Department of Biological Sciences, California State Polytechnic University, Pomona, CA 91768, USA
Received 14 June 2004; returned 19 July 2004; revised 20 August 2004; accepted 12 September 2004
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Abstract |
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Methods: Mice were immunosuppressed with cyclophosphamide every 3 days, beginning day 3 before challenge with log10 5.0 cfu Candida albicans. Treatment was begun 4872 h post-challenge with daily or intermittent dose regimens of AmBisome, followed by determination of kidney cfu for up to 1 month post-treatment.
Results: A single AmBisome dose of 4 mg/kg was as effective as four daily, 1 mg/kg treatments. A total of 8 mg/kg, given as 4 mg/kg on days 2 and 4, or as 5 mg/kg on day 2 followed by 1 mg/kg on days 3, 4, and 5, also produced comparable efficacy. While 20 mg/kg given day 2, 4 and 6 post-challenge as a 1 week loading dose, followed by one 10 mg/kg treatment on day 13, decreased the fungal burden by up to 5 logs compared with controls (log10 2.3 cfu/g and log10 7.5 cfu/g, respectively), 20 mg/kg given Monday, Wednesday and Friday for 5 weeks, reduced the fungal burden to undetectable levels (i.e. log10 1.0 cfu).
Conclusions: Significant reduction or clearance of kidney cfu, following intermittent, high dose AmBisome treatment, indicated that non-daily dosing regimens could be successfully used instead of conventional daily dosing to treat established C. albicans infection in immunosuppressed mice.
Keywords: AmBisome loading dose , antifungal therapy , intermittent AmBisome dosing
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Introduction |
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When compared with other amphotericin B lipid formulations, AmBisome (liposomal amphotericin B) is the least toxic,6,7 and doses of AmBisome as high as 15 mg/kg have been shown to be well tolerated by patients.810 Doses of 4 mg/kg or higher have been reported to be associated with good drug penetration into animal tissues as well as maintenance of therapeutic drug levels in the animal tissues for several weeks following treatment.11 This is probably related to the unique composition and pharmacodynamics of AmBisome (i.e. high AUC, high Cmax and non-linear pharmacokinetics) resulting in elevated levels of amphotericin B concentrations in the blood of patients12 and animals,13 as well as in the reticuloendothelial system (RES) and non-RES tissues of animals11 which include important sites of fungal dissemination, such as the brain14 and the kidneys.15,16
Although treatment regimens for AmBisome, like those for AMB, have nearly always utilized daily dosing,1 some non-clinical studies have shown that AmBisome can be given every other day to significantly reduce fungal burdens17,18 and significantly improve survival compared with untreated controls.1719 Thus, intermittent dosing of AmBisome may be just as effective as conventional daily dosing. In addition, the drug's pharmacokinetic profile suggests that administration of a high loading dose of AmBisome at the beginning of treatment might provide an improved treatment outcome. Loading dose regimens are already routinely used with other antifungal drugs, such as itraconazole,20 and caspofungin.21,22 The focus of this study was to use a severe, systemic Candida mouse infection model to investigate these different approaches to AmBisome dosing. High dose AmBisome (420 mg/kg), was given intermittently and as a 1 week loading dose, to try to reduce the number of drug treatments needed to effectively treat systemic candidiasis in continually immunosuppressed mice.
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Materials and methods |
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Female C57BL/6N mice (1820 g) were obtained from Charles River (Indianapolis, IN, USA) or B&K Universal (Fremont, CA, USA) and maintained in micro-isolator boxes with standard rodent diet (Lab Mouse Diet #5015, PMI Nutrition International, Brentwood, MO, USA) and water ad libitum. All animal research procedures were approved by the Institutional Animal Care and Use Committee of California State Polytechnic University, Pomona.
Test substance
AmBisome (Gilead Sciences, Inc., San Dimas, CA, USA), a lyophilized liposomal preparation of amphotericin B, was reconstituted with 12 mL of sterile water for injection (WFI) and shaken vigorously for 1 min, and filtered according to the manufacturer's instructions. This resulted in a 4 mg/mL solution of amphotericin B.
