1 Sumitomo Pharmaceuticals Research Division, 198, Kasugadenaka 3-chome, Konohana-ku, Osaka 5540022; 2 Division of Respiratory Disease, Department of Medicine, Kawasaki Medical School, 577, Matsushima, Kurashiki 7010192, Japan
Received 17 July 2003; returned 13 September 2003; revised 19 October 2003; accepted 5 November 2003
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Abstract |
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Methods: Mice were infected with the organisms via tail veins. At 4 h after infection, antifungals were administered intravenously. For 30 days the number of mice surviving was recorded.
Results: AmBisome at 1 mg/kg or higher significantly prolonged the survival time of mice infected with five out of seven isolates of Aspergillus compared with the control group. There was no difference in in vivo activity between AmBisome and Fungizone at 1 mg/kg in six isolates of Aspergillus. At the maximum tolerated dose of antifungals, however, AmBisome (10 mg/kg) showed greater efficacy than Fungizone (1 mg/kg).
Conclusions: These results suggest that the overall protective activity of AmBisome against disseminated aspergillosis is superior to that of Fungizone.
Keywords: liposomal amphotericin B, amphotericin B deoxycholate, Aspergillus, tolerability
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Introduction |
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Although amphotericin B is a potent antifungal agent and remains the drug of choice for treatment of invasive aspergillosis, it frequently causes severe kidney damage.7 Hence, its maximal dosage is generally 1.5 mg/kg/day; and less toxic alternatives are strongly sought.
In order to reduce the toxicity of amphotericin B, a liposomal formulation (AmBisome) has been developed. AmBisome is a homogeneous suspension of unilamellar vesicles containing amphotericin B within their hydrophobic membranes. Upon administration, AmBisome remains intact in the blood and distributes to the tissues where fungal infections may occur.8 Disruption of AmBisome is observed after attachment to the outside of fungal cells, resulting in fungal cell death.8 Although AmBisome exhibits less in vitro antifungal activity than amphotericin B in some cases,911 much higher doses of AmBisome can be administered safely, resulting in an improved therapeutic profile.1219 Although in vivo antifungal activity of AmBisome has been evaluated in several animal models, little is known about the efficacies of AmBisome and amphotericin B at maximum tolerated dosages in models of aspergillosis. In the present study, therefore, the efficacies of AmBisome and amphotericin B were compared, at equivalent and maximum dosages, as therapy for both immunocompetent and temporarily leucopenic mice with invasive aspergillosis.
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Materials and methods |
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Liposomal amphotericin B (AmBisome; amphotericin B 50 mg, Gilead Sciences, Inc., San Diams, CA, USA) and amphotericin B deoxycholate (Fungizone; Bristol Pharmaceuticals Y.K., Tokyo, Japan) were reconstituted with sterile water according to the manufacturers instructions to obtain solutions of 4 and 5 g/L, respectively. These solutions were diluted with 5% glucose. In this paper, the concentration of antifungals is expressed as that of amphotericin B.
Animals
Male albino ddY mice, 34 weeks old, were purchased from Japan SLC, Inc. (Shizuoka, Japan) and maintained with chow and water ad libitum for 6 or 7 days. Mice were randomized, by a stratified sampling method, into groups of 10 animals each; those weighing 2531 and 2227 g were used in the immunocompetent and temporary leucopenic models, respectively. The experiments were performed under institutional animal guidelines.
Organisms
Aspergillus fumigatus H11-20 was isolated from a rat dying of spontaneously acquired pulmonary aspergillosis while on steroid treatment.20 Aspergillus flavus CI-3, Aspergillus niger CI-1 and A. niger CI-5 were isolated from different patients in a hospital.21 A. fumigatus IFO 8868, A. fumigatus IFO 9733 and A. flavus IFO 5839 were obtained from the Institute for Fermentation, Osaka, Japan. The Aspergillus conidia were stored at 80°C with silica gel. For each experiment, the fungi were grown at 32°C on Sabouraud dextrose agar (Difco Laboratory, Detroit, MI, USA), and 3- or 4-day-old conidia were harvested. The conidia were overlaid with 0.1% Tween 80 in water and gently removed with a bent plastic rod. The suspension was filtered to remove hyphae. It was then centrifuged at 2000g for 5 min at room temperature. The conidia obtained were re-suspended in sterile saline. The number of conidia was counted on a haemocytometer, and adjusted to the desired concentration.
