Determination of MICs of Aminocandin for Candida spp. and Filamentous Fungi

Candida and Aspergillus spp., as well as other filamentous molds,have increasingly been reported as the causes of severe invasivefungal infections. We evaluated the new echinocandin aminocandin(AMN) for its antifungal activities against a range of fungalpathogens by determination of the MICs for the organisms. TheMICs of the comparator drugs amphotericin B, caspofungin, micafungin,and voriconazole were also determined. The MICs of AMN for 25strains each of non-Candida albicans Candida spp. (includingCandida parapsilosis, Candida krusei, Candida guilliermondii,and Candida tropicalis), Aspergillus fumigatus, Scedosporiumspp., Fusarium spp., and zygomycetes (including Absidia, Mucor,and Rhizopus spp.) were determined by using the Clinical andLaboratory Standards Institute M27-A2 and M38-A methodologiesfor yeasts and filamentous molds, respectively. The MIC rangesof AMN for all yeasts were similar (0.03 to 4.0 µg/ml),while the MIC ranges of AMN for filamentous fungi were speciesspecific. AMN demonstrated potent antifungal activity againstA. fumigatus, limited activity against Scedosporium spp., andno activity against zygomycetes or Fusarium spp. Our data showedthat AMN demonstrated potent antifungal activities against allof the yeasts and Aspergillus isolates tested, suggesting thatAMN could be an important addition to our arsenal of antifungalsfor the treatment of invasive fungal disease.

INTRODUCTION

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Introduction

Invasive candidiasis and aspergillosis remain the most commoninvasive fungal infections, with bloodstream infections withCandida spp. (yeasts) representing the fourth most common bloodstreaminfection in the United States (19). Aspergillus infectionsare becoming more frequent, resulting in significant morbidityand mortality in developing countries (15). The risk of infectionis especially high among the immunocompromised population andin nosocomial settings. Furthermore, other filamentous molds,such as Fusarium, Scedosporium, and zygomycete species, haveincreasingly been reported as the causes of severe invasivefungal infections in these patient populations (9, 18).

Recently developed therapeutic options include the new triazolevoriconazole (VOR) and a new class of antifungal agents, theechinocandins, which inhibit the synthesis of the fungal cellwall component 1,3-beta-D-glucan. Despite these advances, therate of cure of invasive mycoses is still not optimal, hoveringat about 50%. Additionally, the treatment of Candida infectionshas led to a rise in the number of intrinsically resistant speciesand the development of azole resistance in previously susceptiblespecies (1, 2, 11). Furthermore, the echinocandins have demonstratedless activity against strains of Candida parapsilosis and Candidaguilliermondii (7). The frequent failure of monotherapy forinvasive aspergillosis has led to the use of combination therapywith echinocandins and newer azoles (13); and the current therapeuticapproaches for invasive fungal infections caused by Fusarium,Scedosporium, and zygomycete species are suboptimal, resultingin exceedingly high mortality rates (10).

Thus, there is a need for new potent and safe antifungals. Aminocandin(AMN) is a new drug that belongs to the echinocandin class ofcompounds undergoing early clinical development. Establishingthe in vitro antifungal activities of AMN against non-C. albicansspp. and opportunistic filamentous molds is essential. In thisstudy, we evaluated the susceptibilities of non-C. albicansCandida species and filamentous fungi to AMN.

MATERIALS AND METHODS

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Materials and Methods

Test organisms. Test isolates were taken from the culture collection at theCenter for Medical Mycology and included Candida parapsilosis,C. guilliermondii, Candida krusei, Candida tropicalis, Aspergillusfumigatus, Fusarium, Scedosporium, and zygomycete species (Absidia,Mucor, and Rhizopus spp.). Candida isolates were identifiedby using the API 20C system (BioMerieux, Durham, NC), whilefilamentous fungi were identified by their colonial and microscopicmorphologies. The test isolates were subcultured from frozenstocks (–80°C) onto potato dextrose agar (Fisher Scientific,Hampton, NH) and incubated at 35°C for 24 h for the Candidaspp. and approximately 1 week for the filamentous fungi. Twenty-fivestrains of each were tested.

Antifungals. AMN was supplied by Indevus Pharmaceuticals, Inc. (Lexington,MA). Caspofungin (CAS), micafungin (MFN), and VOR were suppliedby Merck & Co., Inc. (Whitehouse Station, NJ), AstellasPharma US, Inc. (Beaver Falls, PA), and Pfizer, Inc. (New York,NY), respectively. Amphotericin B (AMB) was obtained from SigmaChemicals (St. Louis, MO). Antifungal stock solutions were preparedin dimethyl sulfoxide (AMB and VOR) or sterile water (AMN, CAS,MFN) and were stored at –80°C until the day of testing.Drug dilutions were prepared in accordance with the Clinicaland Laboratory Standards Institute (CLSI; formerly NCCLS) M27-A2and M38-A susceptibility standards for the susceptibility testingof the Candida spp. and the filamentous fungi, respectively(4, 5).

MICs. The MICs of AMN and the comparator agents for each isolate weredetermined according to the CLSI standards. Cell counts werestandardized by using a hemacytometer, and the suspensions wereadjusted in RPMI 1640 buffered with 3-(N-morpholino)propanesulfonicacid (Hardy Diagnostics, Santa Maria, CA) to 0.5 x 10³ to 2.5x 10³ CFU/ml and 0.4 x 10⁴ to 5 x 10⁴ CFU/ml for the Candidaspp. and the filamentous fungi, respectively. Microdilutionplates were incubated at 35°C for 24 h for Candida and zygomycetes,48 h for Aspergillus and Fusarium, and 72 h for Scedosporiumisolates.

