Abstract

Editor’s Perspective
What We Already Know about This Topic
  • For most bacterial pneumonia, the lung interstitium is considered to be the site of infection, and adequate antibiotic concentrations are important for drug effect

  • Despite systemic antibiotic therapy, therapeutic failure is common, perhaps due to poor lung penetration, and resulting low interstitial space fluid antibiotic concentrations

  • Increasing systemic antibiotic doses in order to increase interstitial space fluid antibiotic concentrations could lead to toxicities such as nephrotoxicity

What This Article Tells Us That Is New
  • In a mechanically ventilated healthy large animal model, nebulized tobramycin produced higher peak lung interstitial space fluid concentrations, as well as higher initial epithelial lining fluid concentrations, with lower plasma concentrations than were observed after intravenous administration due to more extensive lung penetration

Background

Nebulized antibiotics may be used to treat ventilator-associated pneumonia. In previous pharmacokinetic studies, lung interstitial space fluid concentrations have never been reported. The aim of the study was to compare intravenous and nebulized tobramycin concentrations in the lung interstitial space fluid, epithelial lining fluid, and plasma in mechanically ventilated sheep with healthy lungs.

Methods

Ten anesthetized and mechanically ventilated healthy ewes underwent surgical insertion of microdialysis catheters in upper and lower lobes of both lungs and the jugular vein. Five ewes were given intravenous tobramycin 400 mg, and five were given nebulized tobramycin 400 mg. Microdialysis samples were collected every 20 min for 8 h. Bronchoalveolar lavage was performed at 1 and 6 h.

Results

The peak lung interstitial space fluid concentrations were lower with intravenous tobramycin 20.2 mg/l (interquartile range, 12 mg/l, 26.2 mg/l) versus the nebulized route 48.3 mg/l (interquartile range, 8.7 mg/l, 513 mg/l), P = 0.002. For nebulized tobramycin, the median epithelial lining fluid concentrations were higher than the interstitial space fluid concentrations at 1 h (1,637; interquartile range, 650, 1,781, vs. 16 mg/l, interquartile range, 7, 86, P < 0.001) and 6 h (48, interquartile range, 17, 93, vs. 4 mg/l, interquartile range, 2, 9, P < 0.001). For intravenous tobramycin, the median epithelial lining fluid concentrations were lower than the interstitial space fluid concentrations at 1 h (0.19, interquartile range, 0.11, 0.31, vs. 18.5 mg/l, interquartile range, 9.8, 23.4, P < 0.001) and 6 h (0.34, interquartile range, 0.2, 0.48, vs. 3.2 mg/l, interquartile range, 0.9, 4.4, P < 0.001).

Conclusions

Compared with intravenous tobramycin, nebulized tobramycin achieved higher lung interstitial fluid and epithelial lining fluid concentrations without increasing systemic concentrations.

Ventilator-associated pneumonia due to Pseudomonasvaeruginosa and other Gram-negative organisms occurs frequently,1  and is associated with significant morbidity and mortality.2  Drugs from the aminoglycoside class, such as tobramycin, are recommended for the treatment of ventilator-associated pneumonia3  and are effective against numerous Gram-negative organisms including P. aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumanii.4  Despite systemic antibiotic therapy, therapeutic failure is common.5  One reason for this lack of effect could be poor lung penetration due to low lung epithelial lining fluid concentrations of tobramycin.6  Increasing the systemic doses could lead to toxicities such as nephrotoxicity.

For treatment of ventilator-associated pneumonia, nebulized tobramycin may be used either as monotherapy or as an adjuvant.7,8  However, aerosol delivery and lung deposition (and hence antibiotic concentrations in the lung) can be affected by mechanical ventilation parameters.9  A number of clinical10–12  and experimental studies13,14  have investigated the pharmacokinetics of nebulized antibiotics with mechanical ventilation using plasma, sputum, and epithelial lining fluid samples. However, we are unaware of any data on the interstitial space fluid pharmacokinetics of nebulized antibiotics.15  As there are no data of the lung pharmacokinetics of different sampling and administration techniques, data from noninfected models can provide valuable mechanistic insights. Rat and rabbit model-based studies provide some data on the pharmacokinetics of nebulized and IV tobramycin.16,17  However, interspecies differences in pulmonary anatomy limit the application of these data to humans. Similarly, pharmacokinetic data from studies in spontaneously breathing cystic fibrosis patients18,19  make it difficult to apply the same for ventilator-associated pneumonia therapy. Experimental pneumonia studies have been useful in the understanding of the pharmacokinetics of nebulized antibiotics with epithelial lining fluid or tissue homogenization technique having inherent limitations.14,20  Sheep are commonly used for large animal model respiratory research and for inhalation therapy studies.21  Compared to the human lung, sheep have similar anatomical and physiologic features.22 

