Efficacy of Caspofungin against Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans

Abstract

The echinocandin caspofungin is a potent inhibitor of the activity of 1,3-β-d-glucan synthase from Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans. In murine models of disseminated infection, caspofungin prolonged survival and reduced the kidney fungal burden. Caspofungin was at least as effective as amphotericin B against these filamentous fungi in vivo.

The incidence of life-threatening Aspergillus infections has risen in recent years (21, 23, 26). Although Aspergillus fumigatus accounts for the majority of cases of human aspergillosis, the number of infections caused by other Aspergillus species has increased (4, 28). The emerging threat posed by these species is especially important to understand because of their inherent reduced susceptibilities to many antifungal agents (8, 13, 30).

Caspofungin (CAS) was first approved for the treatment of invasive aspergillosis in patients refractory to or intolerant of other therapies. CAS derives its antifungal activity by inhibiting the synthesis of 1,3-β-d-glucan, an essential cell wall polymer. The FKS gene encodes an integral membrane protein that is part of the 1,3-β-d-glucan synthase (GS) complex (10), and the FKS gene family is highly conserved among a number of clinically relevant fungal pathogens (9). CAS inhibits the growth of A. fumigatus in vitro and has significant efficacy against A. fumigatus in animal models of pulmonary (27), disseminated (5), and central nervous system (17) disease, as well as clinical efficacy (19, 22).

CAS is active in vitro against several Aspergillus species (3, 29). CAS has been demonstrated to have activity against A. terreus in animal models (4, 12), and case reports have described the efficacy of CAS in patients infected with A. flavus (15) or A. terreus (7). Here we describe efforts to better characterize the activity of CAS against A. flavus, A. terreus, and A. nidulans.

(This work was presented in part at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 15 September 2003.)

Clinical isolates of A. flavus (CLF9089), A. terreus (CLF9062), and A. nidulans (CLF14) were used in these studies. Spores were harvested from cultures incubated for 5 to 7 days at 35°C on Sabouraud dextrose agar (SDA). Prior to infection, the viability of the spore suspensions was determined by spreading dilutions onto SDA plates and enumerating the CFU. CAS (Merck & Co., Inc. Rahway, NJ) was solubilized and serially diluted in sterile distilled water. Amphotericin B (AMB; Fungizone; Bristol-Myers Squibb, Princeton, NJ) was reconstituted according to the manufacturer's instructions and serially diluted in sterile distilled water.

Purified GS was prepared from each isolate by using product entrapment (18, 20), and the 50% inhibitory concentration of CAS was determined. Whole-cell susceptibility was measured by performing broth microdilution assays according to method M38-A of the Clinical and Laboratory Standards Institute (6).

Female DBA/2 mice (weight, 18 to 22 g; Taconic or Jackson Laboratories) were used for in vivo studies with A. flavus and A. terreus; female CD-1 mice (weight, 23 to 27 g; Charles River) were used for studies with A. nidulans. For survival studies, disseminated Aspergillus infections were induced by injecting 0.2 ml of a spore suspension containing 1.8 × 10⁵ A. flavus, 3.7 × 10⁶ A. terreus, or 8.6 × 10⁴ A. nidulans CFU into the lateral tail vein. The compounds were administered intraperitoneally (i.p.) once daily for 7 days, beginning 15 to 30 min after infection (10 mice per group). Survival was monitored daily for 28 days. For the studies with A. nidulans, CD-1 mice were rendered chronically pancytopenic as described previously (1).

To assess the effect of therapy on the fungal burden, mice were infected by intravenous injection of 0.2 ml of a spore suspension containing 2.8 × 10⁵ A. flavus, 4.1 × 10⁶ A. terreus, or 9.0 × 10⁴ A. nidulans CFU. A. nidulans-infected CD-1 mice were rendered pancytopenic as described above for the survival studies. CAS, AMB, or vehicle was administered i.p. once daily for 7 days, beginning 15 to 30 min after challenge (n = 10). Animals were euthanized 24 h after the last dose, and the kidney fungal burden was determined by quantitative PCR (qPCR) (5). Sense and antisense primers and a dual-labeled hybridization probe designed for the 18S rRNA gene of A. fumigatus (5) were used for A. flavus and A. terreus. For A. nidulans, the same primers but a different hybridization probe (5′-FAM-AGCCAGCGGCCCGCGGACG-TAMRA-3′, where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine) were used. qPCR values were normalized for genomic DNA recovery and are expressed as conidial equivalents (CE) per gram kidney (14, 16). Statistical significance relative to vehicle-treated animals (P ≤ 0.05) was determined by the log rank test (survival) or Student's t test (kidney burden).

The predicted protein sequence of the full-length A. fumigatus Fks was aligned with Fksp orthologues from A. flavus, A. terreus, and A. nidulans. The alignment suggests strong Fksp sequence conservation (Table 1). Specific amino acids that play a role in the echinocandin susceptibilities of Candida albicans and Saccharomyces cerevisiae (25) are conserved in these Fks proteins (data not shown). The high degree of homology is consistent with the susceptibility of the partially purified GS enzyme activity to inhibition by CAS (Table 1). CAS also demonstrates whole-cell activity against these isolates, with MIC₈₀ values that are comparable to those seen for CAS-susceptible A. fumigatus isolates. The susceptibility to AMB was commensurate with the values reported previously for these species (11).

TABLE 1.

Whole-cell and GS enzyme susceptibilities to CAS and conservation of the FKS target gene for A. flavus, A. terreus, and A. nidulans

Species and strain	MIC (μg/ml)		% Identity to AfFKS1c	GS IC50 (ng/ml)e
	CASa	AMBb
A. fumigatus MF5668	<0.03	0.25		0.03
A. flavus CLF9089	0.125	1	83	0.2
A. terreus CLF9062	0.06	2	88	0.04
A. nidulans CLF14	0.125	0.25	90d	0.21

^aMIC₈₀.

