Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 6. Aminoglycosides

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Respiratory infections in mechanically ventilated patients are frequent (1). Although parenteral administration of aminoglycosides constitutes the standard route of administration, nebulization (NEB) is being used more and more frequently, and tobramycin (TOB) and amikacin (AMK) are the most commonly prescribed agents for NEB (1). Compared with fluoroquinolones, which present high membrane permeability (2), the difficulty of aminoglycosides to permeate membranes may allow for high intrapulmonary concentrations to be obtained by patients after NEB. This was observed in rats, with a ratio of TOB area under the epithelial lining fluid (ELF) concentration-time curve (AUC_ELF) over plasma AUC of 222 after NEB (3). The aim of this new study was to extend this finding to two other aminoglycoside antibiotics, AMK and gentamicin (GEN), using the same standardized protocol (3).

GEN and AMK for parenteral administration were used for all experiments (GEN sulfate Panpharma solution 80 mg · ml⁻¹, expressed in base, and AMK Mylan 500 mg powder). Animal experiments were conducted in compliance with European Community directive 2010/63/EU after approval by the local ethics committee (COMETHEA) and were registered by the French Ministry of Higher Education and Research under authorization number 2015070211159865. Male Sprague Dawley rats (n = 49 for GEN and 40 for AMK; mean weight, 300 g) from Janvier Laboratories (Le Genest-St-Isle, France) were used for the experiments. As previously described, they were divided in two groups corresponding to the route of administration for each molecule (intravenous [i.v.] or NEB) (3, 4). GEN and AMK were administered under anesthesia by i.v. bolus in the tail vein (1 ml) or by intratracheal NEB (100 μl) with a microsprayer 1A-1B (Penn-Century, Wyndmoor, PA) at doses commonly used in clinical practice, after correction for body weight, and equal to 8 and 30 mg · kg⁻¹, respectively. Bronchoalveolar lavage (BAL) fluid and blood sampling were performed until 4 h after administration (4 or 5 sampling times [0.5, 1, 2, 3, 4 h] and 4 to 7 rats per sampling time). Assays were conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The mass spectrometer was operated in the positive mode, and ions were analyzed by multiple-reaction monitoring. An Alliance Waters 2695 system coupled with a Quattro micro API tandem mass spectrometer (Waters SAS, Saint Quentin en Yvelines, France) was used to perform GEN analysis in plasma and BAL fluid, using a previously described method with limited modifications (5). Determination of AMK concentrations in plasma and BAL fluid was performed using a method previously developed by KCAS Bioanalytical and Biomarker Services (Shawnee, Kansas). The system was composed of a Shimadzu LC module (Nexera XR; Shimadzu France, Marne la Vallée, France) coupled with an API 3000 mass spectrometer (Sciex; Life Sciences, Les Ulis, France). For both molecules, the same concentrations were used for BAL fluid and plasma standard curves (0.005 to 2 μg · ml⁻¹); BAL fluid samples were injected directly, whereas plasma samples were deproteinized with trichloroacetic acid 10%. Intra- and interday variabilities were evaluated at four levels of concentration (limit of quantification [LOQ], 2 or 3 times the LOQ, an intermediate concentration in the standard curve, and 75% of the highest standard curve concentration). Precision and accuracy of <15% was tolerated, with the exception of 20% at the LOQ. Urea concentrations in plasma and BAL fluid were measured as previously described (4, 6). ELF antibiotic concentrations were calculated from the measured BAL fluid concentration after correction for dilution by the urea method (4, 6). For each compound, plasma and ELF concentrations versus time were analyzed simultaneously after i.v. and NEB administration with the nonlinear mixed-effect method using S-ADAPT software (7). Plasma protein binding of AMK and GEN was assumed to be negligible. The final structural pharmacokinetic model was the same for both molecules, i.e., a central compartment from which drugs were eliminated and two ELF compartments connected by two distribution clearances, one between plasma and the first ELF compartment and the other between the two ELF compartments. In accordance with previous observations with the Penn-Century microsprayer, complete bioavailability after NEB was hypothesized and was fixed to 100% (3, 8). Areas under plasma and ELF concentration-time curves from time zero to infinity (AUC_plasma, AUC_ELF) and elimination half-lives were estimated from the model (Berkeley Madonna, version 8.3.18, University of California). To compare pulmonary drug exposure after NEB versus after i.v. administration, the targeting advantage of NEB was estimated by the ratio of AUC_ELF after NEB to AUC_ELF after i.v. corrected for doses (9).