For most bacterial pneumonia, the lung interstitium is considered as the site of infection, and adequate antibiotic concentrations are important for drug effect.23  Currently available pharmacokinetics methods used for nebulized antibiotics have significant limitations that affect their ability to sample the interstitial space fluid.24  Lung microdialysis is a validated technique for the sampling of interstitial space fluid and measurement of free antibiotic concentration.24 

The aim of this study was to compare the regional pulmonary and systemic concentrations of tobramycin after nebulization and IV administration in a mechanically ventilated healthy ovine model. We hypothesized that nebulized tobramycin will achieve higher pulmonary concentrations with lesser systemic concentrations compared to IV tobramycin.

Materials and Methods

Ethics approval was obtained from the Queensland University of Technology (Chermside, Australia) animal ethics committee (approval No. 1100000052).

Study Design

The study was performed using 10 merino sheep (10 ewes, 40 to 50 kg), which were anesthetized and mechanically ventilated. Five sheep were administered IV tobramycin and the other five given nebulized tobramycin. Figure 1 shows the study setup.

Fig. 1.

Study methods showing the operating theater setup, the sheep position, and instrumentation. Sheep mechanically ventilated in “prone position” with head support. Microdialysis catheters placed via thoracotomy and perfused using microdialysis pumps. Intercostal catheters inserted after thoracotomy.

Fig. 1.

Study methods showing the operating theater setup, the sheep position, and instrumentation. Sheep mechanically ventilated in “prone position” with head support. Microdialysis catheters placed via thoracotomy and perfused using microdialysis pumps. Intercostal catheters inserted after thoracotomy.

Experimental Method

Anesthesia, Ventilation, and Monitoring.

The sheep were anesthetized with midazolam 0.5 mg/kg and alfaxalone 3 mg/kg (Jurox Pty. Ltd., Australia). Maintenance of anesthesia was with ketamine 3 to 5 mg · kg-1 · h-1, midazolam 0.25 to 0.5 mg · kg-1 · h-1, and alfaxalone 4 to 6 mg · kg-1 · h-1 infusions. Sheep were orotracheally intubated using an endotracheal tube. Tracheostomy was performed, and mechanical ventilation was commenced with initial settings of tidal volume 10 to 12 ml/kg, positive end-expiratory pressure 5 cm H2O, respiratory rate 14 breaths per minute and inspiratory flow 40 l per minute square wave pattern, no inspiratory pause, and inspiratory time 0.75 s with the resulting peak airway pressure 20 to 22 mmHg and subsequently adjusted according to the arterial blood gases to maintain oxygen saturation greater than 90% partial pressure of oxygen greater than 60 cm H20 and arterial carbon dioxide tension 35 to 45 mmHg. The humidifier was turned off for the duration of nebulization. Monitoring was performed using electrocardiogram, arterial blood gas, capnography, pulse oximetry, and central venous pressure monitoring.

Microdialysis Probe Insertion.

Bilateral thoracotomies were performed in the fifth intercostal space. Under direct vision, microdialysis probes (CMA 63; CMA Microdialysis AB, Sweden) were inserted, one probe each in the upper and lower lobes of both lungs. An intercostal catheter was placed on each side, and the thoracotomy incisions was closed. An intravascular microdialysis catheter (CMA 64; CMA Microdialysis AB) was inserted in the left jugular vein through a percutaneously inserted 18G cannula (B. Braun Australia Pty. Ltd., Australia). CMA 107 pumps (CMA Microdialysis AB) were used to perfuse the microdialysis catheters.

Gentamicin (4 mg/l) as a perfusate was used for in vivo microdialysis calibration which was done as per the internal indicator method.25,26  The perfusate flow was maintained at 1 μl/min for 1 h to achieve steady state equilibrium. Thereafter, samples were collected at every 20 min in a microvial (CMA Microdialysis AB) for 8 h, with time 0 h being the commencement of drug administration. Samples were stored at –80°C for further analysis.

Drug Administration

Intravenous.

