In Vitro Activity of a New Oral Glucan Synthase Inhibitor (MK-3118) Tested against Aspergillus spp. by CLSI and EUCAST Broth Microdilution Methods

TEXT

Mold-active azoles (itraconazole, posaconazole, and voriconazole) are the primary class of antifungal agents used for the prevention and treatment of invasive aspergillosis (IA) (1). Echinocandins may play a role as alternatives to the azoles (1, 2); however, the lack of an oral formulation limits the role of these glucan synthase (GS) inhibitors for the prevention or treatment of IA. Echinocandin resistance among clinical isolates of Aspergillus is considered uncommon; however, both increased resistance and breakthrough infections have been reported and may occur in at-risk patient groups with or without long-term exposure to these agents (3–10). In particular, azole resistance in Aspergillus spp. may be associated with a high probability of treatment failure (11), and a recent report from the Netherlands found that the case fatality rate of patients with azole-resistant IA was 88% (9). These observations have prompted a call for an expanded search for new antifungal agents with novel mechanisms of action, as well as an expanded role for antifungal susceptibility testing of Aspergillus spp. (5, 9–17).

MK-3118 is an orally active, semisynthetic derivative of the natural product enfumafungin with in vitro and in vivo activity against Aspergillus spp. (18–22). MK-3118 and other derivatives of enfumafungin are potent inhibitors of fungal GS, yet these compounds are structurally distinct from the echinocandins (20–22). The sites of mutations in fks that are associated with resistance to the echinocandins are distinctly different from those causing decreased susceptibility to the enfumafungin derivatives; likewise, echinocandin-resistant isolates remain susceptible to these agents (21).

In this study, we used a collection of Aspergillus isolates selected to contain both wild-type (WT) as well as azole-resistant strains to examine the activity of MK-3118 as determined by both Clinical and Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) methods (23, 24).

A total of 71 isolates of Aspergillus spp. obtained from centers participating in the 2008 to 2010 ARTEMIS and SENTRY Antimicrobial Surveillance Programs (25, 26) were evaluated. The collection included 23 isolates of Aspergillus flavus species complex (SC), 21 isolates of Aspergillus fumigatus SC, 18 isolates of Aspergillus terreus SC, and 9 isolates of Aspergillus niger SC. Phenotypically resistant isolates (as determined by CLSI methods) (23) included eight itraconazole-resistant isolates (MIC, ≥4 μg/ml), including A. fumigatus SC (6 isolates), A. niger SC (1 isolate), and A. terreus SC (1 isolate). The isolates were obtained from a variety of sources, including sputum samples, bronchoscopy specimens, and tissue biopsy specimens, and represented individual infection episodes. Isolates were identified by standard microscopic morphology (27) and DNA sequencing of 28S and β-tubulin genes, as previously described (28). Before testing, each isolate was subcultured at least twice on potato dextrose agar (Remel, Lenexa, KS) to ensure viability and purity.

All isolates were tested against MK-3118, amphotericin B, and caspofungin using both CLSI and EUCAST broth microdilution method (BMD) methods (23, 24). The MIC values for amphotericin B were determined visually as the lowest concentration of drug that caused complete inhibition of growth (first clear well) relative to that of the growth control. The minimum effective concentration (MEC) values for MK-3118 and caspofungin were determined as described previously (19, 26) and by the CLSI (23). Quality control was ensured by testing the following strains recommended by CLSI and EUCAST (29): Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, and Aspergillus flavus ATCC 204304 (23, 29).

Discrepancies of more than two log₂ dilution steps among MEC results were used to calculate the essential agreement (EA) between the two methods. High off-scale MEC results were converted to the next highest concentration, and low off-scale results were left unchanged.

Although clinical breakpoints for antifungal agents and Aspergillus spp. have not been officially designated by either CLSI or EUCAST, we have used published criteria to classify the strains used in this study as either resistant to itraconazole (MIC, ≥4 μg/ml) (10, 17) or as WT versus non-WT to amphotericin B (MIC, ≤2/>2 μg/ml for A. fumigatus, A. flavus, and A. niger; MIC, ≤4/>4 μg/ml for A. terreus) (30) and caspofungin (MEC, ≤0.5/>0.5 for A. fumigatus, and ≤0.25/>0.25 for A. flavus, A. niger, and A. terreus) (31).