The echinocandin MIC endpoint was defined as the lowest concentrationthat inhibited 50% of fungal growth compared to the growth ofthe growth control. VOR inhibition endpoints were 50% for theCandida spp. and 100% for the filamentous fungi, while the AMBendpoint was 100% inhibition for all strains.

RESULTS

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Results

The MIC data for the Candida spp. are summarized in Table 1.The range of MICs of AMN for all yeasts was 0.03 to 4.0 µg/ml,with each species showing a similar range. The MIC₅₀s of AMNfor C. parapsilosis, C. krusei, C. guilliermondii, and C. tropicaliswere 1.0, 0.12, 0.5, and 0.25 µg/ml, respectively, whilethe MIC₉₀s for these strains were 2.0, 0.5, 1.0, and 1.0 µg/ml,respectively.

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TABLE 1. MIC ranges, MIC₅₀s, and MIC₉₀s of AMN and comparator agents for Candida spp.

VOR demonstrated the most potent activity against the yeaststested, with an MIC range, MIC₅₀, and MIC₉₀ of $≤$ 0.016 to 32,0.03, and 0.12 µg/ml, respectively. One C. tropicalisisolate was resistant to VOR (MIC = 32 µg/ml). AMB andCAS had identical MIC₅₀s and MIC₉₀s of 0.5 and 1.0 µg/ml,respectively. The MICs of MFN for all yeasts tested were generallyhigher than those of AMN, AMB, CAS, and VOR, with an MIC range,MIC₅₀, and MIC₉₀ of 0.001 to 64, 0.5, and 8.0 µg/ml, respectively.

The MIC ranges of AMN for the filamentous fungi were speciesspecific (Table 2). The MIC range, MIC₅₀, and MIC₉₀ of AMN forA. fumigatus were 0.12 to 0.5, 0.25, and 0.5 µg/ml, respectively.The MIC range of AMN for Scedosporium was 4.0 to 8.0, whilethe MIC₅₀ and the MIC₉₀ were both equal to 8.0 µg/ml.The MIC range, MIC₅₀, and MIC₉₀ of AMN for the zygomycetes were4.0 to >16, 16, and >16 µg/ml, respectively. AMNshowed no activity against the Fusarium isolates tested (MICrange = 128 to >256 µg/ml).

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TABLE 2. MIC ranges, MIC₅₀s, and MIC₉₀s of AMN and comparator agents against filamentous fungi

MFN demonstrated the most potent activity against A. fumigatus,with an MIC range, MIC₅₀, and MIC₉₀ of 0.016 to 0.06, 0.03,and 0.06 µg/ml, respectively. VOR had an MIC range, MIC₅₀,and MIC₉₀ of 0.06 to 0.5, 0.12, and 0.25 µg/ml, respectively,for A. fumigatus, while the MIC₉₀s of AMB and CAS were 1.0 and0.5 µg/ml, respectively, for this organism. AMB and VORdemonstrated similar activities (MIC₉₀s = 4.0 µg/ml) againstthe Fusarium and Scedosporium isolates tested, while neitherCAS nor MFN showed activity against isolates of either of thesegenera. Finally, AMB demonstrated the most potent activity againstthe zygomycetes, with an MIC range, MIC₅₀, and MIC₉₀ of 0.06to 1.0, 0.25, and 0.5 µg/ml, respectively. VOR had anMIC₅₀ of 4.0 µg/ml, while neither CAS nor MFN showed activityagainst the zygomycetes tested.

DISCUSSION

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Discussion

Our data showed that AMN demonstrated potent activities againstall of the yeast isolates tested, with overall MIC₅₀s and MIC₉₀sidentical to those of AMB and CAS. These data are in agreementwith published data describing the antifungal activities ofother echinocandins, including CAS, MFN, and anidulafungin (3,8, 14). Furthermore, AMN was active against the C. krusei andC. guilliermondii isolates, which are generally known to havelower susceptibilities to fluconazole. Interestingly, the MIC₉₀of AMN for all yeasts was threefold lower than that of MFN,demonstrating that differences in the activities against non-C.albicans strains exist among members of this drug class.

Furthermore, AMN demonstrated potent activities against theA. fumigatus isolates tested, with MIC₉₀s similar to those ofAMB, VOR, and CAS. Again, the activity of AMN against A. fumigatusis similar to those of the other members of the echinocandinclass (3, 6, 8).

As with the other two echinocandins tested, AMN demonstratedno activity against the zygomycete or Fusarium strains, whichis similar to echinocandin MIC data from earlier studies (6,12, 16, 17). The mechanisms underlying the lack of activityof echinocandins against zygomycetes and Fusarium spp. are believedto be attributable to differences in their cell wall compositions,as these organisms largely contain 1,3-alpha-glucan and glycuronomannoproteinsinstead of 1,3-beta-D-glucan (8). AMN did show limited activityagainst the Scedosporium isolates (MIC₉₀ = 8 µg/ml); thisagrees with the limited in vitro activities of MFN against dematiaceousfungi, including Scedosporium, Cladosporium, Exophiala, andFonsecaea spp., reported by Nakai et al. (12).

These data suggest that AMN possesses potent activities againstnon-C. albicans Candida spp. as well as Aspergillus and couldbe an important addition to our arsenal of antifungals for thetreatment of invasive fungal disease. Further in vivo and clinicaltesting is warranted.

FOOTNOTES

* Corresponding author. Mailing address: Center for Medical Mycology, University Hospitals of Cleveland/Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106. Phone: (216) 844-8580. Fax: (216) 844-1076. E-mail: mahmoud.ghannoum{at}uhhs.com.

Published ahead of print on 4 October 2006.

REFERENCES

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