Tobramycin 400 mg (4 ml; Tobra-day 500 mg/5 ml, Phebra) was injected through the central venous line as a 30-min infusion.

Nebulization.

Tobramycin 400 mg (4 ml; Tobra-day 500 mg/5 mL, Phebra) was nebulized using a disposable vibrating mesh nebulizer (Aeroneb Pro; Aerogen, Ireland) which was inserted in the inspiratory limb of the circuit just before the Y-piece. The nebulization was complete when there was no visible mist.

Bronchoalveolar Lavage.

Bronchoalveolar lavage fluid was collected from both lower lobes using a fiber optic bronchoscope (Olympus; USA) before drug administration and repeated at 1 h and 6 h after the completion of drug administration. Samples were stored at –80°C for further analysis.

Sample Analysis

Bronchoalveolar lavage fluid and plasma urea concentration values were obtained for further calculations of tobramycin in the epithelial lining fluid (Cepithelial lining fluid) with a commercially available assay (UniCel DxC 800; Beckman Coulter, USA).

The gentamicin and tobramycin concentrations in the microdialysis samples and the tobramycin concentrations in the bronchoalveolar lavage samples reported were measured by a liquid chromatography tandem mass spectrometry method: Shimadzu Nexera2 system coupled to a Shimadzu 8030+ triple quadrupole mass spectrometer (Japan). The transitions monitored for tobramycin were 468.1→163.2 and 468.1→324.2, for gentamicin were 464.2→322.3, 478.2→322.3, and 450.2→322.3, and for vancomycin were 746.1→144.2 and 725.6→144.1. For the analysis of tobramycin in bronchoalveolar lavage, 10 μl of sample was diluted with 90 μl of internal standard (50 μg/ml of vancomycin), vortex-mixed, and transferred to an autosampler vial. For the analysis of tobramycin and gentamicin in microdialysis, 10 μl of sample was diluted with 30 μl of internal standard (100 μg/ml of vancomycin), vortex-mixed, and transferred to an autosampler vial. Chromatographic separation was achieved using a SeQuant zic-HILIC analytical column (Merck, Germany) with a gradient elution of mobile phases composed of (A) 10% acetonitrile in 90% of 2 mM ammonium acetate containing 0.2% formic acid and (B) 90% acetonitrile in 10% of 2 mM ammonium acetate containing 0.2% formic acid. The gradient commenced with 80% of mobile phase B for 0.5 min, decreasing down to 10% of mobile phase B for more than 1.5 min, where it was held for 1.5 min. The mobile phase then returned to 80% B for more than 1 min, to allow a final run time of 7.5 min at a flow 0.4 ml/min. The concentration range of the assay was 0.1 to 20 μg/ml for tobramycin and 0.5 to 10 μg/ml for gentamicin with the calibration best fitted as quadratic with a 1/concentration2  weighting. For tobramycin, the precision was 3.9, 5.2, and 1.9% and accuracy was –4.6, –7.0, and –1.6% at 0.25, 1, and 4 μg/ml, respectively, and for gentamicin, the precision was 2.8 and 3.1% and accuracy was –2.0 and 3.9% at 1 and 4 μg/ml, respectively. Using the method described in the data analysis section, the microdialysis recovery data were used to calculate the corrected tobramycin concentrations in the microdialysate.

Data Analysis

Tobramycin Concentration in Epithelial Lining Fluid.

The volume (V) of epithelial lining fluid in each sample was calculated as previously described25,27,28 :

formula

Therefore, the concentration of tobramycin (TOB) in the epithelial lining fluid from the bronchoalveolar lavage fluid is as follows:

formula

where Vepithelial lining fluid equals volume of epithelial lining fluid, Urea (mg/l)bronchoalveolar lavage equals concentration of urea in the bronchoalveolar lavage fluid, Urea (mg/l)Plasma equals concentration of urea in the plasma, Vbronchoalveolar lavage equals volume of bronchoalveolar lavage fluid, and TOBepithelial lining fluid equals concentration of tobramycin in the epithelial lining fluid.

Microdialysis Recovery.

The relative recovery was calculated by loss of gentamicin (Gent) for each microdialysate using the internal reference method,25,26  as per the equation

formula

where Cin is gentamicin concentration in the perfusate and Cout is gentamicin concentration in the dialysate.

Tobramycin Concentration in Interstitial Space Fluid.

