Journal of Antimicrobial Chemotherapy (2000) 45, 673-676
© 2000 The British Society for Antimicrobial Chemotherapy
Efficacy of voriconazole against invasive pulmonary aspergillosis in a guinea-pig model
Division of Infectious Diseases, Department of Internal Medicine, Wayne State University School of Medicine, Detroit, MI 48201, USA
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Abstract |
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We compared the efficacies of amphotericin B and voriconazole against invasive pulmonary aspergillosis in a guinea-pig model. A susceptible isolate of Aspergillus fumigatus was used to produce the infection. Voriconazole-treated animals had significantly better survival and decreased fungal burden in the lungs as compared with controls. Although no statistical difference was seen between the efficacies of voriconazole and amphotericin B, a trend favouring voriconazole was noted. Thus, voriconazole, with its cidal activity, may be an attractive alternative to potentially toxic amphotericin B in the treatment of invasive pulmonary aspergillosis.
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Introduction |
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Invasive pulmonary aspergillosis is an increasingly common fungal infection causing morbidity and mortality in immunocompromised hosts.1–4 Aspergillus fumigatus is the most commonly isolated Aspergillus sp. causing disease. Drugs available for the treatment of invasive aspergillosis are amphotericin B and itraconazole, but neither agent is associated with an optimal response.2,5 Lipid-complexed forms of amphotericin B appear to have efficacy similar to the conventional formulation of amphotericin B.6
Voriconazole is a highly potent, mono-triazole with excellent in vitro activity against a large variety of fungi including Aspergillus spp., other filamentous fungi and yeasts.7–10 It has fungicidal activity against Aspergillus spp. and offers potential advantages over amphotericin B including oral as well as iv administration and reduced toxicity.11 In this study we compared the efficacies of voriconazole and amphotericin B against invasive aspergillosis in a guinea-pig model. We chose this animal for evaluating voriconazole because the drug achieves prolonged systemic exposures in guinea-pigs comparable with those seen in humans.12
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Materials and methods |
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Test organism
The A. fumigatus isolate used in this study was a pathogen obtained from an immunosuppressed patient at the Detroit Medical Center. The original culture obtained on Sabouraud dextrose agar slants was sub-cultured on the same medium to confirm viability and purity of the culture. Working cultures of the isolate were maintained on Sabouraud dextrose agar slants at 4°C.
Antifungal agents
The following drugs were used: amphotericin B (Batch 20-914-29670, Squibb Institute for Medical Research, Princeton, NJ, USA), itraconazole (R 51211, Batch STAN-9304-005-1, Janssen Pharmaceutica, Beerse, Belgium) and voriconazole (Pfizer Pharmaceuticals, New York, NY, USA). Stock solutions were prepared by dissolving the drugs in dimethylsulphoxide (DMSO) to obtain a concentration of 1 mg/mL and were stored at –20°C. The frozen stock was thawed at room temperature before the experiments.
Antifungal susceptibility testing
The in vitro susceptibility of the A. fumigatus isolate to amphotericin B, voriconazole and itraconazole was determined by a broth macrodilution technique as previously described.13 Fresh conidia were collected from the A. fumigatus isolate and suspended in RPMI 1640 medium (American Biorganics, Niagara Falls, NY, USA) to obtain approximately 2 x 104 cfu/mL, twice the final test inoculum.
Stock solutions of the antifungal agents were diluted with RPMI medium in sterile 6 mL polystyrene tubes (Falcon 2054, Becton Dickinson, Lincoln Park, NJ, USA) to obtain twice the final test concentrations for the broth macrodilution assay. The concentrations of the antifungal drugs studied ranged from 0.0625 to 16 mg/L. Equal volumes (0.5 mL) of the conidial suspension and the antifungal drug solution were incubated at 35°C for 48 h. The tubes were then gently vortexed and scored for visible growth. A drug-free growth control and a set of tubes with RPMI alone were used to monitor contamination of the medium. The MIC was defined as the lowest concentration of the drug that produced no visible growth (i.e. 100% inhibition). The MIC determination for the isolate was carried out in duplicate and the experiment was repeated at least once.
Establishment and treatment of invasive pulmonary aspergillosis in the guinea-pig
All animal research procedures were approved by the Institutional Animal Care and Use Committee of Wayne State University. Female Hartley guinea-pigs weighing 300 ± 50 g obtained from Harlan Research Laboratory Animals (Indianapolis, IN, USA) were used. Animals were made neutropenic by ip injections of cyclophosphamide (100 mg/kg) on days –3, –1, +1 and +3, where day 0 represents the day of infection. Leucocyte counts were monitored throughout the experiment. Animals were anaesthetized by ip injections of ketamine 40 mg/kg plus xylazine 5 mg/kg, as described elsewhere.14 The anterior neck area of the animal was shaved with an electric shaver and disinfected by treating with 10% povidone iodine (Betadine solution). The tracheal wall was exposed by blunt dissection and 0.2 mL of the conidial suspension containing 5 x 107 conidia/mL was injected into the trachea via a 25 gauge needle. The tracheal incision was closed aseptically with steel clips.
Guinea-pigs that survived the first 4 h after intratracheal inoculation of the isolate received antifungal therapy once daily for the next six consecutive days. Animals were pre-randomized to therapy with amphotericin B, voriconazole or placebo. Amphotericin B was dissolved in DMSO resuspended in sterile PBS and was given at 1 mg/kg/day (0.5 mL/dose) intraperitoneally. Voriconazole was dissolved in PEG 200 and was given by gavage at 10 mg/kg bd (0.25 mL/dose). The control group received a comparable amount of solvent either intraperitoneally or by gavage.