In vitro susceptibility tests
The MICs of AmBisome and Fungizone for each of the organisms were determined by the broth microdilution method in accordance with NCCLS procedures.22 Two-fold serial dilutions of antifungals were prepared with RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) buffered to pH 7.0 with 0.165 M MOPS (Gibco BRL), and 100 µL of the drug solution was dispensed into the wells of 96-well plates. The conidia were diluted to 2 x 107 conidia/L with MOPS-buffered RPMI 1640 medium. A 100 µL aliquot of the inoculum was added to the drug dilution series, so that the final volume in the well was 200 µL. The final drug concentrations were in the range 0.0311024 and 0.03164 mg/L for AmBisome and Fungizone, respectively. The conidia were plated onto Sabouraud dextrose agar to determine the actual number of living cells inoculated. The 96-well plates were incubated at 35°C for 70 h.11,23 The MIC was defined as the lowest concentration exhibiting no visible growth.
A 160 µL portion was removed from each well showing no growth, mixed with 40 µL of 0.5% Tween 80 in water and plated onto Sabouraud dextrose agar. The minimal fungicidal concentration (MFC) was defined as the lowest drug concentration at which >99% of the original inoculum was killed.
Determination of tolerated dosage
Six doses of AmBisome (10, 20, 30, 40, 60 and 80 mg/kg) and four of Fungizone (1, 2, 3 and 4 mg/kg) were used in this experiment. Control mice were given 5% glucose intravenously (iv). The tolerated dose was defined as the dosage that caused neither death nor a significant change in body weight, renal function [serum creatinine (CRE) and blood urea nitrogen (BUN)] or hepatic function [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] 24 h after intravenous administration.
Efficacies of AmBisome and Fungizone in infected immunocompetent mice
Mice were infected with 0.2 mL of the following saline-suspended organisms via tail veins: 3 x 107 conidia/mouse for A. fumigatus H11-20, A. fumigatus IFO 8868, A. fumigatus IFO 9733, A. niger CI-1 and A. niger CI-5; 2 x 106 conidia/mouse for A. flavus CI-3; and 5 x 106 conidia/mouse for A. flavus IFO 5839. Six doses of AmBisome (0.03, 0.1, 0.3, 1, 3 and 10 mg/kg) and four of Fungizone (0.03, 0.1, 0.3 and 1 mg/kg) were used in the course of the experiment. All doses of AmBisome and Fungizone were administered via tail veins 4 h after infection. Control mice were treated as mentioned above. For 30 days the number of mice surviving was recorded.
Efficacies of AmBisome and Fungizone in infected temporarily leucopenic mice
To induce leucopenia, cyclophosphamide (Shionogi & Co., Ltd, Osaka, Japan) was administered subcutaneously at 200 and 100 mg/kg 2 days before and a day after infection, respectively. Mice were inoculated iv with 0.2 mL of the conidial suspensions of A. fumigatus H11-20 (5 x 105 conidia/mouse). Drug efficacies were evaluated by the method described above.
Statistical analysis
The 50% effective dose (ED50) values were calculated by probit analysis from the number of survivors in each group on the final day. The number of white blood cells was analysed by the Welch test. The renal and hepatic parameters were analysed using Dunnetts test of multiple comparisons. Since the effect of drug on body weight was expected to be monotonic, the Williams test of multiple comparisons was used to compare body weight.24,25 However, since observation of surviving mice was ended 30 days after infection, it was better to consider survival time as non-parametric data. Thus, the ShirleyWilliams testa non-parametric version of the Williams testwas used to compare survival time between the control group and each antifungal treatment group.26,27 For the same reason, the differences in the survival time of AmBisome treatment groups (1, 3, 10 mg/kg) and the Fungizone 1 mg/kg treatment group were evaluated using Steels test of multiple comparisons (equivalent to the parametric Dunnetts test).28,29 Two-sided P values were given, except for the Williams and ShirleyWilliams tests. A P value of <0.05 was considered statistically significant. All statistical analyses described above were performed using the Statistical Analysis System for Windows (SAS Institute Inc., Cary, NC, USA).
