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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, April 2007, p. 1253-1258, Vol. 51, No. 4
0066-4804/07/$08.00+0 doi:10.1128/AAC.01449-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
College of Pharmacy, University of Houston, Houston, Texas,1 Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas M. D. Anderson Cancer Center, Houston, Texas2
Received 18 November 2006/ Returned for modification 29 December 2006/ Accepted 17 January 2007
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ABSTRACT |
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5 mg/kg/day. Clearance of A. fumigatus during the first 24 h was associated with AMB tissue concentrations of >4 µg/g. At 5 mg/kg/day, ABLC produced a more rapid fungal clearance than did L-AMB, but at the end of therapy, fungal burden reductions were similar for both formulations and were not improved with higher dosages. These data suggest that ABLC delivers active AMB to the lung more rapidly than does L-AMB, resulting in faster Aspergillus clearance in an experimental model of IPA. However, pharmacodynamic differences between the two formulations were less apparent when mice were dosed at 10 mg/kg/day. |
INTRODUCTION |
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It is generally accepted that any delay in the initiation of antifungal therapy for IPA contributes to a poorer clinical response (2, 11). In neutropenic hosts, unimpeded growth of Aspergillus hyphae in the lung results in hemorrhage and coagulative tissue necrosis with limited blood flow (27). Hence, it is unlikely that significant amounts of drug are delivered to infarcted tissue containing the sequestered Aspergillus hyphae. Using a neutropenic rat model of IPA, Becker and colleagues found that delaying the start of L-AMB therapy (10 mg/kg of body weight/day) as little as 8 h increased animal mortality (6). Interestingly, L-AMB treatment was significantly more effective if a dose of conventional AMB-deoxycholate (1 mg/kg/day) was administered at the start of L-AMB treatment, suggesting that the availability of active AMB in the lung at early stages of infection was a critical factor in animal survival (6). The investigators did not test other lipid formulations, such as ABLC, which is known to rapidly distribute to the lung after intravenous administration.
The goals of the current investigation were (i) to compare the kinetics of AMB accumulation in lung tissue following intravenous treatment with ABLC and L-AMB and (ii) to determine whether differences in the rates of tissue AMB accumulation between the two lipid formulations correlated with different rates of Aspergillus fumigatus clearance in an experimental model of acute IPA.
(Part of this work was presented previously [R. E. Lewis, G. Liao, J. Hou, G. Chamilos, R. A. Prince, and D. P. Kontoyiannis, Abstr. 46th Intersci. Conf. Antimicrob. Agents Chemother., abstr. M-1689, 2006].)
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MATERIALS AND METHODS |
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Animals. Eight-week-old female BALB/c mice (18 to 25 g; Charles River Laboratories) were used in all experiments. Mice were housed in sterilized filter-top cages and had access to sterile food and water ad libitum. All mice were cared for in accordance with the highest standards for humane and ethical care, as approved by the Institutional Animal Care and Use Committee.
Inoculum preparation. Aspergillus fumigatus 293, the strain used for genome sequencing (21), was grown on potato dextrose agar for 7 days prior to collection of conidia. Conidia were harvested from the slant with 0.1% Tween 20 in phosphate-buffered saline and passed through a syringe with sterile glass wool to remove hyphal fragments. The resulting suspension was then centrifuged for 5 min at 15,000 x g, the supernatant was discarded, and the number of conidia was determined by hemocytometer counting. The final concentration was adjusted to 5 x 107 conidia. Harvested conidia were determined to be >98% viable based on plating of a serially diluted inoculum on Sabouraud dextrose agar. Susceptibility testing was performed using AMB epsilometer strips (AB Biodisk, Solna, Sweden) and established methods for determining the mean fungicidal concentration (MFC) for filamentous fungi (24).