The experiment was carried out for 7 days. The animals were weighed and closely monitored for appearance and activity. Autopsy was performed on the animals that died during the study and on the animals killed at the end of the study. At autopsy, the lungs were removed, weighed and homogenized in 10 mL of sterile PBS supplemented with piperacillin and amikacin each at 100 µg/mL, using a tissue homogenizer. The homogenate was serially diluted 10-fold, and 0.1 mL aliquots were plated on Sabouraud dextrose agar plates. The plates were incubated at 35°C for 48 h, and the number of colony forming units (cfu) per total weight of lung tissue was calculated.
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Results |
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In vitro susceptibility
The A. fumigatus isolate used in the study was susceptible to amphotericin B, voriconazole and itraconazole. The MICs of amphotericin B, voriconazole and itraconazole were 0.5, 0.5 and 0.25 mg/L, respectively.
In vivo efficacy
Among the voriconazole-treated guinea-pigs, all survivors (12 of 15) appeared normal throughout the study. The three voriconazole recipients that died, the amphotericin B-treated animals and the controls showed various signs of distress including hunched back, laboured breathing, reddened eyes, lethargy and in extreme cases, loss of righting ability. Also, whereas the voriconazole-treated survivors showed a weight gain of 10–30%, the remainder lost weight.
Figure 1
shows the survival data for the three groups of animals. Eighty per cent of the voriconazole-treated animals (12 of 15 animals) were alive at the end of study. In contrast, death was most rapid in the control animals; 14 of 15 animals (95%) had died during the 7 day period. Among the amphotericin B-treated animals, 12 of 15 (80%) died during the same period.
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, amphotericin B 1 mg/kg/day; 
, voriconazole 10 mg/kg bd.Figure 2
shows the microbial burden in the autopsied lungs. Lung cultures had minimal or no fungal growth in 12 of 15 voriconazole-treated animals, eight of 15 amphotericin B-treated animals and three of 15 controls. Mean burden of fungal organisms in the lungs (cfu/g lung tissue) in the three groups was 5.3 x 103 (voriconazole group), 1.4 x 104 (amphotericin B group) and 5.0 x 104 (controls). Compared with the control group of animals, voriconazole-treated and amphotericin B-treated animals had a 90% and 75% reduction in fungal burden, respectively. Voriconazole-treated animals had significantly better survival and decreased fungal burden when compared with control animals (P = 0.036), but not with amphotericin B-treated animals (P = 0.398).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The present study demonstrated good in vivo activity of voriconazole against experimental invasive aspergillosis. Marked improvement in survival rate and a reduction in fungal burden in the lungs were noted in voriconazole-treated animals. No statistically significant difference was observed between the efficacies of voriconazole and amphotericin B. However, voriconazole was associated with a favourable trend in survival as compared with amphotericin B (12 of 15 voriconazole-treated animals versus three of 15 amphotericin B-treated animals). Also noteworthy is the fact that, at autopsy, minimal or no fungal growth was seen in the lungs in most voriconazole-treated guinea-pigs (12 of 15 voriconazole-treated versus eight of 15 amphotericin B-treated guinea-pigs). Thus, cidal activity of voriconazole against Aspergillus sp., as observed in vitro, is further confirmed in the present study. Voriconazole was easy to administer orally and was well tolerated. It is unclear why the survival rate among amphotericin B-treated guinea-pigs was no better than that for controls.
Previously reported activity of voriconazole against experimental aspergillosis in the rat and rabbit models has been encouraging.15,16 Voriconazole significantly delayed or prevented mortality in the rat model of invasive pulmonary aspergillosis and the drug eliminated mortality and reduced the fungal burden in various organs in the rabbit model of disseminated aspergillosis. However, the pharmacokinetics of voriconazole in rats or rabbits may not be similar to that in man. On the other hand, the relatively slow metabolism of the drug in guinea-pigs may be more analogous to the human situation and therefore evaluating voriconazole using the guinea-pig model may be more relevant.12 Against endocarditis and disseminated aspergillosis in guinea-pigs, voriconazole has been shown previously to be effective.17,18 The drug was superior to itraconazole in the prevention and treatment of Aspergillus endocarditis. Clinical studies of therapy against invasive aspergillosis are continuing.
As aspergillus infections are increasing in frequency, better alternatives to amphotericin B are urgently needed. Given the in vitro and in vivo cidal activity of voriconazole against Aspergillus sp., the drug is of potential clinical use. Further clinical data are awaited.
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Acknowledgments |
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We thank Dr William Brown for providing the Aspergillus sp. isolate, and Pfizer, Inc. for their financial support. Our grateful thanks go to Ms Eileen Surma for secretarial assistance. This study was presented at the Twenty-First International Congress of Chemotherapy held in Birmingham, UK, during July 1999 (Journal of Antimicrobial Chemotherapy 44, Suppl. A, Abstract P-58).
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Notes |
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* Corresponding author. Tel: +1-313-745-9649; Fax: +1-313-993-0302; E-mail: pchandrasekar{at}oncgate.roc.wayne.edu
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References |
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Received 17 August 1999; returned 2 November 1999; revised 3 December 1999; accepted 20 December 1999
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