The corrected tobramycin concentration in the microdialysate was calculated as

formula

where Cinterstitial space fluid is tobramycin concentration in the interstitial space fluid and Cdialysate is tobramycin concentration in microdialysate.

Systemic bioavailability of the nebulized dose was calculated as follows:

formula

where AUCplasma equals the area under the drug concentration-time curve.

Pharmacokinetic Analysis

The pharmacokinetic analyses of tobramycin were performed for microdialysis and plasma samples using non-compartmental methods (see Supplemental Digital Content 1, http://links.lww.com/ALN/B936, describing the methods used for the pharmacokinetic analyses, and Supplemental Digital Content 2, http://links.lww.com/ALN/B937, showing the derived model and the results obtained). For intravenous tobramycin, the lung penetration was used to describe the percentage drug penetration into the interstitial space fluid and defined as fAUCinterstitial space fluid / AUCplasma × 100 and calculated from the AUC0-last, where AUCinterstitial space fluid and AUCplasma are area under the concentration-time curve from 0 to 8 h (AUClast) for interstitial space fluid and plasma, respectively, and expressed as a percentage. As the AUCepithelial lining fluid cannot be calculated reasonably with two concentration points, this value was estimated by population pharmacokinetics modeling. Plasma, interstitial space fluid, and epithelial lining fluid observations were fitted into a three-compartment model, and AUCepithelial lining fluid was determined using individual estimated parameters for each sheep. This analysis was performed using the nonparametric adaptive grid algorithm available in the Pmetrics R package (Laboratory of Applied Pharmacokinetics and Bioinformatics, USA).29 

Statistical Analysis

The variables were summarized as medians and interquartile range (twenty-fifth and seventy-fifth).

We used boxplots of the data by treatment groups to see what tests were suitable, choosing the parametric t test when the data were relatively well behaved in terms of skew. Where there was strong skew in the observed data, we used a bootstrap test in order to avoid any parametric assumptions. For the Bayesian model, we log-transformed the pharmacokinetic parameters to remove the positive skew. We checked that the predictions (means and 95% credible intervals) from the Bayesian model gave sensible predictions that matched the observed averages. The Bayesian estimates were made using Markov Chain Monte Carlo. We used two chains with a burn-in of 5,000 and sample of 5,000 both thinned by three, and visually checked the convergence of the two chains to the same solution. Vague priors were used for all parameters. The Markov Chain Monte Carlo estimates were also used to estimate Bayesian P values for the probability that the treatment had no effect by calculating the proportion of times that the estimate was positive or negative, and then taking the smallest of these two proportions and multiplying by two to give a two-sided posterior probability for the null hypothesis that the difference is zero. For the tobramycin concentrations in the epithelial lining fluid and interstitial space fluid, we used linear regression as a standard approach, but we log-transformed the dependent variable (concentration) because of a strong positive skew. This helps the model’s validity as it is more likely that the residuals will be normally distributed and hence our inferences in terms of means, CIs, and P values will be more reliable.

Plasma pharmacokinetics parameters between the IV and nebulized route were compared using a simple unpaired t test. When comparing the IV and nebulized route, the mean differences between the lung interstitial space fluid and bronchoalveolar lavage fluid concentrations of tobramycin, Bayesian regression model with a random intercept was used for each sheep and Bayesian two-sided P values obtained. R statistical language version 3.4.3 (R Development Core Team, New Zealand; www.r-project.org) and WinBUGS version 1.4.3 (BUGS project, MRC Biostatistics Unit, United Kingdom)30  for the Bayesian regression analysis were used. For the ratio of AUCepithelial lining fluid and AUCplasma, as well as the ratio of AUCinterstitial space fluid and AUCplasma, an ordinary parametric bootstrap method with 10,000 replications was used. All P values were two-tailed. No statistical power calculation was conducted before the study. Sample size was based on our previous experience with microdialysis-based studies describing the pharmacokinetic variability.31  The regression models were fit for the data described, and the fitness was considered acceptable for the purpose.

Results

Ten healthy merino sheep (ewes, 40 to 50 kg) underwent anesthesia and mechanical ventilation. Data from all the samples was available for analyses, and there were no missing data.

Lung Interstitial Space Fluid Pharmacokinetics

The in vivo recovery values determined were 43% for lung interstitial space fluid. The combined median interstitial space fluid pharmacokinetics parameters with ranges comparing the two routes of administration are given in table 1. The median peak concentration was significantly lower for IV tobramycin than for nebulized.