Immunosuppression and infection. Immune suppression was achieved by intraperitoneal (i.p.) injections of cyclophosphamide (75 mg/kg) 4 days and 1 day prior to infection. This regimen results in total polymorphonuclear neutrophil depletion until 96 h after infection (16). In addition, animals received a single 300-mg/kg i.p. dose of cortisone acetate suspension prepared in phosphate-buffered saline with 0.2% Tween 20 1 day prior to infection (31). Doxycycline HCl (Sigma) was added to the drinking water (0.5 mg/ml) as antibacterial prophylaxis. Additionally, doxycycline-drinking water-soaked mouse chow was placed in the corner of each cage and exchanged daily to reduce animal dehydration.
Prior to inoculation, animals were anesthetized with a single i.p. injection (200 µl) of ketamine (80 mg/kg) plus xylazine (10 mg/kg) and placed on warming pads prior to intranasal inoculation with 1.5 x 106 A. fumigatus conidia. Animal inoculation was performed by slowly instilling a 30-µl droplet on both nares, and the mice were allowed to inhale the inoculum in an upright position until normal breathing resumed (16). After inoculation, animals were returned to the warming pad and observed until full recovery. This protocol results in reproducible infection of the lungs, with untreated animals succumbing to the infection 96 to 120 h after inoculation (16).
Antifungal treatment and sample collection. Groups of immunosuppressed mice (20 per treatment arm) received intravenous antifungal therapy with L-AMB or ABLC, at a dose of 1, 5, or 10 mg/kg, diluted in sterile 5% dextrose water and administered once daily by lateral tail vein injection. Control animals were administered 5% dextrose water alone. In selected experiments, L-AMB and ABLC regimens were also administered as a single intravenous dose of either 5 or 10 mg/kg. All antifungal regimens were started 12 h after inoculation and continued daily for 4 days. At serial time points after infection (0, 24, 72, and 120 h), five mice were euthanized by CO2 narcosis, and blood was immediately collected into a heparinized syringe by cardiac puncture. Blood was then transferred to a sterile capped tube and centrifuged (10,000 x g) for 10 min. Plasma supernatants were subsequently transferred to a beta-glucan (BG)-free cryovial and stored at –80°C until analysis. Lungs were then aseptically removed and stored at –80°C until analysis.
Tissue fungal burden. Pulmonary fungal burdens were determined by real-time quantitative PCR by previously reported methods (9, 30). Briefly, DNA samples isolated from homogenized lungs were assayed in duplicate by use of an ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA), using primers and a dually labeled fluorescent hybridization probe specific for the Aspergillus 18S rRNA gene (9). The cycle threshold of each sample was interpolated from a seven-point standard curve of cycle threshold values prepared by spiking uninfected mouse lungs with known amounts of conidia (101 to 107) from A. fumigatus 293. An internal standard was amplified in separate reactions to correct for the percent difference in DNA recovery (30, 31). Results are reported as conidial equivalents (CE) of A. fumigatus DNA.
AMB tissue concentrations. Determinations of total AMB tissue concentrations in infected lungs were performed by high-performance liquid chromatography (HPLC), using a modification of previously published assays (15, 20, 26). Briefly, AMB and a spiked internal standard (1-amino-4-nitro-naphthalene [ANNP]) were isolated from tissue homogenates by acetonitrile precipitation of proteins, followed by centrifugation. A 50-µl aliquot of the extracted supernatant was injected through a C18 guard column into a Nova-Pak C18 column (3.9 mm by 150 mm by 4 µm). AMB and ANNP (internal standard) were eluted at a flow rate of 1 ml per minute with a gradient program (acetonitrile from 30 to 45% plus 2.5 mM EDTA from 70% to 55% in 8 min) and were detected at 406 nm. The calibration curve was linear over a range of 0.25 to 10 µg/g in tissue. Mean inter- and intra-assay coefficients of variation over the range of the standard curve were <10%. The lower limit of accurately detectable AMB in tissue was 0.25 µg/g.
