1 Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, 69978, Israel; 2 Institute of Pathology, Assaf Harofe Medical Center, Zerifin, Israel
Received 21 April 2005; returned 31 May 2005; revised 15 June 2005; accepted 21 June 2005
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Abstract |
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Methods: ICR mice were immunosuppressed by intraperitoneal administration of cyclophosphamide. Three days later, the mice were inoculated intravenously (iv) with A. fumigatus conidia. Infection and treatment were evaluated during an observation period of 30 days in terms of mortality (survival rate and mean survival time) and morbidity (quantitative determination of fungal burden, histopathology, and detection of serum galactomannan).
Results: Combination of caspofungin + G-CSF or addition of G-CSF to the combination of caspofungin + amphotericin-B-intralipid increased the survival rate of infected mice up to 78.9% and prolonged their mean survival time to 25 days. These combinations also resulted in a reduction in fungal burden in organs, and a decrease in serum galactomannan.
Conclusions: The successful results obtained in the experimental model may possibly open the way to more effective management of aspergillosis in humans.
Keywords: aspergillosis , caspofungin , amphotericin-B-intralipid , granulocyte-colony stimulating factor , combination therapy
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Introduction |
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Our laboratory has shown previously4 that an admixture of amphotericin-B and intralipid obtained by vigorous prolonged shaking produced a stable, non-toxic preparation, which could be of value in view of the high cost of the commercially available amphotericin-B lipid formulations. This admixture demonstrated in vitro activity against Aspergillus fumigatus, and was significantly more effective than conventional amphotericin-B in an experimental model of systemic aspergillosis.5 The echinocandin lipopeptide caspofungin inhibits synthesis of 1,3-ß-D-glucan and causes severe damage to A. fumigatus at the sites of hyphal growth.6
Human granulocyte-colony stimulating factor (G-CSF) is a glycoprotein that regulates the production and release of functional neutrophils from the bone marrow, and may contribute to better efficacy of antifungal drugs.7,8
We hypothesized that inhibition of the synthesis of a major component of the fungal cell wall and an effect on the cell membrane, by combining an echinocandin and a polyene, may result in a synergic effect against A. fumigatus. Therefore, in this study we investigated the combination of caspofungin and amphotericin-B-intralipid, supplemented by the cytokine G-CSF in the treatment of experimental murine aspergillosis. The data are reported here.
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Materials and methods |
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A. fumigatus (ATCC 64026) was used throughout the study. The strain was grown on Sabouraud dextrose agar (SDA) (28°C, 7296 h). Conidia were harvested with sterile saline; the conidial suspension was adjusted to the required concentration by counting in a haemocytometer and by plating on SDA for determination of colony counts.
Animals, induction of an immunosuppressed state and experimental aspergillosis
Female ICR mice, 45 weeks old (2328 g), kept under conventional conditions, were used. The ethics committee of the Faculty of Medicine of Tel-Aviv University granted permission for the animal experiments.
A transiently compromised state in the mice was achieved by intraperitoneal injection of cyclophosphamide (200 mg/kg), 3 days prior to infection. At this time point, the mice were in an immunocompromised state, as previously determined by a decrease in the number of total white blood cells and neutrophils.9
Experimental systemic aspergillosis was obtained by intravenous (iv) inoculation via the lateral tail vein of immunocompromised mice with 0.8 x 106 conidia/mouse of A. fumigatus, administered on day 3 post cyclophosphamide treatment. Infection was followed up for 30 days and evaluated in terms of mortality [survival rate and mean survival time (MST)] and morbidity (quantitative determination of fungal burden, histopathology, and detection of serum galactomannan).
Drugs and administration
Amphotericin-B-intralipid was prepared by a 25-fold dilution of conventional amphotericin-B (Fungizone, Bristol-Myers Squibb, France) in the lipid emulsion intralipid 20% (Kabi Pharmacia, Stockholm, Sweden) to a final amphotericin-B concentration of 0.2 mg/mL, and then agitated vigorously at 24°C for 18 h on a controlled environment incubator shaker (New Brunswick Scientific Co., Edison, NJ, USA) at 280 rpm.4 Caspofungin acetate (Merck & Co., Inc., Whitehouse Station, NJ, USA) was dissolved in 0.9% sodium chloride for injection. Recombinant methionyl human G-CSF (Filgrastim), the product of Roche (F. Hoffmann-La Roche Ltd, Basle, Switzerland), was diluted in 5% dextrose solution to a final concentration of 30 µg/mL.
Infected mice received the following single treatmentsamphotericin-B-intralipid (1 mg/kg per day, iv), caspofungin (0.5 mg/kg per day, iv), G-CSF (300 µg/kg per day, intraperitoneal), and combination therapiescaspofungin + G-CSF, caspofungin + amphotericin-B-intralipid + G-CSF (the same doses as in single agent treatment). Treatment began 2 h after fungal inoculation and consisted of 5 consecutive daily drug injections. Assessment of the treatment was based on the criteria of evaluating mortality and morbidity as in the model of experimental aspergillosis.
Cultures from organ homogenates
Enumeration of Aspergillus cfu in tissue homogenates of kidneys and lungs was performed at days 1, 2, 3, 5, 7, 14, 21 and 28. Kidney and lung tissue homogenates were prepared, diluted and plated on SDA (incubation: 28°C, 48 h) for cfu determination.