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Results |
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Table 1 shows the MICs and MFCs of AmBisome and Fungizone against the test organisms used in this study. The MICs of AmBisome for A. niger were lower than those for A. fumigatus and A. flavus. AmBisome was less active against A. flavus IFO 5839 than the other isolates. In contrast, Fungizone showed almost the same activity against the seven strains. In addition, there was little difference between the MICs and MFCs of AmBisome for A. niger and A. flavus, whereas they varied for A. fumigatus. Meanwhile, there was no distinction between the MICs and MFCs of Fungizone for the Aspergillus strains used. Taken together, AmBisome was as active as Fungizone against A. niger. Against A. fumigatus and A. flavus, however, it was less active than Fungizone.
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In immunocompetent mice, there were no deaths observed in the groups administered AmBisome and Fungizone up to doses of 60 and 2 mg/kg, respectively. Furthermore, body weights were not changed by AmBisome 10 mg/kg or Fungizone 1 mg/kg (data not shown). As a result, AmBisome could be given at an 10 times higher dose than Fungizone. Subsequently, we assessed the biochemical parameters of renal and hepatic functions (CRE, BUN, AST and ALT) in immunocompetent and temporarily leucopenic mice after administration of AmBisome and Fungizone at doses of 10 and 1 mg/kg, respectively. Although a significant increase in AST was only observed in temporarily leucopenic mice after treatment with AmBisome 10 mg/kg, the value was still in the normal range. These results are consistent with that of a previous investigation.17 In addition, empty liposomes had no toxicity (data not shown). Therefore, in subsequent experiments, we regarded the doses of AmBisome 10 mg/kg and Fungizone 1 mg/kg as the maximum tolerated dosages.
Comparative study on efficacies in disseminated aspergillosis
We examined the efficacy of AmBisome in a murine infection model using the seven Aspergillus strains described above. The survival percentage, the median survival time and the ED50 are all shown in Table 2. Representative survival curves of mice infected with A. fumigatus H11-20 are illustrated in Figure 1.
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There was a significant improvement in the survival time of infected mice treated with AmBisome at 1 and
3 mg/kg in comparison with the control group in the A. niger CI-1 and CI-5 models, respectively (P < 0.01). For these two A. niger strains, the ED50s of AmBisome were 1.50 and 2.59 mg/kg, which were almost equal to those of the A. fumigatus infection model, although AmBisome had different in vitro activity against these two species. With A. niger CI-1, AmBisome 10 mg/kg was superior to Fungizone 1 mg/kg (P < 0.001), and there was no difference in the survival time at the same doses (1 mg/kg) between AmBisome and Fungizone. In the case of A. niger CI-5 infection, AmBisome 1 mg/kg had less efficacy than Fungizone 1 mg/kg (P < 0.001).
In our murine model of A. flavus, the in vivo activities of AmBisome were less effective than those against A. fumigatus and A. niger. AmBisome at 1 and
3 mg/kg was superior to the control treatment in the A. flavus CI-3 and IFO 5839 models, respectively (P < 0.01). AmBisome and Fungizone at 1 mg/kg showed a comparable efficacy. However, treatment with AmBisome 10 mg/kg was superior to that with Fungizone 1 mg/kg in terms of survival time (P < 0.05).
Efficacy in a temporarily immunosuppressed mouse model of disseminated aspergillosis
Next, we evaluated the efficacy of AmBisome and Fungizone using a temporarily immunosuppressed mouse model (Table 3 and Figure 2). The administration of cyclophosphamide resulted in leucopenia (<0.8 x 109 cells/L, P < 0.0001) at least until 3 days after infection. All control mice died within 17 days of infection. AmBisome at doses of 1 mg/kg and Fungizone at 1 mg/kg were superior to the control treatment in terms of survival time (P < 0.05). There was no significant difference in efficacy between AmBisome and Fungizone at the same dose (1 mg/kg). Treatment with AmBisome 10 mg/kg resulted in 90% survival 30 days after infection, superior to treatment with Fungizone 1 mg/kg (P < 0.05).