Plasma BG concentration. Plasma BG concentrations in infected animals treated with L-AMB or ABLC were determined using a commercially available assay according to the manufacturer's instructions (Fungitell; Associates of Cape Cod). Plasma samples (5 µl) were pretreated for 10 min at 37°C with an alkaline reagent (20 µl; 0.125 M KOH-0.6 M KCl) to inactivate serine proteases as well as inhibitors in mouse plasma and to enhance the reactivity to activated factor G (28). After the addition of the BG assay reagent, the microtiter plate was inserted into a preincubated microplate spectrophotometer (Powerwave X Select; Biotech Instruments, Winooski, VT), and a kinetic assay was run at 405 nm, with 490-nm correction, using KC4 software (Biotech) on unknowns from infected animals or BG standards (15 to 250 pg/ml) provided by the manufacturer.
Statistical analysis. All data were expressed as means ± standard errors of the means and were compared by the Mann-Whitney test or one-way analysis of variance, with Tukey's posttest for multiple comparisons where appropriate. Differences were considered statistically significant when P values were <0.05. Total AMB tissue concentrations associated with inhibition of A. fumigatus growth (stasis) or a 1-log10 reduction in fungal burden at 24 h were determined by fitting a four-parameter logistic model (Hill equation), using computer curve-fitting software (Prism 4; GraphPad Software, Inc., San Diego, CA), to the experimental data. Goodness of fit was assessed by determining R2 and the standard error of the 50% effective concentration.
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RESULTS |
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Kinetics of fungal burden reduction. Plots of tissue fungal burden at 0, 24, 72, and 120 h versus total concentrations of AMB in lung tissue are presented in Fig. 1. After inoculation, the mean baseline fungal burden in the lung was 5.86 x 105 (range, 1.31 x 105 to 6.60 x 106) A. fumigatus DNA CE across all treatment groups. In control animals (treated with 5% dextrose water), fungal burden increased 1 log10 by 72 h and were associated with the onset of animal mortality at 72 to 120 h, consistent with previous studies (9, 31).
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Animals treated with L-AMB or ABLC at 5 mg/kg/day exhibited significant reduction in fungal burden versus control animals at 72 h (P < 0.05). Clear differences in the pattern of fungal burden reduction were observed between the two lipid AMB formulations, with ABLC producing immediate reductions in fungal burden by the first 24 h that were not seen with L-AMB until 72 h (Fig. 1B). Analysis of AMB tissue concentrations by HPLC confirmed the higher concentrations of AMB in ABLC-treated animals at 24 h than in L-AMB-treated animals (34.2 µg/g versus 2.09 µg/g; P < 0.05). By 72 h, the mean tissue fungal burdens (1.35 x 105 versus 3.23 x 105 A. fumigatus 293 CE) and AMB tissue concentrations (12.7 µg/g versus 8.21 µg/g) were similar for ABLC- and L-AMB-treated animals dosed at 5 mg/kg/day (Fig. 1B). The absolute difference in fungal burdens between the two dosing regimens at 24 h did not quite reach statistical significance (P = 0.06).
ABLC or L-AMB treatment at 10 mg/kg/day achieved higher concentrations of AMB in the lungs at 24 h (55.4 µg/g versus 15.0 µg/g; P < 0.05) and faster reductions in A. fumigatus tissue burdens in L-AMB- but not ABLC-treated animals than those for the 5-mg/kg/day treatment regimens (Fig. 1C). The rates of fungal clearance were similar between the treatment groups receiving L-AMB at 10 mg/kg/day and ABLC at 10 mg/kg/day. Despite improvements in the rate of fungal burden reduction at the 10-mg/kg dose for L-AMB, the extents of fungal burden reduction at the end of the experiment were similar for both formulations at the 5- and 10-mg/kg/day dosing levels.
