Antifungal activity of amphotericin B–lipid admixtures in experimental systemic candidosis in naive mice

Yona Shadkchan and Esther Segal*,

Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have shown previously that admixtures of amphotericin B (AMB) and Intralipid (AMB–IL) obtained by vigorous and prolonged agitation are stable and can be standardized. These preparations exhibited in-vitro activity against various Candida spp., and had significantly lower toxicity. The present study was undertaken to evaluate the activity of AMB–IL admixtures in vivo in comparison with the conventional formulation of AMB (Fungizone), using a murine model of experimental systemic candidosis. ICR female mice (4–6 weeks old) were injected iv with 5 x 104 Candida albicans CBS 562. The animals developed a lethal infection (100%) within 10 days. Systemic candidosis was demonstrated by the presence of fungal elements in kidneys and spleen tissue, and by enumeration of cfu of Candida in the tissue homogenates. AMB–IL or AMB was administered iv 48 h post-Candida inoculation for 5 consecutive days. Four experiments with 108 mice treated with AMB 5 x 0.4 mg/kg and followed up for 6 weeks, showed that the mean survival percentages at the end of the experiment were 0, 24.9 and 52.5% for the untreated group, conventional AMB-treated and AMB–IL-treated groups, respectively. The mean survival time (MST) was 7.4, 25 and 30 days for the untreated, conventional AMB-treated and AMB–IL-treated groups, respectively. Use of increased doses of AMB showed that conventional AMB at doses greater than 5 x 1 mg/kg caused immediate animal death. AMB–IL was used at doses of AMB up to 5 x 2 mg/kg. Experiments with 104 mice revealed that the mean survival percentage at the end of the experiment was 0, 34.5, 58.6 and 97% for the untreated, conventional AMB-treated (5 x 1 mg/kg), AMB–IL-1-treated (5 x 1 mg/kg) and AMB–IL-2-treated (5 x 2 mg/kg) groups, respectively. The MST was 7, 27.8, 34.8 and 41.4 days for the untreated, conventional AMB-treated, AMB–IL-1-treated and AMB–IL-2-treated groups, respectively. The results of this study reveal that AMB–IL is significantly more effective in treating systemic murine candidosis than conventional AMB.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a previous study1 we showed that admixtures of amphotericin B (AMB) and some lipid emulsions, such as Intralipid 20%, prepared by vigorous overnight agitation, constitute stable, standardized preparations with in-vitro activity against various Candida spp., and significantly lower toxicity, as measured by lysis of RBC and leakage of K+. AMB is the drug of choice for infections caused by a wide range of fungi,2,3 but its use is associated with various adverse effects.4,5 A number of strategies have been explored for reducing the toxic effects of AMB, such as the use of different detergents, liposomes and several lipid combinations as carriers.6,7,8 Some of the liposomal preparations are now available for clinical use;9 however, their use is limited owing to their very high cost.

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% (AMB–IL) in comparison with conventional AMB formulation (conventional AMB) would be worthwhile. Toward this aim we investigated the efficacy of the AMB–IL admixtures versus AMB in a systemic candidosis model induced in mice.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal model

Female ICR mice, 4–6 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. AMB–IL 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 AMB–IL at different doses (range 0.4–2 mg/kg). Treatment began 48 h after inoculation of Candida and consisted of five consecutive daily injections of AMB (conventional AMB or AMB–IL). A control group with infected and sham-treated mice (injected with buffer–PBS) was included. Survival was followed up for 42 days. Assessment of the activity of AMB–IL 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 AMB–IL

Acute toxicity was determined for AMB–IL 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 (0.4–2 mg/kg) either as conventional AMB or AMB–IL, and surveyed for mortality.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental murine candidosis

Inoculation of 4–6 week old female ICR mice (20 mice) with 5 x 104 C. albicans organisms/mouse resulted in 100% mortality within 5–10 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 106–3 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 AMB–IL 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 AMB–IL) significantly increased the survival of the mice as compared with the sham-treated controls, with over 50% of mice surviving when treated with AMB–IL. 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 AMB–IL-treated groups, respectively. The follow-up of the course of infection indicated that conventional AMB and AMB–IL 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 AMB–IL-treated groups, respectively (Figure 2).



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Figure 1. Course of infection in mice treated with low dose (0.4 mg/kg) AMB formulations for 5 consecutive days. Data are the mean of four experiments; {blacksquare}, control, C. albicans; •, C. albicans + AMB; {blacktriangleup}, C. albicans + AMB–IL.

 


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Figure 2. Mean survival time (follow up of 42 days) of mice treated with low dose (0.4 mg/kg) AMB formulations for 5 consecutive days.Data are the mean of four experiments.

 
Treatment of systemic murine candidosis with high doses of AMB

To attempt to improve the efficacy of the AMB–IL 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 AMB–IL in doses of 0.4–2 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. AMB–IL 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 AMB–IL 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 AMB–IL (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, AMB–IL-1-treated and AMB–IL-2-treated groups, respectively (Figure 3). The MSTs were 7, 27.8, 34.8 and 41.4 days for the untreated, conventional AMB-treated, AMB–IL-1-treated and AMB–IL-2-treated groups, respectively (Figure 4). These experiments indicated that AMB–IL is more effective in treating systemic murine candidosis than conventional AMB, especially at the higher concentration of 10 mg/kg.