Galactomannan assay
Serum galactomannan concentrations were determined by the Platelia Aspergillus EIA (commercial sandwich ELISA kit) on days 1, 2, 3, 5, 7, 14, 21 and 28, and were carried out according to the protocol recommended by the manufacturer (Platelia Aspergillus 62797, Sanofi Diagnostic Pasteur). Enzyme immunoassay data were expressed as a serum galactomannan index (GMI). Sera with GMIs of less than 1 were considered negative, those greater than 1.5 were positive.
Histopathology
Organ samples (kidneys and lungs) were fixed in 4% buffered formalin overnight, processed and sectioned by routine histological techniques after paraffin embedding. Sections were then stained with haematoxylin and eosin stain.
Statistical analysis
Analysis of survival data was by KaplanMeier test and comparisons between groups were performed by the one-way analysis of variance (ANOVA) multiple comparisons test using the GraphPad Prism 4 software package (GraphPad Software Inc., San Diego, CA, USA). P values of <0.05 were considered significant in these analyses.
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Results |
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Treatment with amphotericin-B-intralipid or with caspofungin significantly increased (P < 0.001) the survival rate of infected mice in comparison with the untreated controls (Figure 1). The MSTs were prolonged from 5.7 days for the untreated controls to 17.9 and 16 days for amphotericin-B-intralipid- and caspofungin-treated mice, respectively.
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Serum galactomannan detection
The detection of galactomannan, as an important indication of therapeutic response, was carried out in serum samples from mice with systemic aspergillosis (Figure 2a). While serum samples from untreated controls, G-CSF-treated mice and caspofungin-treated mice showed high galactomannan levels throughout the experiment, those treated with combination therapy demonstrated a decline starting on day 3 (caspofungin + G-CSF) or on day 5 (caspofungin + amphotericin-B-intralipid + G-CSF). No galactomannan was detected after day 14 of the experiment in serum of animals treated with combined therapies.
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Kidneys. In animals treated with combination therapies, clearance of fungal colonization was noted from day 5 or 7. It is noticeable that the combination of caspofungin + amphotericin-B-intralipid with G-CSF was particularly effective in reducing the fungal burden.
Lungs. Fungal colonization was detected in all groups at 24 and 48 h post-infection. Aspergillus was absent in the lungs of caspofungin + amphotericin-B-intralipid + G-CSF-treated animals from day 3 (Figure 2b), and not detected in most treated groups and untreated controls from day 5 of the experiment.
Histopathology
Histopathological examination of kidneys and lungs from untreated controls, caspofungin- and caspofungin + amphotericin- B-intralipid + G-CSF-treated mice was carried out (data not shown). The examinations revealed foci of acute pyelonephritis within the medulla and fungal hyphae in kidneys of controls. Lung tissue of untreated animals showed diffuse areas of massive bronchopneumonia. In addition, occasional abscesses were scattered within lung tissue, surrounded by oedematous tissue and foci of necrosis.
In caspofungin-treated mice, chronic and acute inflammatory infiltrates within the pelvic mucosa were found in the kidneys, while no evidence of inflammation was noted in kidneys from caspofungin + amphotericin-B-intralipid + G-CSF-treated mice. A diffuse heavy bronchopneumonia accompanied by congestion of blood vessels was seen in lung tissue of animals treated with caspofungin alone. In those mice treated with the combination of caspofungin + amphotericin-B-intralipid + G-CSF, the lungs revealed few areas of bronchopneumonia, and no necrosis of tissue.
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Discussion |
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To the best of our knowledge, the evaluation of efficacy of caspofungin and G-CSF as well as the combination of two antifungals and G-CSF has thus far not been reported.
The combination treatment demonstrated significantly increased efficacy in comparison with either drug alone or to untreated controls, as expressed by all evaluated criteria. The synergic interaction between the polyene and the echinocandin against Aspergillus is apparently due to a simultaneous effect of the amphotericin-B-intralipid on the fungal cell membrane and inhibition by caspofungin of the synthesis of 1,3-ß-D-glucan in the cell wall.
A synergic effect of G-CSF with caspofungin was also observed in this study. The data demonstrated that combination of caspofungin with G-CSF was clearly more effective than caspofungin alone in treatment of experimental systemic aspergillosis (P < 0.001). These data are compatible with our previous study9 that revealed the synergic effect of G-CSF in combination with polyenes.
The results obtained from the experiments indicated that combination of all three agents (caspofungin + amphotericin-B-intralipid + G-CSF) markedly delayed the mortality of mice and significantly increased their survival rates (P < 0.001). This therapy also led to a significant reduction in the fungal burden or even absence of A. fumigatus in tissues of organs, compared with animals treated with single therapy or not treated. However, the experiments showed that the combined therapies consisting of three agents were not more effective than combinations involving only two componentsantifungal drug and the cytokine G-CSF.
In our model of systemic aspergillosis (i.e. via iv inoculation), the fungi disseminated to all internal organs investigated, e.g. kidneys, spleen (data not shown) and lungs. Aspergillus cfu were completely eliminated from the organs of the majority of treated mice at day 14 of infection. Histologically, no hyphal presence was observed in the lungs throughout infection, despite the massive bronchopneumonia found in untreated mice. It should also be noted that other research groups who histologically evaluated Aspergillus-infected mice,10 did not indicate the presence of hyphae in the lungs of the animals with disseminated aspergillosis, while fungi were detected in other organs (e.g. kidneys, liver, spleen).
In summary, our study demonstrated that treatment of experimental aspergillosis by combinations of antifungal agents and immunotherapy is significantly more effective, in comparison with single therapy. Thus, further clinical studies are needed to investigate the benefit of combination therapy for invasive aspergillosis in humans.
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Footnotes |
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References |
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