Because early reductions in lung fungal burden appeared to correlate with a threshold concentration of AMB in the lung in the first 24 h, fungal burden data from all treatment groups at 24 h (n = 60 mice) were plotted in relation to total AMB tissue concentrations measured by HPLC (Fig. 1D). A four-parameter logistic model was then fitted to the data to predict threshold AMB tissue concentrations associated with growth stasis and a 1-log10 reduction in A. fumigatus DNA CE. Variability in fungal burdens across all treatment groups at 24 h could be explained largely by tissue concentrations of AMB (R2 = 0.64). The total AMB tissue threshold concentrations associated with growth stasis and a 1-log10 reduction in fungal burden were 0.53 µg/g and 4.20 µg/g, respectively. At a dosage of 5 mg/kg/day, 100% of ABLC-treated mice versus 20% of L-AMB-treated animals achieved or surpassed the tissue concentrations required for fungal clearance at 24 h (P = 0.04; two-sided Fisher's exact test). At a dosage of 10 mg/kg/day, both formulations consistently (100%) achieved tissue concentrations required for fungal clearance in the first 24 h.
Plasma BG concentrations. Plasma BG concentrations measured in infected neutropenic mice at baseline and at 24, 72, and 120 h correlated with A. fumigatus DNA CE fungal burden measurements determined by quantitative real-time PCR (Fig. 2). Animals treated with L-AMB or ABLC at 1 mg/kg/day exhibited an initial mean plasma BG concentration of 56.9 ± 7.1 pg/ml, which increased to 62.7 ± 8.5 pg/ml by 24 h and to 82.5 ± 19.4 pg/ml by 72 h (Fig. 1C). Similar to the fungal burden data, animals treated with L-AMB at 5 mg/kg/day experienced a higher mean peak BG concentration in the plasma at 24 h than did animals treated with 5 mg/kg/day of ABLC (84.7 ± 8.8 pg/ml versus 38.16 ± 8.1 pg/ml; P = 0.017); however, plasma BG concentrations were similar between the treatment groups at 72 and 120 h (Fig. 1B). No difference in mean plasma BG concentrations was noted between L-AMB- and ABLC-treated animals at a dose of 10 mg/kg/day (Fig. 1C).
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DISCUSSION |
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Early clearance of A. fumigatus from the lung was associated with total AMB tissue concentrations that surpassed the MFC of the infecting isolate. Using logistic regression, we determined that AMB tissue concentrations of 4 µg/g were associated with a significant reduction (1 log10) in the lung fungal burden, as determined by quantitative real-time PCR. This threshold concentration is in agreement with results from Olson et al., who reported that tissue concentrations of AMB in the lungs of mice of >3 µg/g were required for therapeutic efficacy of L-AMB or ABLC treatment in a murine model of pulmonary aspergillosis (22). Unlike our study, the investigators did not report any L-AMB or ABLC treatment regimens (1 to 12 mg/kg/day) that achieved tissue concentrations of >10 µg/g (22). However, the investigators documented AMB biodistribution primarily in noninfected animals, which may be diminished in the absence of the hemorrhaging and residual inflammatory cell recruitment that are seen in infected animals (4). Indeed, in the study by Olson et al., tissue concentrations were disproportionately higher in infected animals treated at 15 mg/kg/day of ABLC or L-AMB (30 to 40 µg/g and 10 to 18 µg/g, respectively) than in uninfected animals who were treated with both lipid formulations at 12 mg/kg/day (10.07 µg/g and 3.16 µg/g, respectively) (22). Fungal burden, as determined by quantitative CFU cultures, were generally lower in ABLC-treated animals, with a 20-mg/kg/day ABLC regimen achieving the greatest reduction in fungal burden among all treatment groups (22).
One unexpected finding of our study was the pattern of decreasing tissue concentrations over the course of the experiment, despite daily dosing. Because the majority of animal studies that have examined the biodistribution of lipid AMB formulations in the lung used only single doses or single time points to assess plasma-to-tissue ratios of drug, we are unable to confirm if this observation is indeed unique to our study (3, 6, 22). Several factors could account for the decreased distribution of AMB during the course of repeated dosing in infected animals, including saturation of pathways involved in drug uptake in the lung and/or decreases in residual (lipid AMB-containing) phagocytic cell recruitment with decreasing fungal burdens. Another possibility is that our method for AMB extraction from tissue becomes less effective as infection persists. If this were the case, we would expect a consistent pattern of decreases in all dosing regimens, which was clearly not observed between the two lipid AMB formulations (Fig. 1B and C). Further studies with radiolabeled AMB would be required to address this issue.
