Treatment of Candida glabrata infection in immunosuppressed mice by using a combination of liposomal amphotericin B with caspofungin or micafungin

Abstract

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.

FIG. 1.

Dose-response profile for liposomal amphotericin B (LAmB) in immunosuppressed mice challenged i.v. with 1.0 × 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).

FIG. 2.

Dose-response profile for caspofungin (A) and micafungin (B) in immunosuppressed mice challenged i.v. with 1.0 × 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).

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 × 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.

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 × 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).

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; Δ, 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 × 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).

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 × 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).

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) (Δ) 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 × 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).