaceae, and nonfermentative bacteria (2). Therefore, adequate antimicrobial concentrations in the ELF, exceeding the MICs of causative pathogens, are required for clinical therapeutic success (3).
Antimicrobials are distributed from plasma to ELF mainly via passive concentration-dependent diffusion across the alveolar capillary membrane (4). This diffusion can be influenced by the physicochemical properties of drugs and by the pharmacokinetic (PK) characteristics, such as the level of protein binding and the permeability of anatomical barriers of the lung (5,–7). Pulmonary fibrosis, which can develop following a variety of acute and chronic lung diseases, is characterized by accumulation of excess connective tissue, distortion of normal lung parenchyma, and severe damage of the capillary bed (8, 9). These histopathological changes of pulmonary fibrosis can impair the pulmonary diffusion function of gases and thus may influence the penetration of many drugs, including antibiotics, into ELF.
In this study, we determined the concentration-time profiles of ciprofloxacin and amikacin in sera and ELF of rats with or without pulmonary fibrosis and investigated the effect of pulmonary fibrosis on the degree of penetration of antimicrobials into the ELF.
Typical pulmonary images and the histopathological appearance of pulmonary tissues of normal rats and rats with pulmonary fibrosis are shown in Fig. 1. In the lungs of normal rats, only a small amount of collagen fibers in the alveolar septum was observed. In the lungs of rats with pulmonary fibrosis, large fibrotic areas and extensive structural damage were observed. Additionally, the degree of pulmonary fibrosis was clearly shown by the significant alveolar-arterial oxygen pressure difference [P(A−a)DO2] between the normal rats and the rats with pulmonary fibrosis. In rats receiving ciprofloxacin, the P(A−a)DO2 values for the normal rats and rats with pulmonary fibrosis were 10.65 ± 12.28 mm Hg and 17.75 ± 11.43 mm Hg, respectively (P = 0.009); in rats receiving amikacin, the P(A−a)DO2 values for f the normal rats and rats with pulmonary fibrosis were 14.22 ± 8.13 mm Hg and 22.40 ± 8.32 mm Hg, respectively (P < 0.001). As shown in Fig. 2, the urea concentrations in plasma and bronchoalveolar lavage fluid (BALF) were not significantly different among the four groups.
The plasma and ELF concentration-time profiles of ciprofloxacin are shown in Fig. 3. In both the normal and pulmonary fibrosis groups, the ciprofloxacin concentrations rose sharply after intravenous bolus injection and then fell progressively over time. At 0.25 h, the mean concentrations in ELF for the two groups were lower than those in plasma. At 0.5 to 2 h, the mean ciprofloxacin concentrations in ELF for the normal group were slightly higher than those in plasma, while the mean concentrations in ELF for the pulmonary fibrosis group were slightly lower than those in plasma. The mean ELF/plasma concentration ratios of ciprofloxacin at 0.25 to 4 h in the normal group and the pulmonary fibrosis group are shown in Fig. 4. Statistical analysis showed no significant difference in the ratios at each time point between the two groups. The calculated PK parameters of amikacin in plasma and ELF are shown in Table 1. The ratio of the area under the concentration-time curve from 0 to 24 h (AUC0–24) in ELF to the AUC0–24 in plasma was 1.02 in the normal group and 0.76 in the pulmonary fibrosis group.
The plasma and ELF concentration-time profiles of amikacin are shown in Fig. 5. The amikacin plasma concentrations at each time point were not significantly different between the normal and the pulmonary fibrosis groups. At 0.25 to 1 h, the mean amikacin concentrations in ELF for the two groups were significantly lower than those in plasma. At 2 to 4 h, the mean concentrations in ELF for the normal group were higher than those in plasma, and the mean concentrations for the pulmonary fibrosis group were still lower than those in plasma. As shown in Fig. 4, the mean of ELF/plasma concentration ratio at each time point in the normal group were higher than those reported for the pulmonary fibrosis group. A statistically significant difference was observed at 1, 2, and 4 h. As shown in Table 1, the ratio of the AUC0–24 in ELF to the AUC0–24 in plasma was 0.49 in the normal group and 0.27 in the pulmonary fibrosis group.