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Discussion |
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This study implied that in vitro activities of AmBisome against A. fumigatus and A. flavus were lower than those of Fungizone, as previously reported.911 However, there was no significant difference in in vivo activity at the same dose between AmBisome and Fungizone in the mouse model of disseminated aspergillosis, except for the A. niger CI-5 infection model. Since AmBisome was 10 times less toxic than Fungizone in mice and able to be administered at up to 10 mg/kg, the protective effect of AmBisome against the Aspergillus infection model was superior to that of Fungizone. Meanwhile, except for the A. niger CI-5 infection model, administration of the maximum tolerated dosage of Fungizone (1 mg/kg) resulted in <50% survival rates. Accordingly, the ED50s of Fungizone were not detectable. On the other hand, the ED50s of AmBisome were lower than the maximum tolerated dosage (10 mg/kg), except in the A. flavus IFO 5839 infection model. These results suggest that the therapeutic window for AmBisome is wider than that for Fungizone. This is consistent with several previous investigations, which found that AmBisome has a slightly lower potency than Fungizone, but that much higher doses of AmBisome can be administered safely resulting in an improved therapeutic profile in some models of candidiasis, cryptococcosis and aspergillosis.1219 By contrast, earlier investigations imply that AmBisome is less toxic than Fungizone, and also less active in several fungal infection models.3336 However, although the maximum dosage of AmBisome used in those studies was higher than that of Fungizone, the dosage was not considered to be the maximum tolerated on the basis of its toxicity in the animals. Thus the therapeutic window of AmBisome might be underestimated.
The correlation between the in vitro activity of AmBisome and its in vivo efficacy against Aspergillus infection is not clear. We found no distinctions in the ED50s of AmBisome between A. niger and A. fumigatus, but the MICs of AmBisome for A. niger were lower than those for A. fumigatus. In addition, the MFCs of AmBisome for the three A. fumigatus strains varied from 8512 mg/L, but the ED50s of AmBisome for these strains were identical. Moreover, the survival rates for Fungizone 1 mg/kg in the A. flavus infection model were apparently lower than those in the A. fumigatus infection model, although it had an equivalent MIC value of 1 mg/L for all five strains. Thus, there may be no correlation between in vitro susceptibility to AmBisome or Fungizone and in vivo outcome in this murine model of Aspergillus infection. Several studies indicated that the in vivo activity of Fungizone in Aspergillus infection models does not depend on its in vitro activity against the isolates used.37,38 On the other hand, a previous study has indicated that a degree of Fungizone response is observed in fungal infection models at MIC 0.25 mg/L, whereas there is no response at MIC > 1 mg/L.39 Lass-Florl et al.40 reported that there is a correlation between the in vitro susceptibility of the organisms and the clinical efficacy of Fungizone in haematological patients with invasive aspergillosis. A difference in in vitro short-term killing activity or the post-antifungal effect against Aspergillus species might be involved in the protective effect.41
It is noteworthy that the in vivo efficacy of the same doses of AmBisome and Fungizone were not significantly different among most of the infection models tested here, although the in vitro activity and pharmacokinetic profiles differ greatly between the two agents. Fungizone has a broad distribution, and tends to be spread in the whole tissue.8 In contrast, the volume of distribution of AmBisome is smaller than that of Fungizone, and AmBisome is distributed to the central compartment.8 Owing to this, AmBisome is less toxic than Fungizone. Moreover, in general, fungi exist in the extracellular space in infected tissue. Adler-Moore & Proffitt42 reported that AmBisome is distributed more directly to the site of fungal infection with systemic candidiasis. Thus, the in vivo efficacy of AmBisome may be due to increased exposure of the organisms to the drug compared with Fungizone.
Even in the immunosuppressed mouse model with disseminated A. fumigatus infection, the efficacy of AmBisome 10 mg/kg was superior to Fungizone 1 mg/kg.
In conclusion, the present findings suggest that the overall protective effect of AmBisome against disseminated aspergillosis is superior to that of Fungizone.
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Acknowledgements |
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Footnotes |
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