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Antimicrobial Agents and Chemotherapy, December 2005, p. 4895-4902, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.4895-4902.2005

Treatment of Candida glabrata Infection in Immunosuppressed Mice by Using a Combination of Liposomal Amphotericin B with Caspofungin or Micafungin

Jon A. Olson, Jill P. Adler-Moore,^* P. J. Smith, and Richard T. Proffitt

Department of Biological Sciences, California State Polytechnic University, Pomona, California 91768

Received 28 June 2005/ Returned for modification 2 August 2005/ Accepted 28 September 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
While Candida albicans remains the most common Candida isolate, Candida glabrata accounts for approximately 15 to 20% of all Candida infections in the United States. In this study we used immunosuppressed mice infected with C. glabrata to investigate the efficacy of liposomal amphotericin B alone or in combination with the echinocandin caspofungin or micafungin. For monotherapy, mice were given six daily doses of liposomal amphotericin B (3 to 20 mg/kg of body weight), caspofungin (1 to 5 mg/kg), or micafungin (2.5 to 10 mg/kg). With concomitant therapy, mice received liposomal amphotericin B (7.5 mg/kg) in addition to caspofungin (2.5 mg/kg) or micafungin (2.5 mg/kg) for 6 days. For sequential therapy, liposomal amphotericin B was administered on days 1 to 3 and caspofungin or micafungin was given on days 4 to 6; conversely, caspofungin or micafungin was administered on days 1 to 3 and liposomal amphotericin B was given on days 4 to 6. Efficacy was based on the number of CFU per gram of kidney 21 days postchallenge. Monotherapy with liposomal amphotericin B (7.5 to 20 mg/kg) was significantly more effective than no drug treatment (control group) (P < 0.05) and demonstrated a dose-dependent response, with 20 mg/kg lowering the CFU/g from 6.3 to 4.2 (significantly different from the value for the control group [P < 0.001]). Monotherapy with all echinocandin doses lowered the CFU/g from 6.0 to 6.4 to 2.7 to 3.3 (significantly different from the value for the control group [P < 0.001]) with no dose-dependent response. Complete clearance of infection could be achieved only when liposomal amphotericin B was given either concomitantly with caspofungin or micafungin or if liposomal amphotericin B was given sequentially with caspofungin. In conclusion, the combination of liposomal amphotericin B with an echinocandin markedly improved the therapeutic outcome in murine C. glabrata systemic infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Candida infections have become the fourth most common cause of hospital-acquired infections, accounting for about 8 to 9% of all such infections (19, 43). While Candida albicans remains the most common Candida isolate, Candida glabrata, generally considered to be a species with low virulence but with a higher mortality rate than C. albicans, now accounts for approximately 15 to 20% of all Candida infections in the United States and is the most common non-C. albicans species isolated (9, 23, 32, 33, 34).

The increased use of prophylactic fluconazole in high-risk patients has played a major role in decreasing the incidence of C. albicans infections without altering the incidence of infections caused by non-C. albicans species, such as C. glabrata and Candida krusei (1, 27). Recent surveys of in vitro sensitivities have shown that 10 to 15% of bloodstream isolates of C. glabrata are resistant to fluconazole (>32 µg/ml). This could be due to the development of fluconazole resistance by these non-C. albicans species (6, 35) as well as the innate resistance to fluconazole that has been observed in a high percentage of C. glabata isolates in surveillance studies (16, 37). In addition, itraconazole sensitivities for up to 50% of C. glabrata isolates fall either in the sensitive dose-dependent (0.25 to 0.50 µg/ml) or resistant (>1.0 µg/ml) categories (34, 36). Despite the fluconazole and itraconazole resistance of some non-C. albicans species, intravenous (i.v.) and oral fluconazole or itraconazole are still widely used as therapy for disseminated Candida infections (4, 7).

Current guidelines for the treatment of hematogenous fungemia in neutropenic patients now recommend the use of amphotericin B (AmB) (0.7 to 1.0 mg/kg of body weight/day) or lipid formulated AmB (3.0 to 6.0 mg/kg/day) for the treatment of infections caused by non-C. albicans species (32). While lipid formulations of AmB have not been investigated in clinical trials specifically for their efficacy against non-C. albicans infections, they do have favorable response rates as first- or second-line therapy against disseminated candidiasis in general (10, 17, 40).

The echinocandins, a different class of drugs, are now also being used to treat non-C. albicans infections. These drugs target the cell wall by inhibiting beta-(1,3)-glucan synthesis, unlike the azoles or the polyenes, which target the ergosterol in the cell membrane. The echinocandins caspofungin (Cancidas) and micafungin (Mycamine) have shown good in vitro and in vivo activity against non-C. albicans infections, including Candida glabrata (12, 20, 28), Candida krusei (12, 24), and azole-resistant Candida albicans (2, 18). Both drugs have been approved for clinical use in the United States (Food and Drug Administration, Center for Drug Evaluation and Research [http://www.fda.gov/cder/]).

While combination drug regimens with AmB and either an echinocandin or an azole are being explored in vitro and in animal models for their efficacy against aspergillosis (11, 14, 25, 26) and cryptococcosis (5), there is limited preclinical combination efficacy data for infections with C. glabrata. Since non-C. albicans infections are becoming more prevalent, we used immunosuppressed mice infected with C. glabrata to investigate the efficacy of liposomal amphotericin B (AmBisome) in combination with the echinocandin caspofungin (Cancidas) or micafungin (Mycamine).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals. Female C57BL/6N mice (18 to 20 g) were obtained from B&K Universal (Fremont, CA) and maintained in microisolator boxes with a standard rodent diet (Lab Mouse Diet 5015; PMI Nutrition International, Brentwood, MO) and water ad libitum. All animal research procedures were approved by the Institutional Animal Care and Use Committee of California State Polytechnic University, Pomona, CA.