When administered as a single dose of 5 or 10 mg/kg, neither L-AMB nor ABLC was as effective at reducing the fungal burden as multiple daily dosing (Fig. 3A and B). Indeed, AMB tissue concentrations were undetectable by 120 h, and fungal clearance reached a plateau after the first 24 to 48 h. These data raise concerns regarding whether dosing of lipid AMB formulations at extended intervals (i.e., greater than 3 to 5 days) would be prudent for the treatment of established IPA. However, higher-dose, infrequently administered regimens may still be effective for low-inoculum infections or in the setting of prophylaxis. Additional studies will be required to confirm the pharmacodynamics of extended interval dosing for the lipid AMB formulations.
Another novel aspect of this study is that we were able to demonstrate a good correlation between A. fumigatus tissue fungal burden measured by quantitative real-time PCR and serum concentrations of BG, including responses to antifungal therapy. The BG test has been reported to be a useful adjunctive diagnostic for aspergillosis in patients with acute myelogenous leukemia, with a sensitivity of 69.9% and a specificity of 87.1% for patients with proven infections when a 60-pg/ml cutoff value is used to define sample positivity (23). However, few preclinical studies have compared serial measurements of BG concentrations in relation to direct measurements of fungal burden in the lungs. We decided to use BG as a surrogate marker over galactomannan due to the greater dynamic range of the test, i.e., BG is reported quantitatively versus the semiquantitative galactomannan test. While the majority of the concentrations tested in this study fell below 60 pg/ml, it is notable that samples that exceeded this threshold were from animals with progressing IPA or delayed responses to antifungal therapy. Additional preclinical and clinical studies are warranted to further explore the utility of serial BG analysis as a surrogate for a response to drug therapy.
In conclusion, our comparative analysis of L-AMB and ABLC for the treatment of acute experimental IPA revealed notable differences in the patterns of early AMB lung distribution and fungal clearance when the formulations were dosed at 5 mg/kg/day but no significant differences in A. fumigatus clearance at a dose of 10 mg/kg/day. Differences in the rate of fungal clearance could be attributed largely to differences between respective formulations in delivering "fungicidal" concentrations of AMB to the lung tissue. While the importance of these findings remains to be determined for more slowly progressing forms of aspergillosis, our data suggest that the biopharmaceutical differences between the lipid formulations could have potentially important clinical implications for rapidly progressing fungal pneumonia (e.g., pulmonary zygomycosis). Currently, studies are under way to confirm these results with more rapidly invasive, less AMB-susceptible molds.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 29 January 2007.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3) beta-D-glucan assay as an aid to diagnosis of fungal infections in humans. Clin. Infect. Dis. 41:654-659.[CrossRef][Medline]3)-beta-D-glucan in human blood. Clin. Chim. Acta 226:109-112.[CrossRef][Medline]This article has been cited by other articles:
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| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
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| J. Clin. Microbiol. | ALL ASM JOURNALS |
, control; 
, ABLC; 
, L-AMB. Line plots represent tissue A. fumigatus fungal burden, expressed as CE, versus time. Each data point is the mean ± standard error for five animals. Total lung AMB tissue concentrations (µg/gram) versus time are plotted on the z axis. Each bar represents the mean tissue concentration for five animals. (A) 1-mg/kg/day dosing; (B) 5-mg/kg/day dosing; (C) 10-mg/kg/day dosing. (D) Relationship of 24-hour A. fumigatus tissue fungal burden and total AMB tissue concentrations from all treatment groups (n = 60 mice). The line depicts the concentration-effective relationship determined by fitting a four-parameter logistic model (Hill equation) to the data, using the GraphPad Prism 4.0 software package. Mean best-fit values and 95% confidence intervals were calculated by the software. *, P < 0.05 for ABLC versus L-AMB tissue concentrations, as determined by the two-sided Mann-Whitney test.