After i.v. bolus, typical plasma elimination half-lives of AMK and GEN were 0.40 and 0.49 h, respectively. Half-lives in ELF were at least 3 times longer than in plasma (2.1 and 1.6 h, respectively), suggesting that elimination from ELF is distribution-rate limited for both molecules (Fig. 1). Because complete bioavailability after NEB was hypothesized, AMK and GEN plasma concentrations were virtually similar regardless of the route of administration. Data were also in accordance with passive diffusion between plasma and ELF, since the ratios of ELF to plasma AUCs after i.v. administration were estimated to be 1.0 for both molecules. After NEB, the maximal plasma concentration (0.5 h) was observed at the first time of sampling (Fig. 1), and the ratio of mean ELF to plasma concentration was ∼5 times higher for AMK than for GEN (871 versus 160) (Table 1). Accordingly, ratios of AUC_ELF after NEB to AUC_ELF after i.v. corrected for doses were estimated by the model to be 874 and 162 for AMK and GEN, respectively (Table 1), attesting to a high targeting advantage with NEB for both molecules. We previously conducted the same study with TOB, but a slightly different model, where an influx clearance from the ELF1 compartment to a central compartment, was used for data fitting (3). To facilitate comparisons, the previous data were reanalyzed using the same model as for AMK and GEN, which led to virtually similar parameter values in particular AUCs. New fits are presented in Fig. 1; the targeting advantage of NEB over TOB was estimated to be 315 (Table 1). From a pharmacokinetic point of view, AMK presents a targeting advantage for NEB over i.v. administration that was 2.8 and 5.4 times higher than those of TOB and GEN, respectively. These observations do not correspond to differences in physiochemical characteristics between aminoglycosides. Indeed, all aminoglycosides are small molecules (molecular weight, 585.6 g · mol⁻¹ for AMK, 477.6 g · mol⁻¹ for GEN, and 467.5 g · mol⁻¹ for TOB), polycationic at a physiological pH (5 net charges for GEN and TOB and 4 charges for AMK), and do not show significant differences in log D values (−8.27 for GEN, −9.81 for AMK, and −9.45 for TOB) (Chemspider, www.chemspider.com). However, GEN solubility in water is at least 3 times lower than the solubility of AMK and TOB (12.6, 49.7, and 53.7 mg · ml⁻¹, respectively) (Drugbank, www.drugbank.ca); but considering concentrations in ELF, this parameter does not explain differences in the pharmacokinetic behavior of aminoglycosides after NEB (Fig. 1).

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FIG 1

Predicted concentration-time profiles of AMK, GEN, and TOB in plasma (solid line) and ELF (dashed line) from simultaneous plasma and ELF pharmacokinetic modeling, after i.v. administration (left panel) and intratracheal nebulization (right panel) of GEN (8 mg · kg⁻¹), AMK (30 mg · kg⁻¹), and TOB (3 mg · kg⁻¹). Closed and open symbols represent experimental mean ± SD concentrations in plasma and ELF, respectively. TOB data obtained previously were reanalyzed using the same model as for AMK and GEN (3). TA, targeting advantage of NEB, corresponding to AUC_ELF after NEB over AUC_ELF after i.v. corrected for doses.

In conclusion, these results cannot be directly extrapolated to the clinical setting, and the choice among aminoglycosides depends on resistance patterns of pathogens. Yet, this new investigation clearly demonstrates a much greater, although unexplained, targeting advantage of NEB for AMK over GEN, with TOB being intermediate. Moreover, this study brings additional information for compiling a biopharmaceutical classification of nebulized antimicrobial agents, especially in this category of low-permeability molecules.