Test substances. A lyophilized liposomal preparation of AmB (AmBisome; Gilead Sciences, Inc., San Dimas, CA), was reconstituted with 12 ml sterile water for injection, shaken vigorously for 1 min, and filtered through a 0.5-µm filter according to the manufacturer's instructions. This resulted in a 4-mg/ml solution of AmB which was diluted in sterile 5% dextrose for i.v. injection and diluted in RPMI 1640 medium containing 0.165 M morpholinepropanesulfonic acid (RPMI-MOPS) for in vitro testing. Caspofungin (Cancidas; Merck and Co., Inc., Whitehouse Station, NJ) was rehydrated in 10.5 ml sterile water to produce a 7-mg/ml solution, which was then diluted in 0.9% sodium chloride (sterile saline) for i.v. injection and diluted in RPMI-MOPS for in vitro testing. Micafungin (Mycamine; Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) was rehydrated in 7.5 ml sterile water to give a 10-mg/ml solution that was diluted in sterile saline for i.v. injection and diluted in RPMI-MOPS for in vitro testing. Both caspofungin and micafungin were protected from light according to the manufacturers' instructions.

Fungal inocula. Beginning 3 days prior to challenge, C. glabrata (strain ATCC 90030) was subcultured daily in Sabouraud's dextrose broth. On the day of challenge, the subculture was pelleted and rinsed twice with 0.01 M phosphate-buffered saline (PBS), pH 7.2. The final pellet was resuspended in PBS, the concentration of blastospores was counted with a hemacytometer, and the blastospore suspension was adjusted with RPMI-MOPS to give 2 x 10⁴ blastospores/ml for the in vitro assays. For the in vivo experiments, the suspension was adjusted with PBS to produce 1 x 10⁸ blastospores/ml.

In vitro assay. A microtiter dilution assay (13) was used to determine the MIC of C. glabrata (strain ATCC 90030) for each of the test agents. The yeast cells were prepared as described above, and 100 µl of the suspension (i.e., 2 x 10³ blastospores) was dispensed into each well of a 96-well flat-bottom plate. A series of twofold dilutions of each drug were made in RPMI-MOPS, and 100 µl of each dilution was added to the appropriate wells. The drug concentrations were as follows: caspofungin, 0.06 to 70 µg/ml; micafungin, 0.03 to 32 µg/ml; liposomal amphotericin B, 0.04 to 20 µg/ml. Alamar blue (Serotec Ltd., Oxford, United Kingdom) (20 µl/well) was added to each well, and the plate was incubated at 35°C for 48 h. The MIC was defined as the lowest concentration of drug preventing the development of a red color.

Immunosuppression. Mice were immunosuppressed with 100 mg/kg cyclophosphamide (Sigma Chemical Co, St. Louis, MO) given intraperitoneally 3 days prior to yeast challenge. Maintenance intraperitoneal doses of 100 mg/kg cyclophosphamide were given on the day of challenge and every third day for the duration of the 21-day study. Blood cell analysis using a Serono Diagnostics Blood Cell Counter System 9000 with appropriate controls showed that the number of white blood cells of cyclophosphamide-treated animals (–3, 0, and +3 days) was reduced by 62% on day +7 compared to nontreated mice (1,600 cells/ml versus 4,200 cells/ml, respectively).

In vivo treatment regimens. To assess the in vivo activity of each of the test agents as monotherapy against systemic infection with C. glabrata (strain ATCC 90030) and to select the drug doses to be used in the combination regimens, C57BL/6N female mice, immunosuppressed with cyclophosphamide, were challenged intravenously via the tail vein with 1.0 x 10⁷ C. glabrata. Twenty-four hours later, daily drug treatment with one of the following agents (given i.v.) was initiated (n = 5 to 7/group) and continued for 6 days: liposomal amphotericin B (3.0, 7.5, 10, 15, or 20 mg/kg), caspofungin (1.0, 2.5, or 5.0 mg/kg), micafungin (2.5, 5.0, or 10 mg/kg), or 5% dextrose (controls). Animals were monitored for morbidity (e.g., weight loss, activity level) for 21 days and then sacrificed. Their kidneys, the target of this severe but nonlethal infection (15), were harvested, weighed, homogenized with a mechanical homogenizer with a 7-mm-diameter probe (Tissue Tearor; Biospec Products, Inc., Bartlesville, OK), and diluted with PBS. Aliquots of each dilution (200 µl) were plated on Inhibitory Mold agar (Hardy Diagnostics, Santa Maria, CA) and incubated at 35°C for 24 h to determine the number of CFU per gram of kidney. The lower limit of detection in this assay was 15 CFU/g kidney. Using the results of the monotherapy experiments, we selected the drug doses for the combination studies. In the combination experiments, the cyclophosphamide-immunosuppressed C57BL/6 female mice were challenged i.v. with 1.0 x 10⁷ C. glabrata, and daily antifungal drug treatment (n = 6 or 7/group) was begun 24 h postchallenge. When the drugs were given sequentially, 7.5 mg/kg i.v. liposomal amphotericin B was given on days 1 through 3 or days 4 through 6. Conversely, the other drugs (2.5, 5.0, or 7.5 mg/kg i.v. caspofungin or 2.5 mg/kg i.v. micafungin) were administered on days 4 through 6 or days 1 through 3. When the drugs were given on the same day (i.e., concomitant therapy) as separate injections, the regimens were as follows: 7.5 mg/kg i.v. liposomal amphotericin B and 2.5 mg/kg i.v. caspofungin daily for 6 days or 7.5 mg/kg i.v. liposomal amphotericin B and 2.5 mg/kg micafungin daily for 3 or 6 days. Animals were monitored for morbidity for 21 days, and the number of CFU per gram of kidney was determined on day 21 as described above for the monotherapy experiments.