Fluoroquinolones and aminoglycosides are widely used for the clinical treatment of pulmonary infections. The pharmacokinetic characteristics of these two antimicrobials in plasma and the lungs have been thoroughly investigated in healthy volunteers and in patients with chronic obstructive pulmonary disease or lower respiratory tract infections (10,–13). However, very few studies have assessed the intrapulmonary concentrations of these agents in patients with pulmonary fibrosis, whose lung structure changes likely influence the intrapulmonary concentration of drugs. In this study, we used rats with bleomycin-induced pulmonary fibrosis, the most common animal model with similarities to human idiopathic pulmonary fibrosis (IPF), to evaluate the effect of pulmonary fibrosis on the penetration of ciprofloxacin and amikacin into the ELF.
The PKs of antimicrobials differ between small animals and humans. The half-lives (t1/2) of these drugs in rats are shorter and the elimination rates much faster than those in humans (14). Because the purpose of this study was to investigate the effect of pulmonary fibrosis on the intrapulmonary concentrations of antimicrobials, we did not take these differences into consideration and simply chose doses similar to those clinically used in patients according to the corresponding surface area-dosage conversion between humans and rats. In this study, in normal rats the maximum concentration (Cmax) of ciprofloxacin in ELF was 77.6% ± 34.9% of that in plasma (Fig. 3) and the penetration ratio of AUC0–24 between ELF and plasma was 1.02, indicating that ciprofloxacin can penetrate well into ELF. Similar results have been noted in critically ill patients with chronic obstructive pulmonary disease. Kontou et al. found that ciprofloxacin penetrated well into bronchial secretions, achieving a Cmax that was 60% of that in plasma and an AUC0–24 penetration ratio of 1.06 (10). However, for amikacin, the Cmax and AUC0–24 in ELF were much lower than those in plasma (Fig. 5), and the PKs in ELF were different from those in plasma. A clinical study that investigated the PKs of gentamicin in patients with ventilator-associated pneumonia showed that the ratio of Cmax in ELF to that in serum was 0.32 (13). These results suggested that the penetration of aminoglycosides through the alveolar-capillary barrier was generally poor.
The results of our study showed that the ELF/plasma ciprofloxacin concentration ratios at each time points were not significantly different between the normal rats and rats with pulmonary fibrosis. However, the AUC0–24 ratios in the pulmonary fibrosis group were lower than those in the normal group. In a preliminary clinical study, Huang et al. compared the difference in intrapulmonary concentration of levofloxacin between patients with normal lung function and patients with IPF (15). Though they did not compare the ELF/plasma concentration ratios between the two groups, they found that the mean ELF concentration at 3 h in IPF patients (n = 10) was 50% lower than that in patients with normal lung function (n = 10) (15). Thus, these results indicate that the distribution of fluoroquinolones from plasma to ELF may be influenced by pulmonary fibrosis to some extent. The ELF/plasma amikacin concentration ratios reached a statistically significant difference at 1 to 4 h, and the AUC0–24 ratio of ELF/plasma amikacin concentration in the pulmonary fibrosis group was nearly half of that in the normal group, suggesting that pulmonary fibrosis has a greater effect on the penetration of amikacin than on that of ciprofloxacin.
Antimicrobials can be roughly classified into hydrophilic (β-lactams, glycopeptides, aminoglycosides, et al.) and lipophilic (fluoroquinolones, macrolides, tetracyclines, et al.) agents according to their physicochemical characteristics (16). The results of our study showed that amikacin does not distribute well into tissues in comparison to ciprofloxacin and that the concentrations of amikacin in ELF are more likely to be influenced by pulmonary fibrosis. Therefore, although plasma concentrations of hydrophilic antibiotics may appear to be therapeutic, the ELF concentrations of antibiotics in areas of fibrosis may be insufficient. Lipophilic antibiotics may be the first choice for treating pneumonia in patients with severe pulmonary fibrosis. For the use of hydrophilic antibiotics, higher doses or special administration approaches, such as extended or continuous infusion of β-lactam antibiotics or administration via nebulization, can be used to raise drug concentrations in ELF (17).
