Antifungal efficacy of caspofungin (MK-0991) in experimental pulmonary aspergillosis in persistently neutropenic rabbits: pharmacokinetics, drug disposition, and relationship to galactomannan antigenemia

Abstract

The antifungal efficacy, pharmacokinetics, and safety of caspofungin (CAS) were investigated in the treatment and prophylaxis of invasive pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Antifungal therapy consisted of 1, 3, or 6 mg of CAS/kg of body weight/day (CAS1, CAS3, and CAS6, respectively) or 1 mg of deoxycholate amphotericin B (AMB)/kg/day intravenously for 12 days starting 24 h after endotracheal inoculation. Prophylaxis (CAS1) was initiated 4 days before endotracheal inoculation. Rabbits treated with CAS had significant improvement in survival and reduction in organism-mediated pulmonary injury (OMPI) measured by pulmonary infarct score and total lung weight (P < 0.01). However, animals treated with CAS demonstrated a paradoxical trend toward increased residual fungal burden (log CFU per gram) and increased serum galactomannan antigen index (GMI) despite improved survival. Rabbits receiving prophylactic CAS1 also showed significant improvement in survival and reduction in OMPI (P < 0.01), but there was no effect on residual fungal burden. In vitro tetrazolium salt hyphal damage assays and histologic studies demonstrated that CAS had concentration- and dose-dependent effects on hyphal structural integrity. In parallel with a decline in GMI, AMB significantly reduced the pulmonary tissue burden of A. fumigatus (P < or = 0.01). The CAS1, CAS3, and CAS6 dose regimens demonstrated dose-proportional exposure and maintained drug levels in plasma above the MIC for the entire 24-h dosing interval at doses that were > or =3 mg/kg/day. As serial galactomannan antigen levels may be used for therapeutic monitoring, one should be aware that profoundly neutropenic patients receiving echinocandins for aspergillosis might have persistent galactomannan antigenemia despite clinical improvement. CAS improved survival, reduced pulmonary injury, and caused dose-dependent hyphal damage but with no reduction in residual fungal burden or galactomannan antigenemia in persistently neutropenic rabbits with invasive pulmonary aspergillosis.

FIG. 1.

Hyphal damage to A. fumigatus by CAS versus AMB by MTT assay. In vitro MTT assays demonstrated significant (P < 0.0001 by ANOVA) concentration-dependent effects on hyphal damage. Values are given as means ± SEMs (error bars). As the SEMs were small for several time points, the error bars may not always be apparent in the hyphal damage curves.

FIG. 2.

Response of primary pulmonary aspergillosis in persistently neutropenic rabbits to antifungal therapy measured by survival, mean lung weight, mean pulmonary infarct score, and mean pulmonary tissue concentration of residual organisms (log CFU/gram) in untreated controls (n = 12) and rabbits treated with CAS (CAS1 [n = 12], CAS3 [n = 12], CAS6 [n = 12]) or AMB (1 mg/kg/day) (n = 6). Values are given as means ± SEMs (error bars). P values are indicated as follows (in comparison to untreated controls, calculated by using ANOVA with Bonferroni’s correction for multiple comparisons): *, P < 0.05; §, P < 0.01; ¶, P < 0.001. For the measure of survival, the values on the x axis are days following inoculation and the values on the y axis are probability of survival.

FIG. 3.

Pulmonary infiltrate scores determined by image analysis of serial CT scans of untreated control and CAS-treated rabbits from all groups. Animals treated with CAS demonstrated significant resolution of pulmonary infiltrates in comparison to untreated controls (P < 0.0001 by ANOVA). Pulmonary infiltrates increased during the first 7 days in untreated control and treated rabbits. The pulmonary infiltrate curve ends on day 7 due to mortality in untreated control rabbits. Pulmonary infiltrates declined following day 7 in the CAS treatment cohort.

FIG. 4.

Expression of galactomannan antigenemia in persistently neutropenic rabbits with pulmonary aspergillosis in the treatment (A) and prophylaxis (B) groups.

FIG. 5.

Effects of CAS and AMB in vivo on hyphal structures and microbiological clearance of Aspergillus fumigatus in experimental pulmonary aspergillosis. All specimens were stained with Grocott-Gomori’s methenamine-silver. Panels A to D demonstrate dose-dependent fragmentation, reduction in length, and increasing swelling and vacuolization of hyphal elements in a representative section of lung tissue from each dosage group. Panel E is a high magnification of the chlamydospore-like structure formed in CAS-treated hyphae. Panels: A, control; B, CAS1; C, CAS3; D and E, CAS6; F, AMB at a dose of 1 mg/kg/day. Magnification for panel E, ×800. Magnification for all other panels, ×320.

FIG. 6.

Response of primary pulmonary aspergillosis in persistently neutropenic rabbits to prophylaxis as measured by survival, mean lung weight, mean pulmonary infarct score, and mean pulmonary tissue concentration of residual organisms (log CFU/gram) in untreated controls (n = 10) and rabbits treated with CAS1 (n = 13). Values are given as means ± SEMs (error bars). §, P < 0.01 (Mann-Whitney U test). For the measure of survival, the values on the x axis are days following inoculation and the values on the y axis are probability of survival.

FIG. 7.

Concentration-time plots after multiple daily doses CAS1, CAS3, and CAS6 over 5 days. Each point plots the mean concentration ± SEM for 12 rabbits at each time point.