Statistical analysis. Statistics for comparisons of CFU/g kidney were performed using GraphPad Prism, version 4.0 (GraphPad Software, Inc., San Diego, CA). A one-way analysis of variance was applied to compare the control to all groups in each experiment, and where differences occurred, a two-tailed Tukey's multiple-comparison test was done between paired groups. A P value of <0.05 was considered significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mean in vitro MICs for C. glabrata (strain ATCC 90030) from three repetitions of the experiment were 0.625 µg/ml for liposomal amphotericin B, 2.2 µg/ml for caspofungin, and 0.25 µg/ml for micafungin, with micafungin having the lowest MIC. All doses of liposomal AmB, except for the 3-mg/kg dose (i.e., 20 mg/kg, 15 mg/kg, 10 mg/kg, and 7.5 mg/kg) were significantly more effective than no drug treatment (P < 0.001, P < 0.001, P < 0.01, and P < 0.05, respectively) when used as 6-day monotherapy for treating murine systemic C. glabrata infection (Fig. 1), with the highest dose (20 mg/kg) lowering the CFU/g kidney by 100-fold. When caspofungin or micafungin was used as 6-day monotherapy, both drugs performed similarly, reducing the CFU/g kidney values by 1,000-fold compared to the values for the untreated controls (P < 0.001) (Fig. 2). There was no dose-dependent response for the echinocandins (P > 0.05) (Fig. 2), but liposomal AmB demonstrated a dose-dependent response, with 20 mg/kg being more efficacious than 10 mg/kg, 7.5 mg/kg, and 3 mg/kg (P < 0.05, P < 0.05, and P < 0.001, respectively) (Fig. 1).

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FIG. 1. Dose-response profile for liposomal amphotericin B (LAmB) in immunosuppressed mice challenged i.v. with 1.0 x 107 C. glabrata/mouse. Beginning twenty-four hours postchallenge, daily i.v. treatments with 5% dextrose (•, control) or the indicated dose of LAmB () were given for 6 days (n = 5/group). Twenty-one days postchallenge, mice were sacrificed and evaluated for fungal burden (log10 CFU/g) in the kidneys. For each group, the black bar represents the mean log10 CFU/g kidney. The values for groups of mice given 7.5, 10, 15, and 20 mg/kg LAmB were significantly different from the values for the control group (P < 0.05, P < 0.01, P < 0.001, and P < 0.001, respectively).

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FIG. 2. Dose-response profile for caspofungin (A) and micafungin (B) in immunosuppressed mice challenged i.v. with 1.0 x 107 C. glabrata/mouse. Beginning twenty-four hours postchallenge, daily i.v. treatments with 5% dextrose (•, control) or the indicated dose of caspofungin () or the indicated dose of micafungin () were given for 6 days (n = 5 or 6/group). Twenty-one days postchallenge, mice were sacrificed and evaluated for fungal burden (log10 CFU/g) in the kidneys. For each group, the black bar represents the mean log10 CFU/g kidney. The values for groups of mice given caspofungin or micafungin were significantly different from the values for the respective control groups (P < 0.001 for all treatment groups).

A sequential approach to drug combination therapy (i.e., 3 days with one drug followed by 3 days with the other drug) was investigated. As all the doses of echinocandins were efficacious with no difference in efficacy among the various doses, we selected 2.5 mg/kg echinocandin for the combination studies (Fig. 2). We used 7.5 mg/kg as the maximum dose of liposomal amphotericin B for the combination studies because the monotherapy experiments had shown that it was the lowest dose of liposomal amphotericin B that was significantly more effective than no drug treatment (controls) (Fig. 1). To determine whether a lower dose of liposomal amphotericin B would be effective when combined with an echinocandin, mice were treated with 2.5 mg/kg caspofungin on days 1 through 3 and then given 2.5, 5.0, or 7.5 mg/kg liposomal AmB on days 4 through 6. Sequential treatment with 2.5 mg/kg caspofungin and then liposomal AmB at 2.5 mg/kg, 5.0 mg/kg, or 7.5 mg/kg was significantly more effective than 3 days of monotherapy with 2.5 mg/kg, 5.0 mg/kg, or 7.5 mg/kg liposomal AmB (P < 0.001 for all comparisons) (Fig. 3). Similarly, the combination of 2.5 mg/kg caspofungin with 2.5 mg/kg, 5.0 mg/kg, or 7.5 mg/kg liposomal AmB was also significantly more effective than monotherapy with caspofungin (P < 0.001 for all comparisons). All of the combination regimens of liposomal AmB and caspofungin, as well as monotherapy with 2.5 mg/kg caspofungin, were significantly more effective than no drug treatment (control group) (P < 0.001 for all comparisons). Repetition of this experiment produced the same results, with clearance of the infection in all mice given 2.5 mg/kg caspofungin and then 7.5 mg/kg liposomal AmB (data not shown). Thus, these doses were selected for all subsequent combination studies.