Table 1.

Comparative Lung Pharmacokinetic Parameters between Intravenous and Nebulized Tobramycin

Comparative Lung Pharmacokinetic Parameters between Intravenous and Nebulized Tobramycin
Comparative Lung Pharmacokinetic Parameters between Intravenous and Nebulized Tobramycin

The regional lung interstitial space fluid pharmacokinetics parameters for IV and nebulised tobramycin are given in tables 2 and 3, respectively. The mean interstitial space fluid pharmacokinetics parameters for each of the four lobes were not significantly different between the IV and nebulized tobramycin. However, the study was not powered to detect a difference. Figure 2 shows the regional concentration-time profile in a graphical form.

Table 2.

Pharmacokinetic Parameters of Tobramycin in the Right and Left, Upper and Lower Lobes as well as Plasma (Intravenous Group)

Pharmacokinetic Parameters of Tobramycin in the Right and Left, Upper and Lower Lobes as well as Plasma (Intravenous Group)
Pharmacokinetic Parameters of Tobramycin in the Right and Left, Upper and Lower Lobes as well as Plasma (Intravenous Group)
Table 3.

Pharmacokinetic Parameters of Tobramycin in the Right and Left, Upper and Lower Lobes as well as Plasma (Nebulized Group)

Pharmacokinetic Parameters of Tobramycin in the Right and Left, Upper and Lower Lobes as well as Plasma (Nebulized Group)
Pharmacokinetic Parameters of Tobramycin in the Right and Left, Upper and Lower Lobes as well as Plasma (Nebulized Group)
Fig. 2.

Log scaled plot showing regional concentration-time profile for lung interstitial space fluid and plasma with nebulized and intravenous (IV) tobramycin showing high lung interstitial space fluid and low plasma levels of nebulized tobramycin versus IV tobramycin.

Fig. 2.

Log scaled plot showing regional concentration-time profile for lung interstitial space fluid and plasma with nebulized and intravenous (IV) tobramycin showing high lung interstitial space fluid and low plasma levels of nebulized tobramycin versus IV tobramycin.

Plasma Pharmacokinetics

The median plasma and interstitial space fluid pharmacokinetics parameters with ranges (minimum to maximum) for the upper and lower lung areas of both lungs are given in tables 2 and 3 for IV and nebulized tobramycin, respectively. Comparison of results between the IV and nebulized administration groups shows differences for some pharmacokinetics parameter estimates including higher values in the IV group for plasma maximum concentration. The median systemic bioavailability of the nebulized dose was 78.2% (interquartile range, 76%, 98.5%).

Epithelial Lining Fluid Concentrations and the Ratio of Epithelial Lining Fluid Concentration and Plasma Concentration between IV and Nebulized Tobramycin

See table 4. The median epithelial lining fluid concentration for tobramycin was significantly lower for IV compared to nebulized tobramycin group at 1 h (P < 0.001) and at 6 h. Similarly, at 1 h and at 6h, the median ratio of epithelial lining fluid concentration and plasma concentration was significantly lower for IV compared to nebulized.

Table 4.

Comparative Tobramycin Concentrations in Epithelial Lining Fluid and Ratio of Epithelial Lining Fluid to Plasma Concentration for Intravenous and Nebulized Tobramycin

Comparative Tobramycin Concentrations in Epithelial Lining Fluid and Ratio of Epithelial Lining Fluid to Plasma Concentration for Intravenous and Nebulized Tobramycin
Comparative Tobramycin Concentrations in Epithelial Lining Fluid and Ratio of Epithelial Lining Fluid to Plasma Concentration for Intravenous and Nebulized Tobramycin

Comparative Epithelial Lining Fluid and Interstitial Space Fluid Pharmacokinetics Parameters between IV and Nebulized Tobramycin

See table 1. The median ratio of AUCepithelial lining fluid and AUCPlasma was significantly higher for nebulized tobramycin than IV. The median AUCepithelial lining fluid/AUCPlasma was significantly higher than AUCinterstitial space fluid/AUCPlasma for nebulized tobramycin and reduced for IV. The median AUCinterstitial space fluid/AUCPlasma was not significantly different for nebulized and IV tobramycin.