Immunosuppression
Mice to be immunosuppressed were given cyclophosphamide at 100 mg/kg intraperitoneally (Sigma Chemical Co., St Louis, MO, USA) 3 days before fungal challenge. Maintenance doses of 75 mg/kg cyclophosphamide were given on the day of challenge and then every third day for the duration of each study (3233 or 6265 days). Although the animals transiently lost an average of 0.5 g on the day following each cyclophosphamide treatment, no secondary bacterial infections were observed. Blood cell counts in mice immunosuppressed with 75 mg/kg cyclophosphamide every 72 h for 1 week were compared with non-immunosuppressed mice and the white blood cells (WBC) in the non-treated mice were 2.6 times greater than the WBC in the cyclophosphamide-treated animals (4.2x106 cells/mL versus 1.6x106 cells/mL, respectively; P < 0.05). Cyclophosphamide is reported to produce decreased numbers of granulocytic cells, monocytic cells, lymphoid cells and myeloid blast cells.23
Fungal inocula
Candida albicans (California State Polytechnic University, strain CP620) was passaged through mouse kidneys as used in our previous studies of systemic candidiasis.15 It was maintained as a frozen stock culture in Sabouraud's dextrose broth containing 20% glycerol. Three days before challenge, a 0.5 mL aliquot of the stock was thawed and subcultured initially at 35°C and then at room temperature, for two more days in Sabouraud's dextrose broth without shaking. On the day of challenge, the blastospore subculture was pelleted, and rinsed twice with 0.01 M phosphate buffered saline, pH 7.2 (PBS). The final pellet was resuspended in 15 mL of PBS, the concentration of viable blastospores determined by 1% Methylene Blue staining and the suspension adjusted with PBS to approximately 1 x 107 cells/mL for non-immunosuppressed mice and 1 x 106 cells/mL for cyclophosphamide immunosuppressed mice. At the time of challenge, non-immunosuppressed mice were injected intravenously (iv) via the tail vein with approximately 0.7 x 106 cells and immunosuppressed mice were injected intravenously with approximately 0.61 x 105 cells.
Treatment regimens
To compare daily dosing with intermittent dosing, immunosuppressed Candida-infected mice were divided into the following four AmBisome treatment groups (n=7 mice/group): 1 mg/kg (4 mg/kg total dose) for four consecutive days (days 2, 3, 4 and 5); 4 mg/kg given once on day 2 (4 mg/kg total dose); alternate day dosing (days 2 and 4) at 4 mg/kg (8 mg/kg total dose); and 5 mg/kg given once on day 2 followed by 1 mg/kg on days 3, 4 and 5 (8 mg/kg total dose). AmBisome was given intravenously via the tail vein. Seven control infected mice were treated intravenously with 5% dextrose (D5W) on days 2, 3, 4 and 5. An additional seven infected mice were killed 48 h post-challenge to determine the fungal burden in the kidneys at the initiation of treatment (log10 6.13 cfu/g, range 5.186.78). Thirty-two days post-treatment, surviving animals in the AmBisome groups and the control group were killed, and then both kidneys were removed from each mouse, weighed and the pair of kidneys homogenized in 1.0 mL of PBS using a Tissue Tearor (Biospec Products Inc., Bartlesville, OK, USA). The kidneys were assayed for cfu because the primary site of infection in murine systemic candidiasis is the kidneys.24 Following serial dilutions of the kidney homogenates, 0.2 mL aliquots of each dilution were plated in duplicate on Sabouraud dextrose agar plates and incubated at 35°C for 24 h to determine cfu per gram of kidneys; the diluting procedures eliminated drug carryover. The cfu for each treatment group was expressed as the mean log10 cfu for that group of mice.
To determine the efficacy of high dose, intermittent therapy, groups of immunocompetent (n=20) and immunosuppressed (n=20) Candida-infected mice were given 20 mg/kg AmBisome intravenously on Mondays, Wednesdays and Fridays for 5 weeks, beginning 48 h post-challenge. Control groups of Candida-infected immunocompetent (n=25) and immunosuppressed (n=25) mice were given D5W using the same injection schedule. The mice in each group (immunocompetent or immunosuppressed) were preassigned to a subgroup of five mice, and the surviving mice in a given subgroup were killed on days 9, 24, 38 or 65 (1 month after the last treatment). The extra five mice in the immunocompetent and immunosuppressed control mouse groups were killed 48 h post-challenge to determine the fungal burden in the kidneys at the initiation of treatment (log10 5.25 cfu/g; range, 4.666.68 for the immunocompetent mice and log10 5.68 cfu/g; range, 5.256.04 for the immunosuppressed mice). On the day of sacrifice, both kidneys from each mouse were collected and processed as described above for determination of cfu/g kidneys.
To study the efficacy of a 1 week loading dose, we intravenously administered 5, 10 or 20 mg/kg AmBisome on days 2, 4 and 6, or on days 3, 5 and 7 post-challenge to immunosuppressed mice (n=7/group), followed by once a week dosing for 2 weeks with 5, 10 or 20 mg/kg. The total dose of drug delivered to the 5, 10 and 20 mg/kg groups was 25, 50 or 100 mg/kg, respectively. Control mice were treated with D5W on days 2, 4, 6, 13 and 20 or on days 3, 5, 7, 14 and 21 post-challenge. Immunosuppression was maintained throughout the studies as described above. Surviving mice were killed on day 32 when the loading dose was given on days 2, 4 and 6, or on day 33 when the loading dose was given on days 3, 5 and 7. In these experiments, an additional seven control mice were killed 48 or 72 h post-challenge to determine the fungal burden in the kidneys at the initiation of treatment (log10 5.95 cfu/g, range, 5.306.94 for 48 h and log10 6.45 cfu/g, range, 5.947.76 for 72 h). On the day of sacrifice, both kidneys from each mouse were collected and processed as described above for determination of cfu/g kidneys.