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FIG. 3. Three-day (3d) monotherapy with liposomal amphotericin B (LAmB) () (2.5 mg/kg, 5.0 mg/kg, and 7.5 mg/kg, given i.v.) or caspofungin (Cas) (, 2.5 mg/kg, i.v.) compared to sequential therapies (, 2.5 mg/kg Cas for 3 days followed by 2.5 mg/kg LAmB for 3 days; , 2.5 mg/kg Cas for 3 days followed by 5.0 mg/kg LAmB for 3 days; , 2.5 mg/kg Cas for 3 days followed by 7.5 mg/kg LAmB) in immunosuppressed mice challenged i.v. with 1.0 x 107 C. glabrata/mouse. Twenty-four hours postchallenge, daily treatments were initiated (n = 5/group). Controls (•) received daily i.v. treatments with 5% dextrose for 6 days. For each group, the black bar represents the mean log10 CFU/g kidney. The values for groups of mice given 2.5 mg/kg LAmB, 5.0 mg/kg LAmB, or 7.5 mg/kg LAmB were not significantly different from the value for the control group (P > 0.05 in all cases). The value for the group of mice given 2.5 mg/kg caspofungin was significantly different from the value for the control group (P < 0.001). The values for groups of mice given 2.5 mg/kg Cas followed by 2.5 mg/kg LAmB, 5.0 mg/kg LAmB, or 7.5 mg/kg LAmB were significantly different from the value for the control group (P < 0.001 in all cases). The value for the group of mice treated sequentially with 2.5 mg/kg Cas followed by 2.5 mg/kg LAmB was not significantly different from the value for the group given 2.5 mg/kg Cas followed by 5.0 mg/kg LAmB (P > 0.05), but these values were significantly different from the value for the group given 2.5 mg/kg Cas followed by 7.5 mg/kg LAmB (P < 0.05). The data are from one representative experiment of two replicate experiments.

To determine whether the sequence of drug administration would alter the efficacy, we varied which drug was given first. When liposomal amphotericin B was given first followed by caspofungin, six of seven mice were cured, and when caspofungin was given first followed by liposomal amphotericin B, all seven mice had no detectable fungi in their kidneys (P < 0.001 compared to the monotherapies and P < 0.001 compared to the untreated control) (Fig. 4). Although there were no cures in the mice given sequential therapy with liposomal AmB and micafungin, administration of micafungin before liposomal AmB was significantly better than no drug treatment (P < 0.05) (Fig. 5), and the mean fungal burden for this sequential treatment was lower than for either liposomal AmB monotherapy or micafungin monotherapy. However, the difference in fungal burden did not reach significance. Compared with the controls, there was no significant reduction in fungal burden in the kidneys (P > 0.05) when liposomal AmB was given prior to micafungin or when liposomal AmB or micafungin was used as 3-day monotherapy.

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FIG. 4. Three-day monotherapy with liposomal amphotericin B (LAmB) (, 7.5 mg/kg, i.v.) or caspofungin (Cas) (, 2.5 mg/kg, i.v.) compared to sequential therapies (, 7.5 mg/kg LAmB for 3 days [3d] followed by 2.5 mg/kg Cas for 3 days; , 2.5 mg/kg Cas for 3 days followed by 7.5 mg/kg LAmB for 3 days) in immunosuppressed mice challenged i.v. with 1.0 x 107 C. glabrata/mouse. Twenty-four hours postchallenge, daily treatments were initiated (n = 7/group). Controls (•) received daily i.v. treatments with 5% dextrose for 6 days. For each group, the black bar represents the mean log10 CFU/g kidney. The values for groups of mice given 7.5 mg/kg LAmB or 2.5 mg/kg Cas were significantly different from the value for the control group (P < 0.05 and P < 0.001, respectively); the values for groups of mice given both sequential treatments with LAmB and Cas were significantly different from the value for the control group (P < 0.001).

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FIG. 5. Three-day monotherapy with liposomal amphotericin B (LAmB) (, 7.5 mg/kg, i.v.) or micafungin (Mc) (, 2.5 mg/kg, i.v.) compared to sequential therapies (, 7.5 mg/kg LAmB for 3 days [3d] followed by 2.5 mg/kg Mc for 3 days; , 2.5 mg/kg Mc for 3 days followed by 7.5 mg/kg LAmB for 3 days) in immunosuppressed mice challenged i.v. with 1.0 x 107 C. glabrata/mouse. Twenty-four hours postchallenge, daily treatments were initiated (n = 7/group). Controls (•) received daily i.v. treatments with 5% dextrose for 6 days. For each group, the black bar represents the mean log10 CFU/g kidney. The values for groups of mice given 7.5 mg/kg LAmB or 2.5 mg/kg Mc as monotherapy were not significantly different from the value for the control group (P > 0.05). The value for the group of mice given sequential treatment with LAmB prior to Mc was not significantly different from the value for the control group (P > 0.05). The value for the group of mice given sequential treatment with Mc prior to LAmB was significantly different from the value for the control group (P < 0.05).

