Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
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Abstract |
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Introduction |
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This body of knowledge and the data obtained in our previous in-vitro study suggested that assessment of in-vivo activity of admixtures of AMB and Intralipid 20% (AMBIL) in comparison with conventional AMB formulation (conventional AMB) would be worthwhile. Toward this aim we investigated the efficacy of the AMBIL admixtures versus AMB in a systemic candidosis model induced in mice.
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Materials and methods |
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Female ICR mice, 46 weeks old were used in all experiments. Experimental systemic candidosis was produced as in previous studies,10 by iv inoculation of Candida albicans CBS 562 using an inoculum of 5 x 104 organisms/mouse (determined by microscopic counts in a haemocytometer). Infection was followed up for 6 weeks and evaluated in terms of mortality and morbidity, the latter being assessed by determination of fungal colonization of viscera (kidneys, spleen and liver).
Fungal infection was demonstrated qualitatively by histopathology of tissue sections of organs removed from mice killed at various time intervals after inoculation. Tissues were fixed in 10% formalin (at least 24 h), embedded in paraffin wax, and cut sections were stained with Periodic Acid Schiff (PAS) for light microscopy. In addition, unstained or calcofluor-stained tissue homogenates were also examined by light or fluorescence microscopy for the presence of fungal elements. (Calcofluor is a flurochrome with affinity for chitin and used therefore as a method for demonstration of fungal elements).
Enumeration of Candida cfu in tissue homogenates from infected animals provided a quantitative measurement of infection. Kidneys and spleens of killed animals were excised aseptically and homogenized in 1 mL sterile saline using a homogenizer (Power Control Unit Drehzahlegler, Switzerland). The tissue homogenates were diluted in phosphate-buffered saline (PBS) and plated on Sabouraud agar (Difco) supplemented with chloramphenicol. Culture plates were incubated for 48 h at 28°C and the enumerated colonies were expressed as cfu/mL/organ.
Antifungal agents
A stock solution of conventional AMB (Bristol-Myers Squibb Pharmaceuticals Ltd, Dublin, Eire) (5 mg/mL) was prepared in 5% dextrose. AMBIL was prepared as described previously,1 by a 25-fold dilution of conventional AMB in the lipid emulsion Intralipid 20% (Kabi Pharmacia, Stockholm, Sweden) to a final AMB concentration of 0.2 mg/mL and then agitated vigorously at 24°C for 18 h on an Orbit Environmental Shaker, Lab Line (orbital diameter = 4 cm) at 280 rpm.
Treatment of mice with intralipid admixtures or conventional AMB
Infected mice were treated based on the report of Polacheck et al.11 with either conventional AMB (as a control) or AMBIL at different doses (range 0.42 mg/kg). Treatment began 48 h after inoculation of Candida and consisted of five consecutive daily injections of AMB (conventional AMB or AMBIL). A control group with infected and sham-treated mice (injected with bufferPBS) was included. Survival was followed up for 42 days. Assessment of the activity of AMBIL was based on the parameters described above for characterization of the systemic candidosis model and was evaluated by mortality and/or morbidity of untreated animals in comparison with animals treated with conventional AMB [expressed as survival rate and mean survival time (MST)]
Evaluation of in-vivo toxicity of AMBIL
Acute toxicity was determined for AMBIL in comparison with conventional AMB, in Candida-infected and non-infected animals. Mice were injected into the tail vein for 5 consecutive days with various doses of AMB (0.42 mg/kg) either as conventional AMB or AMBIL, and surveyed for mortality.
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Results |
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Inoculation of 46 week old female ICR mice (20 mice) with 5 x 104 C. albicans organisms/mouse resulted in 100% mortality within 510 days. Organs examined macroscopically for pathological signs before further processing, showed microabscesses. Microscopic observation of organ homogenates stained with calcofluor demonstrated the presence of hyphae and budding yeasts. Sections of the kidneys stained by PAS revealed large fungal lesions, which contained yeast cells and hyphal elements.