In a subsequent study of the 1 week loading dose, we treated 56 Candida-infected, continually immunosuppressed mice with 20 mg/kg AmBisome intravenously on days 2, 4 and 6 post-challenge and then divided the mice into four AmBisome treatment groups (n=14/group) which were further subdivided into two subgroups (n=7/subgroup). Each subgroup for a given AmBisome treatment was to be killed on a different day (i.e. day 32 or day 62). The four AmBisome regimens were as follows: 2.5 mg/kg on days 13, 20, 27 and 34; 5 mg/kg on days 13 and 20; 10 mg/kg on day 13; and 20 mg/kg on days 13, 20, 27 and 34. The first three regimens delivered the same total dose of drug to the mice (i.e. 70 mg/kg) and the latter regimen gave a total dose of 140 mg/kg. On day 32 post-challenge, surviving mice in one subgroup from each AmBisome treatment were killed, and surviving mice in the remaining four AmBisome subgroups were killed approximately 1 month after treatment termination (day 62 post-challenge). Control Candida-infected, immunosuppressed mice were divided into two subgroups of seven mice each to be killed on day 32 and day 62 following treatment with D5W on days 2, 4, 6, 13 and 20. An additional seven immunosuppressed control mice were killed 48 h post-challenge (day 2) to determine the fungal burden in the kidneys at the initiation of treatment (log10 5.95 cfu/g; range, 5.306.94). For all killed mice, both kidneys from each mouse were collected and processed as described above for determination of cfu/g kidneys.
Statistical analysis
For cfu/g kidneys, a KruskalWallis test (analogous to a one-way ANOVA) was applied to all groups and where differences occurred, a two-tailed MannWhitney test was done on paired groups.
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Results |
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Discussion |
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One of the reasons that daily treatments with polyenes and other antifungal drugs have become the convention,25 is that patients with fungal infections are often immunosuppressed requiring intensive therapy to treat any established infection. However, when we used a Monday, Wednesday, Friday dosing schedule with high dose (20 mg/kg) AmBisome to treat an established systemic Candida infection in continually immunosuppressed mice, the regimen was effective in significantly reducing infection after only 1 week of treatment. After 5 weeks of treatment, it had reduced the infection to undetectable levels. The absence of fungi in the kidneys 1 month after treatment had terminated, indicated that this intermittent high dose regimen had probably cleared the infection. It is likely that there were therapeutic levels of AmBisome remaining in the tissues at this time since there are several reports of therapeutic levels of AmBisome being detected in infected tissues 7 days11 and even 103 days26 post-treatment. The sustained presence of AmBisome has also been noted in several prophylactic studies which measured the amount of AmBisome in the target tissues (including kidneys) at the time of challenge15,27,28 as well as in other experiments where treatment was terminated and yet the cfu continued to decrease in the kidneys29 and spleens30 of AmBisome-treated animals.
To reach therapeutic drug levels quickly, loading doses are used with the antifungal drugs itraconazole20 and caspofungin21,22 and the data in this study indicate that this may also be an effective strategy for AmBisome. In our study, a 1 week loading dose of 5, 10 or 20 mg/kg, was able to significantly reduce the fungal burden in immunosuppressed mice even when the Candida infection was well-established at the start of treatment. We also observed that if a 60 mg/kg loading dose of AmBisome was followed by weekly treatment(s) with 2.5, 5, 10 or 20 mg/kg of AmBisome, the fungal burden in the kidneys was significantly lower with follow-up doses of 10 or 20 mg/kg when compared with the lower, follow-up drug dosing regimens. This observation is consistent with the non-linear pharmacokinetics of AmBisome12,31,32 which is associated with more drug being delivered to non-reticuloendothelial system tissues, such as the kidneys, when the dose is higher.
In conclusion, intermittent high dose treatments with AmBisome, given over a 5 week period, were able to clear the Candida infection from the kidneys of immunosuppressed mice. These data indicate that alternative dosing regimens, like those discussed in this paper have the potential to decrease the clinical cost and time required to administer the drug by reducing the total number of treatments needed. Given AmBisome's markedly better safety profile compared with other amphotericin B formulations, the results of the present experiments support the idea of further investigation of alternative dosing schedules which take advantage of the non-linear pharmacokinetics and the sustained drug tissue levels of high dose (420 mg/kg) AmBisome.
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Acknowledgements |
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Footnotes |
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Present address. 2095 Lavender Hill Ct., Lincoln, CA 95648, USA
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References |
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