Having demonstrated that sequential therapy was effective, we tested simultaneous dosing of these drugs. Concomitant administration of 7.5 mg/kg liposomal amphotericin B with 2.5 mg/kg of either caspofungin or micafungin showed markedly improved efficacy compared to monotherapy (Fig. 6 and 7). Six days of treatment with liposomal AmB and caspofungin resulted in undetectable levels of yeast in six of seven mice with a P value of <0.001 compared to monotherapy or no drug treatment (Fig. 6). Liposomal AmB (7.5 mg/kg) and micafungin (2.5 mg/kg) administered concomitantly for 6 days produced undetectable levels of fungi in four of seven mice and was also significantly more effective than 3 days of monotherapy with liposomal AmB (P < 0.001) or micafungin (P < 0.001) or no drug treatment (controls) (P < 0.001) (Fig. 7). When the combination of liposomal AmB and micafungin was given for only 3 days, three of seven mice had no detectable fungus, and this combination was significantly better than 3 days of monotherapy with liposomal AmB (P < 0.01) or micafungin (P < 0.001).

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FIG. 6. Six-day monotherapy with liposomal amphotericin B (LAmB) (, 7.5 mg/kg, i.v.) or caspofungin (Cas) (, 2.5 mg/kg, i.v.) compared to 6 days (6d) of concomitant therapy (, 7.5 mg/kg LAmB and 2.5 mg/kg Cas, i.v.) in immunosuppressed mice challenged with 1.0 x 107 C. glabrata/mouse. Twenty-four hours postchallenge, daily treatments were initiated and continued for 6 days (n = 7/group). Controls (•) received daily i.v. 5% dextrose treatments. For each group, the black bar represents the mean log10 CFU/g kidney. The values for groups of mice given 7.5 mg/kg LAmB or 2.5 mg/kg Cas as monotherapy or concomitant therapy of LAmB and Cas were significantly different from the values for the control group (P < 0.001 in all cases). The value for the group of mice given concomitant therapy of LAmB and Cas for 6 days was significantly different from the values for the groups given either drug alone for 6 days (P < 0.001 in all cases).

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FIG. 7. Three-day monotherapy with liposomal amphotericin B (LAmB) (, 7.5 mg/kg, i.v.) or micafungin (Mc) (, 2.5 mg/kg, i.v.) compared to 3-day (x3d) () day or 6-day (x6d) () concomitant therapy with LAmB and Mc (7.5 mg/kg, i.v., and 2.5 mg/kg, i.v., respectively) in immunosuppressed mice challenged with 1.0 x 107 C. glabrata/mouse. All treatments were initiated twenty-four hours postchallenge and continued daily for the indicated time periods (n = 7/group). Controls (•) received daily i.v. treatments with 5% dextrose. For each group, the black bar represents the mean log10 CFU/g kidney. The values for groups of mice given 3-day monotherapy with 7.5 mg/kg LAmB or 2.5 mg/kg Mc were not significantly different from the value for the control group (P > 0.05). The values for groups of mice given 3 or 6 days of concomitant therapy of LAmB and Mc were significantly different from the value for the control group (P < 0.001).

Top Abstract Introduction Materials and Methods Results Discussion References

 
With the increased importance of treating non-C. albicans yeast infections, including those caused by C. glabrata, there has been more focus on the use of antifungal agents, such as the lipid AmB formulations and the echinocandins, which are active against non-C. albicans yeast species (21, 22, 30). As shown in our murine model of C. glabrata infection, 6 days of monotherapy with either the polyene liposomal amphotericin B or an echinocandin (caspofungin or micafungin) was significantly effective in reducing the fungal burden in infected kidneys, although none of these monotherapies were able to clear the infection from the kidneys. In comparison, by combining liposomal amphotericin B with caspofungin or micafungin as concomitant therapy for 6 days, we observed no detectable fungi in the kidneys of many of the treated animals. For the sequential therapies in which each drug was given for a total of only 3 days, there was more efficacy for the sequential treatments with caspofungin and liposomal amphotericin B than for those with micafungin and liposomal amphotericin B. This was probably due to the higher efficacy of 3 days of monotherapy with caspofungin, which reduced the CFU/g kidney by 1,000-fold compared to the controls. With 3 days of liposomal amphotericin B or micafungin monotherapy, there was an approximately 10-fold reduction in the mean CFU/g kidney for either therapy compared to the controls.

Liposomal amphotericin B is the least toxic of the commercial amphotericin B formulations (39, 41, 42), and repeated treatments with doses up to 20 mg/kg have been reported to be minimally toxic in experimental animals (3, 8, 29). For the purposes of the present study, however, we selected a dose of 7.5 mg/kg liposomal amphotericin B for the combination studies, as this was the lowest dose to produce significantly more efficacy compared to no drug treatment (control group). Since the monotherapy experiments with liposomal amphotericin B showed that doses of 15 or 20 mg/kg were more effective in treating C. glabrata-infected mice, it is possible that by using higher doses of liposomal amphotericin B for the combination therapies with the echinocandins (concomitant or sequential), we could further improve the efficacy.

Other studies have been done to evaluate combination therapies with an amphotericin B formulation and an echinocandin in different murine models of fungal infections. Hossain et al. (18) reported that concomitant use of amphotericin B and caspofungin significantly prolonged the survival of mice infected with Candida albicans compared to infected, untreated controls (P = 0.006) and was better than amphotericin B alone, although the increased survival compared to mice treated with amphotericin B did not reach significance (P = 0.36). The combination was also the only treatment regimen that resulted in a significant reduction of CFU in the kidneys (P = 0.05). Serena and coworkers (38) using a murine model of disseminated Trichosporon asahii found that concomitant administration of amphotericin B (1.0 mg/kg) and micafungin (5.0 mg/kg) produced a significantly higher survival rate than did no drug treatment (controls) (P < 0.0001) or monotherapy of either drug (P < 0.05).