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Figure 3. Course of infection in mice treated with high dose AMB formulations. Data are the mean of four experiments. {blacksquare}, control, C. albicans; •, C. albicans + AMB (1 mg/kg x 5); {blacktriangleup}, C. albicans + AMB–IL (1 mg/kg x 5); {diamondsuit}, C. albicans + AMB–IL (2 mg/kg x 5)

 


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Figure 4. Mean survival time (follow up of 42 days) of mice treated with high dose AMB formulations. Data are the mean of four experiments;

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of the present study was to evaluate in vivo the efficacy of amphotericin B–Intralipid admixtures in comparison with conventional AMB. Towards this aim we used experimental systemic candidosis induced in mice, the most widely used experimental animal model for studying systemic mycoses.12 The evaluation was based on accepted criteria, including mortality and morbidity, the latter being assessed primarily by quantitive determination of fungal infection of the kidneys.13 We chose an experimental model in which the development of infection was not too rapid—mortality began at day 5, which enabled a follow-up of the course of infection. The fungal inoculum adopted—5 x 104 organisms/mouse—produced 100% mortality within 10 days. This in turn permitted reliable evaluation of the treatment.

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 AMB–IL) significantly increased the survival of animals and affected the course of infection in comparison with untreated controls. The data also showed that AMB–IL is more effective than conventional AMB in treatment of murine candidosis, demonstrating a significant difference between the survival rate of conventional AMB-treated versus AMB–IL-treated animals. However, the AMB–IL 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 AMB–IL admixtures, since conventional AMB at such doses was lethal. The higher dose of AMB (5 x 2 mg/kg) in the AMB–IL 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.


    Acknowledgments
 
This work was in partial fulfillment towards the Ph.D. degree (Y.S.). The authors wish to express their gratitude to Ms Altermann Pearl and Abrahamer Hana (Department of Pathology, Tel-Aviv University) for skilful technical assistance in the preparation of the histopathological material.


    Notes
 
* Corresponding author. Tel: +972-3-6409069 or +972-3-6409870; Fax: 972-3-6409160; E-mail: segale{at}post.tau.ac.il Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Shadkhan, Y., Segal, E., Bor, A., Gov, Y., Rubin, M. & Lichtenberg, D. (1997). The use of commercially available lipid emulsions for the preparation of amphotericin B–lipid admixtures. Journal of Antimicrobial Chemotherapy 39, 655–8.[Abstract]

2 . Gallis, H. A. (1996). Amphotericin B: a commentary on its role as an antifungal agent and as a comparative agent in clinical trials. Clinical Infectious Diseases 22, Suppl. 2, s145–7.[ISI][Medline]

3 . Graybill, J. R., Revankar, S. G. & Patterson, T. F. (1998). Systemic antifungal drugs in current use. In Microbiology and Microbial Infections. 9th edn, (Topley & Wilson, Eds), pp. 163–5. Arnold, Great Britain.

4 . Brajtburg, J. & Bolard, J. (1996). Carrier effects on biological activity of amphotericin B. Clinical Microbiology Reviews 9, 512–31.[Abstract]

5 . Sabra, R. & Branch, R. A. (1990). Amphotericin B nephrotoxicity. Drug Safety 5, 94–108. [ISI][Medline]

6 . Gates, C. & Pinney, R. J. (1993). Amphotericin B and its delivery by liposomal and lipid formulations. Journal of Clinical Pharmacy and Therapeutics 8, 147–53.

7 . Brajtburg, J., Powderly, W. G., Kobayashi, G. S. & Medoff, G. (1990). Amphotericin B: Delivery systems. Antimicrobial Agents and Chemotherapy 34, 381–4.[ISI][Medline]

8 . Janoff, A. S. (1990). Liposomes and lipid structures as carriers of amphotericin B. Journal of Clinical Microbiology and Infectious Diseases 9, 146–51.

9 . Hiemenz, J. W. & Walsh, T. J. (1996). Lipid formulations of Amphotericin B: recent progress and future directions. Clinical Infectious Diseases 22, Suppl. 2, s133–44.[ISI][Medline]

10 . Levy, R., Segal, E. & Eylan, E. (1981). Protective immunity against murine candidiasis elicited by Candida albicans ribosomal fractions.Infection and Immunity 31, 874–8.[ISI][Medline]

11 . Levy, M. Y., Polachek, I., Barenholz, Y. & Benita, S. (1993). Efficacy evaluation of a novel submicron Amphotericin B emulsion in murine candidiasis. Journal of Medical and Veterinary Mycology 31, 207–18. [ISI][Medline]

12 . Ryley, J. F. (1990). Screening and evaluation in vivo. In Handbook of Experimental Pharmacology: Chemotherapy of Fungal Diseases. Vol 96, (Ryley, J. F., Ed.) pp. 129–36. Springer-Verlag, Germany.

13 . Anaissiae, E.J., Pinczowski, H. & Louria, D. B. (1993). Candida infections in experimental animals. In Candidiasis: Pa6thogenesis, Diagnosis and Treatment. 2nd edn, (Bodey, Ed.), pp. 43–57. Raven Press, New York.

Received 16 February 1999; returned 9 July 1999; accepted 19 July 1999