Comparative Epithelial Lining Fluid and Interstitial Space Fluid Tobramycin Concentrations 1 and 6 h after Administration

See table 5 and figure 3. At 1 h after administration, for nebulized tobramycin, the median concentration in the epithelial lining fluid was significantly higher than the interstitial space fluid concentration (1,637 vs. 16 mg/l, P < 0.001). In comparison, for IV tobramycin, the median interstitial space fluid concentration was significantly higher compared to that of the epithelial lining fluid (18.5 vs. 0.19, P < 0.001). At 6 h after administration, for nebulized tobramycin, the median epithelial lining fluid concentration was significantly higher than the interstitial space fluid (48 vs. 4 mg/l, P < 0.001). However, for IV tobramycin, the median interstitial space fluid concentration was significantly higher than that of epithelial lining fluid (3.2 vs. 0.34, P < 0.001).

Table 5.

Comparative Tobramycin Concentrations in Epithelial Lining Fluid and Interstitial Space Fluid for Intravenous and Nebulized Tobramycin

Comparative Tobramycin Concentrations in Epithelial Lining Fluid and Interstitial Space Fluid for Intravenous and Nebulized Tobramycin
Comparative Tobramycin Concentrations in Epithelial Lining Fluid and Interstitial Space Fluid for Intravenous and Nebulized Tobramycin
Fig. 3.

Comparative box plots showing higher epithelial lining fluid (ELF) than interstitial space fluid (ISF) tobramycin concentrations for nebulized tobramycin compared to intravenous (IV) tobramycin. The dots indicate the sample points that are outside the box plots.

Fig. 3.

Comparative box plots showing higher epithelial lining fluid (ELF) than interstitial space fluid (ISF) tobramycin concentrations for nebulized tobramycin compared to intravenous (IV) tobramycin. The dots indicate the sample points that are outside the box plots.

Discussion

This study quantitatively characterizes and compares the concentration and disposition of tobramycin in regional lung interstitial space fluid, epithelial lining fluid, and plasma after IV and nebulized administration using a microdialysis technique. The main finding of this study is that nebulized tobramycin achieves significantly higher peak concentrations with more extensive lung penetration in the lung interstitial space fluid and epithelial lining fluid with low plasma concentrations, thus potentially minimizing systemic side effects as compared to IV. The epithelial lining fluid concentrations 1 h after nebulization were 100-fold higher than those of the interstitial space fluid of tobramycin. This could be due to bronchial contamination during the bronchoalveolar sampling. Conversely, the epithelial lining fluid concentrations 1 h after IV administration were less than 1% of those of the interstitial space fluid of tobramycin, suggesting that epithelial lining fluid is poorly representative of interstitial space fluid concentrations.

After nebulization, the peak plasma concentration was lower compared to IV (median, 17.8, range, 14.4–18.2 mg/l for nebulized; median, 24.1, range, 22.8–27.6 mg/l for IV; P = 0.089). Previous studies have described low plasma trough concentrations less than 0.5 mg/l after administration of 300 mg of IV tobramycin.11  The authors have even suggested that drug level monitoring may not be necessary with nebulized tobramycin. Although our study did not complete the 24-h dosing interval period for tobramycin therapy, the low end-study concentrations would indicate reduced trough concentrations with nebulized tobramycin. Thus, systemic toxicity is likely less with nebulized antibiotics, although further validation is required with this and other nebulized antibiotics.

For tobramycin, which displays a concentration-dependent bactericidal activity, the interstitial space fluid maximum concentration is the central pharmacokinetics parameter defining effectiveness. In the lung interstitial space fluid, the peak tobramycin concentrations were significantly higher when tobramycin was nebulized compared to when given IV (median, 48.3 mg/l vs. 20.2 mg/l, P = 0.002). For tobramycin, the maximum concentration/minimum inhibitory concentration ratio 7 to 10 is the pharmacodynamic index associated with increased clinical cure32  and greater than 10 for resistance suppression.33  With the European Committee on Antimicrobial Susceptibility Testing minimum inhibitory concentration for Pseudomonas of 4 mg/l, the desired maximum concentration is > 40 mg/l. The data from our study indicate that it is possible to achieve these targets with nebulized tobramycin. Taken together, the low plasma concentrations and higher interstitial space fluid concentrations seen after nebulized tobramycin confer an advantage for this route by potentially minimizing systemic side effects as compared to IV.