Our data support the results of previous studies by demonstrating that liposomal amphotericin B, used concomitantly or sequentially, with micafungin or caspofungin markedly improved the therapeutic outcome for murine C. glabrata systemic infection compared to monotherapy. Additional studies are warranted to further investigate this combination regimen for treating other non-C. albicans infections.

 
This work was supported by a research grant from Gilead Sciences, Inc., Foster City, CA.

 
* Corresponding author. Mailing address: Department of Biological Sciences, California State Polytechnic University, 3801 West Temple Ave., Pomona, CA 91768. Phone: (909) 869-4047. Fax: (909) 869-4048. E-mail: jpadler{at}csupomona.edu.

Present address: RichPro Associates, 2095 Lavender Hill Ct., Lincoln, CA 95648.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Abi-Said, D., E. Anaissie, O. Uzun, I. Raad, H. Pinzcowski, and S. Vartivarian. 1997. The epidemiology of hematogenous candidiasis caused by different Candida species. Clin. Infect. Dis. 24:1122-1128.[Medline]
2
2. Abruzzo, G. K., A. M. Flattery, C. J. Gill, L. Kong, J. G. Smith, V. B. Pikounis, J. M. Balkovec, A. F. Bouffard, J. F. Dropinski, H. Rosen, H. Kropp, and K. Bartizal. 1997. Evaluation of the echinocandin antifungal MK-0991 (L-743,872): efficacies in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis. Antimicrob. Agents Chemother. 41:2333-2338.[Abstract]
3
3. Adler-Moore, J. P., J. A. Olson, and R. T. Proffitt. 2004. Alternative dosing regimens of liposomal amphotericin B (AmBisome) effective in treating murine systemic candidiasis. J. Antimicrob. Chemother. 54:1096-1102.[Abstract/Free Full Text]
4
4. Anaissie, E. J., R. O. Darouiche, D. Abi-Said, O. Uzun, J. Mera, L. O. Gentry, T. Williams, D. P. Kontoyiannis, C. L. Karl, and G. P. Bodey. 1996. Management of invasive candidal infections: results of a prospective, randomized, multicenter study of fluconazole versus amphotericin B and review of the literature. Clin. Infect. Dis. 23:964-972.[Medline]
5
5. Barchiesi, F., E. Spreghini, A. M. Schimizzi, M. Maracci, D. Giannini, F. Carle, and G. Scalise. 2004. Posaconazole and amphotericin B combination therapy against Cryptococcus neoformans infection. Antimicrob. Agents Chemother. 48:3312-3316.[Abstract/Free Full Text]
6
6. Bennett, J. E., K. Izumikawa, and K. A. Marr. 2004. Mechanism of increased fluconazole resistance in Candida glabrata during prophylaxis. Antimicrob. Agents Chemother. 48:1773-1777.[Abstract/Free Full Text]
7
7. Buchner, T., W. Fegeler, H. Bernhardt, N. Brockmeyer, K. H. Duswald, M. Herrmann, D. Heuser, U. Jehn, G. Just-Nubling, M. Karthaus, G. Maschmeyer, F. M. Muller, J. Muller, J. Ritter, N. Roos, M. Ruhnke, A. Schmalreck, R. Schwarze, G. Schwesinger, G. Silling, and Panel of Interdisciplinary Investigators. 2002. Treatment of severe Candida infections in high-risk patients in Germany: consensus formed by a panel of interdisciplinary investigators. Eur. J. Clin. Microbiol. Infect. Dis. 21:337-352.[CrossRef][Medline]
8
8. Clemons, K. V., R. A. Sobel, P. L. Williams, D. Pappagianis, and D. A. Stevens. 2002. Efficacy of intravenous liposomal amphotericin B (AmBisome) against coccidioidal meningitis in rabbits. Antimicrob. Agents Chemother. 46:2420-2426.[Abstract/Free Full Text]
9
9. Colombo, A. L., J. Perfect, M. DiNubile, K. Bartizal, M. Motyl, P. Hicks, R. Lupinacci, C. Sable, and N. Kartsonis. 2003. Global distribution and outcomes for Candida species causing invasive candidiasis: results from an international randomized double-blind study of caspofungin versus amphotericin B for the treatment of invasive candidiasis. Eur. J. Clin. Microbiol. Infect. Dis. 22:470-474.[CrossRef][Medline]
10
10. Coukell, A. J., and R. N. Brogden. 1998. Liposomal amphotericin B. Therapeutic use in the management of fungal infections and visceral leishmaniasis. Drugs 55:585-612.[CrossRef][Medline]
11
11. Cuenca-Estrella, M., A. Gomez-Lopez, G. Garcia-Effron, L. Alcazar-Fuoli, E. Mellado, M. J. Buitrago, and J. L. Rodriguez-Tudela. 2005. Combined activity in vitro of caspofungin, amphotericin B, and azole agents against itraconazole-resistant clinical isolates of Aspergillus fumigatus. Antimicrob. Agents Chemother. 49:1232-1235.[Abstract/Free Full Text]
12
12. Ernst, E. J., E. E. Roling, C. R. Petzold, D. J. Keele, and M. E. Klepser. 2002. In vitro activity of micafungin (FK-463) against Candida spp.: microdilution, time-kill, and postantifungal-effect studies. Antimicrob. Agents Chemother. 46:3846-3853.[Abstract/Free Full Text]
13