To characterize the extent of fungal colonization of the kidney, a target organ in systemic candidosis, we enumerated cfu in kidneys of 20 mice on the day of death. We found that their kidneys were infected with greater than 106 C. albicans organisms (range 1.6 x 1063 x 107; mean 8.6 ±0.86 x 106).
Treatment of systemic murine candidosis with low doses of AMB
The experiments involved naive ICR mice inoculated iv with 5 x 104 C. albicans organisms and treated 48 h later with either AMBIL or conventional AMB at a concentration of 0.4 mg/kg, or buffer. Treatment was administered for 5 consecutive days (total AMB dose 2 mg/kg). Animals were surveyed for up to 42 days. Data obtained from four experiments with 108 mice are summarized in Figures 1 and 2. Both formulations (conventional AMB and AMBIL) significantly increased the survival of the mice as compared with the sham-treated controls, with over 50% of mice surviving when treated with AMBIL. Specifically, the mean percentages of surviving mice at day 42 were 0, 24.85±1.84 and 52.48±3.38% for the untreated, conventional AMB-treated and AMBIL-treated groups, respectively. The follow-up of the course of infection indicated that conventional AMB and AMBIL increased the survival time of the treated mice. Thus, the MSTs in those mice that succumbed to infection were 7.38±0.57; 25±1.77 and 30±2.09 days for the untreated control, conventional AMB-treated and AMBIL-treated groups, respectively (Figure 2).
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To attempt to improve the efficacy of the AMBIL preparations we planned experiments with higher doses of AMB. Towards this aim we first had to establish the maximum doses of AMB to which the mice would be tolerant. We injected 84 mice with either conventional AMB or AMBIL in doses of 0.42 mg/kg for 5 consecutive days. We found that the maximum tolerated dose was 1 mg/kg x 5 for conventional AMB, with higher doses causing immediate death. AMBIL at a concentration of 2 mg/kg (total 10 mg/kg) did not cause death during an observation period of 6 weeks.
Based on these results we performed four additional treatment experiments with 104 mice, using higher doses of AMB in the AMBIL preparations. We thus had four groups in each experiment: sham (buffer-treated) controls, conventional AMB-treated group (1 mg/kg x 5) and two groups (1 and 2) treated with AMBIL (1 mg/kg x 5 and 2 mg/kg x 5). These experiments revealed that the mean survival percentages at day 42 were 0, 34.5, 58.6 and 97% for the untreated, conventional AMB-treated, AMBIL-1-treated and AMBIL-2-treated groups, respectively (Figure 3). The MSTs were 7, 27.8, 34.8 and 41.4 days for the untreated, conventional AMB-treated, AMBIL-1-treated and AMBIL-2-treated groups, respectively (Figure 4). These experiments indicated that AMBIL is more effective in treating systemic murine candidosis than conventional AMB, especially at the higher concentration of 10 mg/kg.
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Discussion |
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The initial AMB treatment, which consisted of five consecutive injections of 0.4 mg/kg, was based on methods described in the literature.11 The experiments revealed that both formulations of AMB (conventional AMB and AMBIL) significantly increased the survival of animals and affected the course of infection in comparison with untreated controls. The data also showed that AMBIL is more effective than conventional AMB in treatment of murine candidosis, demonstrating a significant difference between the survival rate of conventional AMB-treated versus AMBIL-treated animals. However, the AMBIL treatment did not save almost half of the animals from succumbing to the experimental candidosis.
In order to attempt to increase the efficacy of the treatment, an increase in the dose of AMB was needed. An increase in the dose of the AMB beyond the level of 5 x 1 mg/kg was possible only for AMBIL admixtures, since conventional AMB at such doses was lethal. The higher dose of AMB (5 x 2 mg/kg) in the AMBIL admixtures was very effective, leading to almost 100% survival. To follow up these promising results we plan to evaluate this treatment regimen in immunocompromised animals.
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Acknowledgments |
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Notes |
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References |
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Received 16 February 1999; returned 9 July 1999; accepted 19 July 1999