For IV tobramycin, the maximum concentration and the other pharmacokinetics parameters in the left lower lobe appeared lower as compared to the other lung lobes. The differences did not achieve statistical significance, possibly due to small sample size. The reduced maximum concentration in the left lower lobe could represent differences in blood flow or could also be due to atelectasis34  by cardiac and other mediastinal structures that could not be excluded.

There was a higher maximum concentration and other pharmacokinetics parameters of drug exposure in both lower lobes compared to upper lobes with nebulized tobramycin. In the left upper lobe, the interstitial space fluid maximum concentration values were lower with nebulized compared to IV tobramycin. In the right upper lobe, the interstitial space fluid maximum concentration was higher with nebulized tobramycin compared to IV. Although these differences were not statistically significant, small sample size may have limited these differences from achieving statistical significance. In the supine position in healthy spontaneously breathing humans, alveolar deposition of aerosols is seen to be directly proportional to ventilation with increased deposition in the lower lobes.35  Our study shows similar deposition patterns in mechanically ventilated sheep.

The low epithelial lining fluid concentration and the low epithelial lining fluid/serum tobramycin concentration ratio with IV tobramycin compared to nebulized tobramycin are expected as evidenced in the critically ill setting6  and in pneumonia.12  The high water solubility feature of aminoglycosides impedes their ability to cross cell membranes, leading to poor epithelial lining fluid penetration with IV administration.36  The concentrations with IV tobramycin were lower in our study (epithelial lining fluid 0.53 mg/ml and epithelial lining fluid/serum ratio 2%) than those reported in the studies where the reported epithelial lining fluid concentrations range from 1.6 mg/ml to 4 mg/l and the ratios from 12 to 30%.6,12  Some of the studies did not measure free tobramycin and were not in a mechanically ventilated setting, which may affect the results. Nebulized tobramycin resulted in higher epithelial lining fluid concentration in all studies. In our study, the concentrations were higher (1,270 mg/l) than most reported studies and more than 1,000-fold higher than that of IV tobramycin. This could be due to a higher nebulized dose (400 mg in our study vs. 300 mg in most other studies). Interspecies variation could account for the differences as well. The possibility of bronchoscope contamination during sampling needs further investigation with a targeted study. At 6 h, the epithelial lining fluid concentrations decreased to 54 mg/l for nebulized and 0.42 mg/l for IV tobramycin. Thus, compared to the values at 1 h, the epithelial lining fluid concentrations for nebulized tobramycin seems to decrease more than those of IV. This could be a result of high lung deposition of nebulized tobramycin due to effective aerosol delivery and concentration gradient–based transport into the interstitial space fluid and blood and less with the mucociliary clearance mechanism. This is further likely due to the high water solubility, low protein binding, small molecular mass, and negative charge on the tobramycin molecule. With IV administration, however, due to high plasma concentrations, the clearance could be predominantly due to the mucociliary escalator mechanism.

Notably, there were significant differences in the epithelial lining fluid and interstitial space fluid concentrations with nebulized tobramycin (at 1 h, epithelial lining fluid, 1,270 mg/l, and interstitial space fluid, 16.90 mg/l; at 6 h, epithelial lining fluid, 54.1 mg/l, and interstitial space fluid, 3.97 mg/l). The ratio of AUCepithelial lining fluid and AUCPlasma was also significantly higher than AUCinterstitial space fluid and AUCPlasma for the nebulized route and lower for the IV route. The poor cell membrane penetration of aminoglycosides36  could explain the differences wherein there are lower epithelial lining fluid concentrations compared to interstitial space fluid with intravenous tobramycin, whereas with nebulized tobramycin, the epithelial lining fluid concentrations are higher than the interstitial space fluid. Thus, depending on the site of infection, either route or combined routes could be used for therapy.

Apart from reasons mentioned above, contamination of the bronchoscope from the airway secretions, which may have higher tobramycin deposition with nebulized tobramycin compared to IV, may result in such differences. Similar high epithelial lining fluid antibiotic concentrations compared to plasma concentrations with inhaled administration could be a result of bronchial contamination as demonstrated in other studies.20  The high epithelial lining fluid concentrations reported in such studies may not translate into high efficacy of inhaled antibiotics. Further studies with other antibiotics are required to confirm these findings before definite recommendations could be made regarding the validity of epithelial lining fluid sampling techniques in the pharmacokinetics evaluation of nebulized antibiotics in general.