13. Espinel-Ingroff, A., J. L. Rodriguez-Tudela, and J. V. Martinez-Suarez. 1995. Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans. J. Clin. Microbiol. 33:3154-3158.[Abstract]
14
14. Graybill, J. R., R. Bocanegra, G. M. Gonzalez, and L. K. Najvar. 2003. Combination antifungal therapy of murine aspergillosis: liposomal amphotericin B and micafungin. J. Antimicrob. Chemother. 52:656-662.[Abstract/Free Full Text]
15
15. Graybill, J. R., R. Bocanegra, M. Luther, A. Fothergill, and M. J. Rinaldi. 1997. Treatment of murine Candida krusei or Candida glabrata infection with L-743,872. Antimicrob. Agents Chemother. 41:1937-1939.[Abstract]
16
16. Hajjeh, R. A., A. N. Sofair, L. H. Harrison, G. M. Lyon, B. A. Arthington-Skaggs, S. A. Mirza, M. Phelan, J. Morgan, W. Lee-Yang, M. A. Ciblak, L. E. Benjamin, L. T. Sanza, S. Huie, S. F. Yeo, M. E. Brandt, and D. W. Warnock. 2004. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J. Clin. Microbiol. 42:1519-1527.[Abstract/Free Full Text]
17
17. Herbrecht, R., V. Letscher, E. Andres, and A. Cavalier. 1999. Safety and efficacy of amphotericin B colloidal dispersion. An overview. Chemotherapy 45(Suppl. 1):67-76.
18
18. Hossain, M. A., G. H. Reyes, L. A. Long, P. K. Mukherjee, and M. A. Ghannoum. 2003. Efficacy of caspofungin combined with amphotericin B against azole-resistant Candida albicans. J. Antimicrob. Chemother. 51:1427-1429.[Abstract/Free Full Text]
19
19. Jarvis, W. R. 1995. Epidemiology of nosocomial fungal infections, with emphasis on Candida species. Clin. Infect. Dis. 20:1526-1530.[Medline]
20
20. Ju, J. Y., C. Polhamus, K. A. Marr, S. M. Holland, and J. E. Bennett. 2002. Efficacies of fluconazole, caspofungin, and amphotericin B in Candida glabrata-infected p47^phox–/– knockout mice. Antimicrob. Agents Chemother. 46:1240-1245.[Abstract/Free Full Text]
21
21. Karyotakis, N. C., and E. J. Anaissie. 1994. Efficacy of escalating doses of liposomal amphotericin B (AmBisome) against hematogenous Candida lusitaniae and Candida krusei infection in mice. Antimicrob. Agents Chemother. 38:2660-2662.[Abstract/Free Full Text]
22
22. Karyotakis, N. C., E. J. Anaissie, R. Hachem, M. C. Dignani, and G. Samonis. 1993. Comparison of the efficacy of polyenes and triazoles against hematogenous Candida krusei infection in neutropenic mice. J. Infect. Dis. 168:1311-1313.[Medline]
23
23. Kauffman, C. A., J. A. Vazquez, J. D. Sobel, H. A. Gallis, D. S. McKinsey, A. W. Karchmer, A. M. Sugar, and The National Institute for Allergy and Infectious Diseases (NIAID) Mycoses Study Group. 2000. Prospective multicenter surveillance study of funguria in hospitalized patients. Clin. Infect. Dis. 30:14-18.[CrossRef][Medline]
24
24. Krishnarao, T. V., and J. N. Galgiani. 1997. Comparison of the in vitro activities of the echinocandin LY303366, the pneumocandin MK-0991, and fluconazole against Candida species and Cryptococcus neoformans. Antimicrob. Agents Chemother. 41:1957-1960.[Abstract]
25
25. Lewis, R. E., R. A. Prince, J. Chi, and D. P. Kontoyiannis. 2002. Itraconazole preexposure attenuates the efficacy of subsequent amphotericin B therapy in a murine model of acute invasive pulmonary aspergillosis. Antimicrob. Agents Chemother. 46:3208-3214.[Abstract/Free Full Text]
26
26. Luque, J. C., K. V. Clemons, and D. A. Stevens. 2003. Efficacy of micafungin alone or in combination against systemic murine aspergillosis. Antimicrob. Agents Chemother. 47:1452-1455.[Abstract/Free Full Text]
27
27. Marr, K. A., K. Seidel, T. C. White, and R. A. Bowden. 2000. Candidemia in allogeneic blood and marrow transplant recipients: evolution of risk factors after the adoption of prophylactic fluconazole. J. Infect. Dis. 181:309-316.[CrossRef][Medline]
28
28. Mikamo, H., Y. Sato, and T. Tamaya. 2000. In vitro antifungal activity of FK463, a new water-soluble echinocandin-like lipopeptide. J. Antimicrob. Chemother. 46:485-487.[Abstract/Free Full Text]
29
29. Ortoneda, M., J. Capilla, F. J. Pastor, I. Pujol, and J. Guarro. 2002. Efficacy of liposomal amphotericin B in treatment of systemic murine fusariosis. Antimicrob. Agents Chemother. 46:2273-2275.[Abstract/Free Full Text]
30
30. Ostrosky-Zeichner, L., J. H. Rex, P. G. Pappas, R. J. Hamill, R. A. Larsen, H. W. Horowitz, W. G. Powderly, N. Hyslop, C. A. Kauffman, J. Cleary, J. E. Mangino, and J. Lee. 2003. Antifungal susceptibility survey of 2,000 bloodstream Candida isolates in the United States. Antimicrob. Agents Chemother. 47:3149-3154.[Abstract/Free Full Text]
31