^bMIC₁₀₀.

^cThe full-length AfFks1p protein sequence (GenBank accession no. U79728) was used to query GenBank and the A. flavus (http://www.aspergillusflavus.org/) and A. terreus (http://www.broad.mit.edu/annotation/fungi/aspergillus_terreus/) genome databases. Percent identity to exon 2 of AfFKS1 (amino acids 47 to 1805) was determined on the basis of a comparison to Fksp from A. flavus (1,853 amino acids), A. terreus (1,711 amino acids), or A. nidulans (1,757 amino acids) by using the tBLASTN algorithm (2).

^dAnFKS1, GenBank accession no. U51272 (20).

^eIC₅₀, the concentration of CAS that inhibited partially purified 1,3-β-d-glucan synthesis activity by 50%.

CAS prolonged survival in mice infected with A. flavus, A. terreus, or A. nidulans in a dose-dependent manner (Fig. 1). CAS was effective (P ≤ 0.05) in the A. flavus mouse model at doses of 0.25, 0.5, and 1 mg per kilogram of body weight, while AMB provided no protection. In A. terreus-infected mice, CAS was efficacious (P ≤ 0.05) at doses greater than 0.06 mg/kg (Fig. 1B). AMB was ineffective at 1 mg/kg but did provide a modest survival benefit (≥60%) at doses from 0.125 to 0.5 mg/kg (data not shown). In A. nidulans-infected mice, CAS at 1.0 and 0.5 mg/kg provided a moderate to significant improvement in survival (P = 0.11 and 0.03, respectively) (Fig. 1C), while AMB was inactive.

FIG. 1.

Survival of mice infected with A. flavus, A. terreus, or A. nidulans. Therapy (1, 0.5, 0.25, 0.125, or 0.06 mg/kg CAS or 1 mg/kg AMB administered i.p. once daily) was initiated immediately after infection and was continued for 7 days. Survival was monitored for 28 days, and Kaplan-Meier plots were generated. A. nidulans-infected mice remained immunosuppressed throughout the study. There were 10 mice in each group. *, P ≤ 0.05; mpk, milligram per kilogram.

The kidney fungal burden was significantly reduced by treatment with CAS at several dose levels (Fig. 2). CAS reduced the tissue burden in A. flavus-infected mice compared to that in vehicle-treated mice, with a peak reduction of ca. 3.5 log₁₀ CE/g kidney (Fig. 2A). A reduction of the A. flavus burden was also seen in animals given AMB, with a peak reduction of ca. 3 log₁₀ CE/g kidney when AMB was administered at 1 mg/kg (Fig. 2A). The A. terreus kidney burden was reduced by CAS, with a dose-dependent trend (Fig. 2B), but the peak burden reduction was only ca. 1 log₁₀. As in the survival study, some AMB doses were efficacious in A. terreus-infected mice (Fig. 2B); however, significant mortality and large standard errors were observed in these AMB-treated animals. In A. nidulans-infected mice, all doses of CAS that we tested reduced the burden ca. 0.8 to 1 log₁₀ CE/g kidney, while there was no apparent titration with AMB (Fig. 2C).

FIG. 2.

Reduction of kidney burden in mice infected with A. flavus, A. terreus, or A. nidulans and treated with CAS or AMB. Infected mice were treated with vehicle or drug (CAS or AMB; 1, 0.5, 0.25, 0.125, 0.06, or 0.03 mg/kg per day administered i.p. once daily), and the kidney fungal burden at 7 days postinfection was determined by qPCR. The mean burdens (log₁₀ conidial equivalents/g kidney ± standard error) for vehicle-treated animals were as follows: A. flavus, 6.70 ± 0.15; A. terreus, 6.72 ± 0.17; A. nidulans, 5.68 ± 0.19. For each drug-treated group, the reduction in the mean burden (± standard error) relative to that in mice that received vehicle treatment is given. Values were derived from 10 mice per therapy group except when specified by numbers within or just above or below the bars. The y-axis scale for the A. flavus plot is different from those for A. terreus and A. nidulans. *, P ≤ 0.05; mpk, milligram per kilogram.

Recently, Graybill et al. (12) reported that CAS prolonged survival in A. terreus-infected neutropenic mice at doses of 0.5, 5, and 10 mg/kg and reduced the spleen CFU at 10 and 15 mg/kg. Barchiesi et al. (4) observed that CAS therapy provided protection in neutropenic mice infected with either of two A. terreus strains, with a significant fungal burden reduction in the kidneys achieved with 1 and 5 mg/kg CAS and a significant fungal burden reduction in the brain achieved with 5 mg/kg CAS. Of note, the peak kidney CFU reduction was comparable to the kidney CE reduction (∼1 log₁₀) shown here. The higher signal in vehicle-treated mice from our study is consistent with the increased sensitivity of the qPCR assay (5, 24, 31).

Here we report the results of both in vitro and in vivo studies which support the potential utility of CAS as treatment for infections with A. flavus, A. terreus, or A. nidulans. FKS, the likely molecular target of the echinocandins, is conserved in these organisms. The activity of the GS enzyme prepared from each isolate is sensitive to inhibition by CAS, consistent with growth inhibition in liquid MIC assays. Finally, CAS is at least as effective as AMB in murine models of disseminated infection, measured both by survival prolongation and by a reduction in fungal burden. Perhaps most importantly, reports of efficacy in patients (7, 22) suggest a clinical role for CAS against these Aspergillus species.