Several limitations of this study exist. Because we produced the model using a single intratracheal instillation of bleomycin, the distribution of fibrosis in rats was not homogeneous. We collected the BALF of the whole lung, but not from the area with fibrosis. The drug concentration in the area with fibrosis should be lower than that determined in this study. Moreover, the pulmonary surface area of rats is much smaller than that of humans. Therefore, the effect of fibrosis on drug penetration in humans may be underestimated by using the results obtained from the rat model. In addition, we used urea as an endogenous marker for the determination of ELF volume and dilution. Urea appears to be a reliable marker, as it is a small and nonpolar molecule that can freely travel across membranes (18). However, it has been shown that if lavage fluid dwell times are prolonged, urea can diffuse from the interstitium and blood, which therefore falsely elevates the urea concentration in the BALF (18). Therefore, in order to reduce these influences, the lavage fluid dwell time in this study was only 20 s.
In conclusion, our study suggests that pulmonary fibrosis can influence the penetration of antimicrobials into the ELF in rats. Pulmonary fibrosis may have a greater effect on the penetration of amikacin than on that of ciprofloxacin. These findings need to be verified in further clinical studies.
Specific-pathogen-free Sprague-Dawley male rats weighing 250 ± 10 g (Charles River, Beijing, China) were used in this study. This animal study was approved by the Research Animal Care and Use Committee of the Chinese PLA General Hospital. Rats were maintained and utilized according to the Protocol for the Protection and Welfare of Animals.
The pulmonary fibrosis model was constructed as described previously (19). The rats were fully anesthetized by an intraperitoneal injection of 30 mg/kg pentobarbital sodium. Then, a midline incision was made in the neck and the trachea was exposed by blunt dissection. A tracheal cannula was inserted under direct visualization into the trachea, and a single dose of 0.1 ml of bleomycin (5 mg/kg in 0.9% NaCl) was intratracheally administered. To induce pulmonary fibrosis, rats were maintained for 1 month while extensive structural changes occurred in the lungs (20, 21). Since body weight is a key factor that may influence the pharmacokinetics (PKs) of antimicrobials (16), rats in control groups were neither subjected to the same protocol nor maintained for 1 month before the administration of antimicrobials. We chose healthy rats with body weights similar to those of the rats with pulmonary fibrosis (the difference in weight between the two groups was less than 20 g) for the control groups. Bleomycin-induced pulmonary fibrosis was confirmed by histopathological examination of 10 randomly selected rats and by comparing the alveolar-arterial oxygen pressure difference [P(A−a)DO2] between the normal rats and rats with pulmonary fibrosis. The P(A−a)DO2 was calculated using the formula P(A−a)DO2 = (Pa − PH2O) × FiO2% − PaCO2/R − PaO2 (22), where Pa is atmospheric pressure (the standard state is 760 mm Hg), PH2O is saturated water vapor pressure (the standard state is 47 mm Hg) FiO2% is the fraction of inspired O2 (0.21 for inhalation of air); R is the respiration quotient (R = 1 was used in this study), PaO2 is the arterial pressure of oxygen, PAO2 is the alveolar partial pressure of oxygen, and PaCO2 is the arterial partial pressure of carbon dioxide.
Rats with pulmonary fibrosis and the healthy control rats were administered, twice daily, either ciprofloxacin (Zhaoyi Biological Co., Ltd., Shanxi, China) at a dose of 60 mg/kg or amikacin (Qilu Pharmaceuticals, Shandong, China) at a dose of 50 mg/kg. According to the manufacturers' recommendations, the intravenous doses of ciprofloxacin and amikacin for pneumonia are 1,200 mg/day [17.14 mg/(kg · day) in a 70-kg patient] and 15 mg/(kg · day), respectively. Because the body weights of the rats with pulmonary fibrosis ranged from 270 to 300 g after 1 month of maintenance, the corresponding surface area-dosage conversion factor from humans to rats used in this study was 7 as recommended (23), and the equivalent human doses for ciprofloxacin and amikacin in rats would be 120 mg/(kg · day) and 105 mg/(kg · day), respectively. Therefore, the aforementioned doses were chosen for this study.