31. Pappas, P. G., J. H. Rex, J. Lee, R. J. Hamill, R. A. Larsen, W. Powderly, C. A. Kauffman, N. Hyslop, J. E. Mangino, S. Chapman, H. W. Horowitz, J. E. Edwards, W. E. Dismukes, for the NIAID Mycoses Study Group. 2003. A prospective observational study of candidemia: epidemiology, therapy, and influences on mortality in hospitalized adult and pediatric patients. Clin. Infect. Dis. 37:634-643.[CrossRef][Medline]
32
32. Pappas, P. G., J. H. Rex, J. D. Sobel, S. G. Filler, W. E. Dismukes, T. J. Walsh, and J. E. Edwards. 2004. Guidelines for treatment of candidiasis. Clin. Infect. Dis. 38:161-189.[CrossRef][Medline]
33
33. Pfaller, M. A., R. N. Jones, G. V. Doern, H. S. Sader, R. J. Hollis, and S. A. Messer. 1998. International surveillance of bloodstream infections due to Candida species: frequency of occurrence and antifungal susceptibilities of isolates collected in 1997 in the United States, Canada, and South America for the SENTRY Program. J. Clin. Microbiol. 36:1886-1889.[Abstract/Free Full Text]
34
34. Pfaller, M. A., R. N. Jones, G. V. Doern, H. S. Sader, S. A. Messer, A. Houston, S. Coffman, and R. J. Hollis. 2000. Bloodstream infections due to Candida species: SENTRY antimicrobial surveillance program in North America and Latin America, 1997-1998. Antimicrob. Agents Chemother. 44:747-751.[Abstract/Free Full Text]
35
35. Redding, S. W., W. R. Kirkpatrick, S. Saville, B. J. Coco, W. White, A. Fothergill, M. Rinaldi, T. Eng, T. F. Patterson, and J. Lopez-Ribot. 2003. Multiple patterns of resistance to fluconazole in Candida glabrata isolates from a patient with oropharyngeal candidiasis receiving head and neck radiation. J. Clin. Microbiol. 41:619-622.[Abstract/Free Full Text]
36
36. Safdar, A., V. Chaturvedi, E. W. Cross, S. Park, E. M. Bernard, D. Armstrong, and D. S. Perlin. 2001. Prospective study of Candida species in patients at a comprehensive cancer center. Antimicrob. Agents Chemother. 45:2129-2133.[Abstract/Free Full Text]
37
37. Safdar, A., V. Chaturvedi, B. S. Koll, D. H. Larone, D. S. Perlin, and D. Armstrong. 2002. Prospective, multicenter surveillance study of Candida glabrata: fluconazole and itraconazole susceptibility profiles in bloodstream, invasive, and colonizing strains and differences between isolates from three urban teaching hospitals in New York City (Candida Susceptibility Trends Study, 1998 to 1999). Antimicrob. Agents Chemother. 46:3268-3272.[Abstract/Free Full Text]
38
38. Serena, C., F. J. Pastor, F. Gilgado, E. Mayayo, and J. Guarro. 2005. Efficacy of micafungin in combination with other drugs in a murine model of disseminated trichosporonosis. Antimicrob. Agents Chemother. 49:497-502.[Abstract/Free Full Text]
39
39. Walsh, T. J., R. W. Finberg, C. Arndt, J. Hiemenz, C. Schwartz, D. Bodensteiner, P. Pappas, N. Seibel, R. N. Greenberg, S. Dummer, M. Schuster, J. S. Holcenberg, and the National Institute of Allergy and Infectious Diseases Mycoses Study Group. 1999. Liposomal amphotericin B for empirical therapy in patients with persistent fever and neutropenia. N. Engl. J. Med. 340:764-771.[Abstract/Free Full Text]
40
40. Walsh, T. J., J. W. Hiemenz, N. L. Seibel, J. R. Perfect, G. Horwith, L. Lee, J. L. Silber, M. J. DiNubile, A. Reboli, E. Bow, J. Lister, and E. J. Anaissie. 1998. Amphotericin B lipid complex for invasive fungal infections: analysis of safety and efficacy in 556 cases. Clin. Infect. Dis. 26:1383-1396.[Medline]
41
41. White, M. H., R. A. Bowden, E. S. Sandler, M. L. Graham, G. A. Noskin, J. R. Wingard, M. Goldman, J. A. van Burik, A. McCabe, J. S. Lin, M Gurwith, and C. B. Miller. 1998. Randomized, double-blind clinical trial of amphotericin B colloidal dispersion versus amphotericin B in the empirical treatment of fever and neutropenia. Clin. Infect. Dis. 27:296-302.[Medline]
42
42. Wingard, J. R., M. H. White, E. Anaissie, J. Raffalli, J. Goodman, and A. Arrieta. 2000. A randomized, double-blind comparative trial evaluating the safety of liposomal amphotericin B versus amphotericin B lipid complex in the empirical treatment of febrile neutropenia. Clin. Infect. Dis. 31:1155-1163.[CrossRef][Medline]
43
43. Wisplinghoff, H., H. Seifert, R. P. Wenzel, and M. B. Edmond. 2003. Current trends in the epidemiology of nosocomial bloodstream infections in patients with hematological malignancies and solid neoplasms in hospitals in the United States. Clin. Infect. Dis. 36:1103-1110.[CrossRef][Medline]

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Antimicrobial Agents and Chemotherapy, December 2005, p. 4895-4902, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.4895-4902.2005

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