Table 1 z summarizes the in vitro susceptibilities of tested Aspergillus spp. to MK-3118 as determined by the CLSI and EUCAST BMD methods. The MEC endpoint was chosen for MK-3118 due to the fact that, similar to the echinocandins, MK-3118 inhibits GS and hyphal extension of Aspergillus spp., resulting in aberrant hyphal growth (short, stubby, highly branched hyphae) but not complete growth inhibition. Preliminary data (19) found that as with the echinocandins, when a MIC for MK-3118 is determined (complete growth inhibition), an endpoint was not achieved for Aspergillus spp.

All Aspergillus spp. were inhibited by ≤0.25 μg/ml as determined by the EUCAST BMD method, and 69/71 (97.2%) were inhibited at this MEC value as determined by the CLSI BMD method (23, 24). The EA between the two reference methods was 94.3% for MK-3118 across all four Aspergillus spp. (range, 85.7 to 100.0%) (Table 1). The MEC values generated by the EUCAST method tended to be slightly lower than those obtained by the CLSI method for most species. These results demonstrate a high level of concordance (same modal MIC, 0.06 μg/ml) between the MEC results produced by both methods when testing Aspergillus spp.

The activity of MK-3118 against this collection of Aspergillus spp. was less than that of caspofungin (MEC₉₀,0.12 to 0.25 μg/ml versus 0.03 to 0.06 μg/ml, respectively [Table 2]), but all of these strains exhibited WT MIC/MEC results for amphotericin B (30) and caspofungin (31).

MK-3118 and caspofungin were both quite active against the itraconazole-resistant isolates (MIC, ≥4 μg/ml) in the collection (Table 3). The MEC results for caspofungin ranged from 0.015 to 0.06 μg/ml, and those for MK-3118 ranged from 0.03 to 0.5 μg/ml. Whereas cross-resistance may be observed among the mold-active azoles (5, 9, 14, 17), the distinctively different mechanisms of action represented by MK-3118, amphotericin B, and caspofungin result in activity of these antifungal agents against this subset of Aspergillus species isolates that are of great concern worldwide (5, 9, 14, 15).

The notable observations to be taken from this study include the excellent in vitro potency of MK-3118 tested against contemporary isolates of Aspergillus spp., including triazole-resistant strains, and the high level of agreement between the CLSI and EUCAST methods for testing MK-3118 against Aspergillus spp. Preliminary data obtained using the CLSI BMD method (23) have demonstrated excellent MK-3118 spectrum and potency against both yeasts and molds, including Aspergillus spp. (19). With regard to Aspergillus spp., those studies are limited by the small numbers of isolates tested, the lack of antifungal comparators, and the lack of a comparison of the MK-3118 potency as determined by the reference BMD methods of the CLSI and EUCAST (23, 24). Although we have shown previously that both methods provide concordant results when testing Aspergillus spp. against itraconazole, posaconazole, voriconazole, anidulafungin, and caspofungin (32, 33), similar data have not been available for MK-3118. Given the important role that both methods currently play in antifungal resistance surveillance and regulatory evaluations of new agents, it is important to demonstrate the comparability of their results in the preclinical development of this new antifungal agent.

In conclusion, MK-3118 is a potent, novel antifungal agent with impressive activity against both WT and antifungal-resistant strains of Aspergillus species. Echinocandin-resistant Aspergillus strains have been rarely observed in the clinical setting and/or reported in the literature and were not tested during this study (34–36). The oral bioavailability of this compound coupled with mechanistic studies that suggest a lack of cross-resistance with the echinocandins suggest that it may provide a valuable benefit for the treatment and prophylaxis of invasive fungal infections (21) and can be tested with confidence by the two most used reference BMD methods.