Antimicrobials were administered in rats with or without pulmonary fibrosis for 3 consecutive days via tail vein injection. At 0, 0.25, 0.5, 1, 2, 4, 8, 12, or 24 h after the last injection on the third day, rats were sacrificed for collecting blood samples and bronchoalveolar lavage fluid (BALF). Separate groups of ≥5 rats were studied for each time point. In brief, after the rats were anesthetized, two blood samples from each rat were obtained using aseptic puncture of the abdominal aorta. The first blood sample was immediately sent for arterial blood gas analysis (ABL 90 flex; Radiometer, Copenhagen, Denmark), and the second blood sample was centrifuged (2,000 rpm at 0°C for 10 min). The tracheas were immediately cannulated. The lungs were lavaged three times with 2 ml sterile saline of each time, and each lavage lasted for 20 s. The BALF was immediately centrifuged (1,000 rpm at 0°C for 10 min). Both the plasma and BALF were frozen at −80°C until assay.
Total ciprofloxacin concentrations in serum and BALF were determined by high-performance liquid chromatography (HPLC) according to a previously described method (10). The lower limit of quantification was 0.05 mg/liter. Coefficients of determination (r2) over the standard curve concentrations of 1 to 10 mg/liter for plasma and of 0.05 to 2 mg/liter for BALF were >0.99. Intra- and interday coefficients of variation for the HPLC measurement were 0.49 to 4.46% and 7.72 to 8.83%, respectively, for plasma samples, and 2.21 to 5.0% and 6.84 to 9.51%, respectively, for BALF samples. The recovery of ciprofloxacin in plasma was 100 to 101.2%, and that in BALF was 95 to 99.6%.
Total amikacin concentrations in serum and BALF were determined by liquid chromatography-tandem mass spectrometry based on the method described by Dijkstra et al. (24). The quantification limit for amikacin was 0.1 mg/liter. Good linearity was obtained, with a correlation coefficient of >0.99 for both plasma in the concentration range of 0.1 mg/liter to 10 mg/liter and BALF in the concentration range of 0.1 mg/liter to 10 mg/liter. Intra- and interday coefficients of variation for the measurement were 3.6 to 8.9% and 5.3 to 9.28%, respectively, for plasma samples and 3.6 to 7.8% and 4.5 to 7.5%, respectively, for BALF samples. The recovery of amikacin in plasma was 86.9 to 120.38%, and that in BALF was 93.0 to 128.5%.
As urea can diffuse readily through the body (25), we used it as an endogenous marker to determine the actual concentrations of antimicrobials in ELF. Urea concentrations in serum and BALF samples were tested using an enzymatic assay (urea colorimetric assay kit no. K375-100; BioVision, Milpitas, CA, USA) in a microplate reader (Diagnostics, Montpellier, France) according to the manufacturers' specifications. In brief, standard curve was first prepared, and the standard curves were linear (r2 = 0.99) over a concentration range of 0 to 5 mmol/liter. Then, 25-μl BALF samples or 10-fold-diluted plasma samples, 25 μl assay buffer, and 50 μl reagents were added to a 96-well plate. The 96-well plate was protected from light and incubated for 60 min at 37°C. The levels of antimicrobials in ELF were calculated using the following formula: antimicrobial (ELF) = antimicrobial (BALF) × [urea (plasma)/urea (BALF)].
Total antimicrobial concentrations in plasma and ELF were plotted against time. The pharmacokinetic (PK) parameters were calculated based on the mean drug concentration at each time point by noncompartmental analysis using the WinNonlin software program (version 6.2; Pharsight Corporation, Mountain View, CA). The area under the curve from 0 to 24 h (AUC0–24) was computed by the linear-up-log-down approach. The antimicrobial penetration into the ELF was evaluated by the ELF/serum concentration ratio at each time point and the ratio of the AUC0–24 in ELF to the AUC0–24 in plasma. Statistical analysis was performed using a statistical software package (SPSS 20.0; SPSS, Chicago, IL, USA). When data were normally distributed, an independent t test was used for individual data, a paired-sample t test was used for paired data, and analysis of variance was used to compare the concentrations of urea in plasma and ELF in the four groups. The nonparametric Mann-Whitney U test was used to compare data that were not normally distributed. A significance level of 0.05 was applied to all tests.
This work was supported by the National Natural Science Foundation of China (no. 81371855).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We declare no conflicts of interest.