Due to low correlation coefficients between interstitial space fluid and epithelial lining fluid concentrations (r2 = 0.03 for IV and r2 = 0.04 for nebulized), there is poor predictability of interstitial space fluid concentration from epithelial lining fluid concentration and vice versa.

Limitations

The study was conducted only with ewes. Although sex difference is unlikely to influence the results of this study, it is possible that there might be some unforeseeable effects. The ventilator settings were not optimized according to consensus conference recommendations.37,38  It is possible that inspiratory flow turbulences due to the large tidal volumes led to a high extrapulmonary and bronchial deposition. This could cause the interstitial space fluid tobramycin concentrations to decrease and epithelial lining fluid tobramycin concentrations to increase because of a high contamination of the bronchoscope during epithelial lining fluid sampling. Therefore, a part of the very high epithelial tobramycin concentrations could be in part related to nonoptimized ventilator settings. The study was conducted in a large animal model, and extrapolation of these data to humans may not yield the same results. The animals were not randomized to the two routes of administration, and the study was not blinded. However, we attempted to mitigate possible bias due to this by assessor-blinding, i.e., drug concentrations were measured by a person unaware of the allocation. The effect of different ventilator settings on aerosol delivery is well known, but its effect on the antibiotic pharmacokinetics is relatively unknown. It is possible that nebulization through a tracheostomy tube may result in differences in distal drug delivery compared to that with an endotracheal tube. However, recent in vitro studies indicate that these differences are insignificant.39  The study was not powered to measure the regional lung tobramycin pharmacokinetics differences. The study was performed in healthy conditions, and pneumonia and other lung conditions could influence the pharmacokinetics parameters in an unpredictable fashion. Although the tidal volumes (10 to 12 ml/kg) were in keeping with other studies in a sheep model, these are not recommended as lung protective ventilation guidelines.40  Optimized ventilator settings are likely to improve the interstitial space fluid concentrations as well as epithelial lining fluid concentrations. However, further in vivo studies are required to study the effects of ventilator settings using this model.

Although there was no visible trauma to the lung, there is a possibility of undetected lung trauma, which may impact the microdialysis results. No radiographic investigations were performed to exclude atelectasis and other lung abnormalities that may influence lung antibiotic pharmacokinetics. As the insertion of the microdialysis probe is a blind procedure, issues such as malposition or effect of respiratory movement of the lungs on the microdialysis membranes could impact the microdialysis results as well, although we believe this was unlikely in our study as our relative recovery values were similar to results reported in previously published studies. It is possible that the microdialysis technique may underestimate the parenchymal concentrations in comparison to that obtained using the tissue homogenate method.

Further studies are required to study the effect of other parameters such as synchronized nebulization, different formulations, and ventilator settings. Patient-related factors such as effect of posture, airways disease, and mucus need consideration in these studies. Lung microdialysis could be used in evaluating the pharmacokinetics of other nebulized antibiotics. Pharmacodynamic studies and clinical studies are required to determine clinical effectiveness.

Conclusions

In a mechanically ventilated healthy large animal model, nebulized tobramycin results in higher lung interstitial space fluid concentrations and achieves high lung penetration without increasing systemic concentrations. Immediately after nebulization, very high tobramycin epithelial lining fluid concentrations were observed, suggesting heavy bronchial contamination. Further studies are necessary to inform tobramycin pharmacokinetics in the mechanically ventilated patients with lung infection.

Acknowledgments

The authors would like to acknowledge the contributions by the staff and administrators of the Medical Engineering and Research Facility, Queensland University of Technology, Chermside, Australia, who provided the valuable resources and facilities to successfully conduct this study.

Research Support

Dr. Dhanani would like to acknowledge funding from The Prince Charles Hospital Foundation (Chermside, Australia; grant No. MS2011-40) and The Royal Brisbane and Women’s Hospital Foundation grants (Brisbane, Australia; 2012 and 2016). Dr. Roberts wishes to recognize funding from the Australian National Health and Medical Research Council for Center of Research Excellence (grant No. APP1099452) and a Practitioner Fellowship (Australia; grant No. APP1117065). Dr. Fraser acknowledges a Queensland Health Research Fellowship (OHMR Qld Health Fellowship, Queensland, Australia: grant No. 6375141/2 JF) and funding from the Australian National Health and Medical Research Council for Center of Research Excellence (Australia; CRE ACTIONS NHMRC Application: grant No. APP1079421).

Competing Interests

The authors declare no competing interests.

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