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Voriconazole concentrations were determined in autopsy samples of eight patients who had been treated for a median of 7 days (interquartile range [IQR], 5 days). Voriconazole penetrates well into various tissues, with median levels of 6.26 μg/g ((interquartile range [IQR], 7.87 μg/g) in the lung, 3.41 μg/g (IQR, 16.72 μg/g) in the brain, 6.89 μg/g (IQR, 24.16 μg/g) in the liver, 6.47 μg/g (IQR, 6.19 μg/g) in the kidneys, 5.60 μg/g (IQR, 11.49 μg/g) in the spleen, and 7.55 μg/g (IQR, 16.91 μg/g) in the myocardium.
Voriconazole (VRC) is an expanded-spectrum triazole with a broad antimycotic spectrum, including Aspergillus and non-albicans Candida species. It is the drug of choice for treatment of invasive aspergillosis (12, 27). The bioavailability after oral intake of VRC is reported to be almost complete (96%) in healthy volunteers, but lower and variable in patients (23.7 to 63.3%) (11). Since data on VRC distribution into human tissues have been sparse so far, we determined concentrations in autopsy samples.
The study was approved by the local ethics committee. Consent was granted by the patients' relatives. Tissue samples were obtained during routine autopsy from eight patients who had died at the medical intensive care unit (ICU) during VRC treatment. The patients' characteristics and data on VRC therapy are displayed in Table Table1;1; routine laboratory values are shown in Table Table22.
VRC (Vfend; Pfizer) had been administered for possible, probable, or proven invasive aspergillosis intravenously (six patients) or as an oral suspension (two patients) at the standard dose. Aliquots (~7 g) were taken from lung, brain, liver, spleen, kidneys, and myocardium and were stored at −80°C. After thawing, samples were homogenized (Ultraturrax T25; Germany) and purified by C18 solid-phase extraction. VRC was quantified by the high-performance liquid chromatography method by Khoschsorur et al. (14), with some modifications, using a Zorbax 300SB-C18 analytical column, UV detection (λ = 255 nm), and a mixture of sodium dihydrogen phosphate buffer, acetonitrile, and methanol (35:45:20 [vol/vol]) as the mobile phase. The detection limit was 0.25 μg/g. The intraday precision, interday precision, and accuracy (mean ± standard deviation) were 4.2% ± 3.7%, 8.8% ± 5.2%, and 7.4% ± 9.6%, respectively. The concentrations were assessed by means of a linear standard curve (R = 0.999), obtained by external standards comprising respective bovine tissues spiked with VRC. The mean VRC recovery was 80%.
Table Table33 displays the VRC concentrations in the different tissue samples. VRC was shown to penetrate well into tissues, with high variability. In patient 1, VRC was detectable even after a low single dose in all tissues but brain and myocardium. Tissue drug levels of all the other patients exceeded those achieved in patient 1. In patient 7 (daily dose of 600 mg for 6 days of VRC treatment), the highest concentrations were achieved in most tissue samples. He also exhibited the highest values in liver and renal function tests. No difference in VRC levels was found between different areas of the lung, nor were there discrepancies between cerebral cortex, hippocampus, nucleus caudatus, medulla oblongata, and cerebellum.
After multiple dosing, VRC levels in the liver correlated with the daily doses (R = 0.79, P = 0.03; in the other organs, R was between 0.38 and 0.64, P > 0.05), but not with the cumulative dose or the interval between the last administration and death. There was no significant difference in tissue concentrations between patients on and off renal replacement therapy. No signs of VRC toxicity could be observed (Table (Table22).
Invasive aspergillosis most frequently affects the respiratory tract and the central nervous system. The myocardium, the liver, and the spleen are further target organs of invasive fungal infections. We found mean VRC tissue concentrations that exceed therapeutic plasma levels (~2.0 to 5.5 μg/ml) (19, 22) and the MICs for Aspergillus species (~0.25 to 2 μg/ml) (15). However, the significance of in vivo target site concentrations in relation to in vitro MICs is controversial considering clinical efficacy.
The lack of therapeutic VRC monitoring precluded a comparison with plasma drug levels. The limited number of patients, differences in underlying diseases, including hepatic and renal function, as well as varying cumulative VRC doses and variable intervals between the last VRC administration and death of the patient are further limitations of our study. Agonal or postmortem changes in VRC tissue concentrations cannot be ruled out completely, although VRC was found to be stable in tissue at 4°C for at least 72 h. Between the tissue drug concentrations and death-to-sampling interval, no correlation was observed. Free VRC and protein-bound VRC were not separated by our assay. The tissue samples consisted of various compartments that are potential targets of fungal invasion, such as different cells, extracellular matrix, and blood vessels (18). However, with our method we could not study the VRC distribution on a cellular level.
In pulmonary epithelial lining fluid (ELF) of lung transplant recipients, VRC levels were between 0.3 and 83.3 μg/ml (3). In ELF of healthy volunteers, mean levels amounted to 10.1 to 48.3 μg/ml; in alveolar macrophages, mean levels were 10.3 to 20.6 μg/ml (5). Thus, the mean VRC concentration we found in lung tissue was somewhat lower than that in ELF. In vitro incubation of polymorphonuclear leukocytes with 2 μg/ml of VRC resulted in intracellular levels of ~15 μg/ml (1). Relatively small amounts of VRC (0.7 to 4.4 μg/ml) were recovered from pleural fluid (17, 21, 23). VRC penetration into cerebrospinal fluid appears to be variable, yielding levels of 0.08 to 3.93 μg/ml (6, 16). Cerebral VRC concentrations had been determined previously in autopsy samples from two patients where 11.8 and 58.5 μg/g were measured and, thus, which exceed the levels we measured in our study population (16). In a brain abscess, 1.2 to 1.4 μg/g was reached by oral intake of 4 mg/kg twice daily (8). In contrast, none of our patients presented with cerebral mycosis. Studies of chickens and horses revealed remarkable interspecies differences in tissue penetration of VRC (2, 4, 20). In rats, free extracellular lung concentrations reached ~2.5 μg/ml after a single dose (13).
Fluconazole, the ancestor drug of VRC, has been found to achieve high levels in various tissues, including the brain (25). In contrast, amphotericin B preparations accumulate in liver and spleen but achieve only low levels in brain and myocardium (26). Unlike with VRC, amphotericin B levels in lung tissue are much higher than in ELF (28). In comparison with amphotericin B, VRC therapy of invasive aspergillosis achieved a superior clinical outcome (12). Echinocandins displayed a tissue distribution similar to that of amphotericin B in animal studies (9, 10, 24).
In conclusion, treatment with VRC at standard doses yields concentrations above the MICs of relevant fungal pathogens in various frequently affected tissues. The significance of target site levels of antifungals for the clinical response should be addressed by adequately powered clinical trials.
We are indebted to Gregor Mikuz, Andrea Brunner, Christian Ensinger, Jens Krugmann, Hans Maier, Patrizia Moser, Ralf Rieker, Consolato Sergi, Irmgard Verdorfer, and Bettina Zelger, Institute of Pathology, Innsbruck Medical University, for providing the tissue samples.
We thank Pfizer, Austria, for financial support.
Published ahead of print on 15 November 2010.