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Amphotericin B (AMB) concentrations were determined in pulmonary epithelial lining fluid (ELF) of 44 critically ill patients, who were receiving treatment with liposomal AMB (LAMB) (n = 11), AMB colloidal dispersion (ABCD) (n = 28), or AMB lipid complex (ABLC) (n = 5). Mean AMB levels (± standard errors of the means) in ELF amounted to 1.60 ± 0.58, 0.38 ± 0.07, and 1.29 ± 0.71 μg/ml in LAMB-, ABCD-, and ABLC-treated patients, respectively (differences are not significant).
Invasive pulmonary mycoses exhibit a high mortality, particularly in critically ill patients (19). Amphotericin B (AMB) lipid formulations—liposomal AMB (AmBisome; Gilead) (LAMB), AMB colloidal dispersion (Amphotec [Three Rivers] and Amphocil [Torrex-Chiesi]) (ABCD), and AMB lipid complex (Abelcet; Zeneus) (ABLC)—display differences in plasma pharmacokinetics and tissue distribution (15, 26, 28). During treatment with AMB lipid formulations, AMB concentrations were investigated in epithelial lining fluid (ELF), which is a well-established model for pulmonary drug penetration (1-4, 6-10, 12, 17).
This study was approved by the local ethics committee. Patients on lipid-formulated AMB requiring bronchoalveolar lavage (BAL) were enrolled (Table (Table1).1). AMB concentrations were assessed in 8-ml aliquots of BAL samples obtained by a standard procedure (16). BAL fluid was concentrated by evaporation, and AMB was quantified by high-performance liquid chromatography as described previously, with modifications for BAL samples (13). The concentrations were assessed by using a linear standard curve (R between 0.995 and 0.999), obtained from standards comprising 0.9% saline solution spiked with AMB. The lower detection limit of AMB in BAL fluid was 0.005 μg/ml. The assay has been found to be linear over the concentration range of 0.005 to 2.5 μg/ml for AMB in BAL fluid. The intraday and interday precisions were 3.2% and 4.7%, respectively. AMB concentrations in ELF were calculated by the urea dilution method (23), AMBELF = AMBBAL × (ureaPLA/ureaBAL), where AMBELF is the AMB concentration in ELF, AMBBAL is the AMB concentration in BAL fluid, ureaPLA is the urea concentration in plasma, and ureaBAL is the urea concentration in BAL fluid (23). Two ml of the BAL fluid was separated for urea quantification, which was performed using an enzymatic assay (urea/blood urea nitrogen; Roche) as with plasma.
Arterial blood samples were simultaneously taken for measurement of plasma AMB and urea concentrations. In patients on LAMB or ABCD therapy, the lipid-associated fractions were separated from AMB that had been liberated from its lipid encapsulation. AMB was measured with high-performance liquid chromatography as described previously (13).
Statistical analysis was performed using the Statistica software program, version 5. The differences between total AMB concentrations in plasma and in ELF were analyzed by using the Wilcoxon matched pairs test. For comparisons between the lipid formulations, the Mann-Whitney U test was applied.
Forty-four patients were enrolled: 11 patients on LAMB, 28 on ABCD, and 5 on ABLC. Table Table22 displays the ELF and plasma concentrations of AMB and the penetration ratios. In the entire study population and in LAMB-treated patients, ELF concentrations correlated with plasma levels (r = 0.68, P < 0.001, and r = 0.66, P = 0.04, respectively). In the LAMB group, this correlation was even more significant when liberated AMB was considered (r = 0.89; P < 0.001). A positive correlation between the time from last infusion to sampling and the penetration ratio was found during LAMB (r = 0.75; P = 0.01) and ABLC (r = 0.95; P = 0.01) treatments.
Inhalation of fungal conidiae is the most common route of infection with molds. During treatment with AMB lipid formulations at standard doses, mean AMB levels in ELF were below 2 μg/ml. For Aspergillus species, the MIC of AMB has been reported to range from 0.25 to 4 μg/ml (14). Thus, in some cases, the MIC exceeds the AMB concentration in ELF. This may contribute to unsatisfying responses sometimes observed, though the impact of target site concentrations in relation to MICs is controversial. ELF concentrations are markedly lower than AMB levels in whole lung tissue (32.6 μg/g after ABCD treatment) (26). Whole tissue samples, however, comprise various compartments and potential targets of fungal invasion, such as different cells, extracellular matrix, and blood vessels.
The differences in the underlying diseases, the limited number of patients that differed between the groups, slight differences in doses, and various intervals between AMB infusion and BAL are limitations of our study. In the LAMB group and in the ABLC group, penetration of ELF increased with this interval. Similarly, a slow increase in concentrations in lung tissue over 25 h was observed after LAMB infusion (11).
A study of rabbits revealed ELF concentrations comparable to our human data (2.28, 0.68, and 0.90 μg/ml after LAMB, ABCD, and ABLC treatment, respectively) (17).
In pleural effusion and ascites, where mainly liberated AMB is found, concentrations were even lower than those in ELF (27, 28). In vitro investigations suggest an influence of phosphatidylcholine liposomes within ELF on membrane oxidation and nitration that could potentially affect the activity of lipid-associated antimicrobial agents in vivo (25). Unlike the case with plasma and with body fluids, separation of liberated and lipid-encapsulated AMB was not feasible in ELF. For LAMB and ABCD, the penetration ratios of liberated AMB were similar, suggesting that mainly liberated AMB penetrates ELF.
Lung transplant recipients on prophylaxis with nebulized LAMB (several 25-mg doses) displayed concentrations in ELF of ~10 μg/ml 2 days after inhalation and 3 to 4 μg/ml after 2 weeks. AMB was undetectable in plasma of all but one patient, suggesting a poor systemic absorption and penetration into deeper lung compartments (20).
Penetration of ELF by voriconazole was studied for lung transplant recipients on prophylactic treatment, revealing various concentrations (0.29 to 83.32 μg/ml; mean penetration ratio, 1,100%) (5). In healthy volunteers who had received posaconazole at the standard dosage for 8 days, a mean concentration in ELF of 1.86 μg/ml was measured (10). Treatment with the high-molecular-weight lipopeptide micafungin (150 mg daily for 3 days) resulted in concentrations in ELF of ~0.5 μg/ml and an accumulation in alveolar macrophage cells (8.4 to 14.6 μg/ml) (21). Similarly, AMB in either a deoxycholate or a lipid formulation accumulates in cells of the reticuloendothelial system, particularly in alveolar macrophage cells, as shown in animal and in vitro experiments (17, 18, 22, 24). In the present study, AMB was not separately quantified in alveolar macrophages.
In conclusion, treatment with AMB lipid formulations at standard doses yields ELF concentrations moderately above or even below MICs of relevant fungal pathogens. ELF levels are much lower than AMB concentrations in lung tissue samples. Further investigations should address the impact of target site penetration of antifungals on the therapeutic outcome in invasive pulmonary mycoses.
This study was supported by the Tiroler Wissenschaftsfonds and by Torrex-Chiesi Pharma, Vienna, Austria.
Published ahead of print on 24 August 2009.