ABSTRACT
The antifungal activity of BMS-207147 (also known as ER-30346) was compared to those of itraconazole and fluconazole against 250 strains of fungi representing 44 fungal species. MICs were determined by using the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth macrodilution method for yeasts, which was modified for filamentous fungi. BMS-207147 was about two- to fourfold more potent than itraconazole and about 40-fold more active than fluconazole against yeasts. With the NCCLS-recommended resistant MIC breakpoints of ≥1 μg/ml for itraconazole and of ≥64 μg/ml for fluconazole against Candida spp., itraconazole and fluconazole were inactive against strains of Candida kruseiand Candida tropicalis. In contrast, all but 9 (allC. tropicalis) of the 116 Candida strains tested had BMS-207147 MICs of <1 μg/ml. The three triazoles were active against about half of the Candida glabrata strains and against all of the Cryptococcus neoformans strains tested. The three triazoles were fungistatic to most yeast species, except for BMS-207147 and itraconazole, which were fungicidal to cryptococci. BMS-207147 and itraconazole were inhibitory to most aspergilli, and against half of the isolates, the activity was cidal. BMS-207147 and itraconazole were active, though not cidal, against most hyaline Hyphomycetes (with the exception ofFusarium spp. and Pseudallescheria boydii), dermatophytes, and the dematiaceous fungi and inactive againstSporothrix schenckii and zygomycetes. Fluconazole, on the other hand, was inactive against most filamentous fungi with the exception of dermatophytes other than Microsporum gypseum. Thus, the spectrum and potency of BMS-207147 indicate that it should be a candidate for clinical development.
In the past two decades, the number of immunocompromised patients has increased significantly. Immunocompromised patients include patients with neoplasm on chemotherapy, organ transplant recipients on immunosuppressive therapy, and patients infected with human immunodeficiency virus (HIV). More than 95% of HIV-infected individuals suffer from oropharyngeal candidiasis (OPC) (7). Since its introduction, fluconazole (FLU) has been used extensively for the treatment of OPC. ThoughCandida albicans remains the most prevalent fungal pathogen causing human disease, other Candida spp. (such as C. krusei, C. tropicalis, and C. glabrata) have increased in frequency as causative agents of candidiasis. The increased isolation of C. krusei in patients on FLU therapy is likely due to its intrinsic resistance to FLU.
The only other triazole marketed for systemic fungal infections is itraconazole (ITR). Unlike FLU, the antifungal spectrum of ITR includes some strains of C. krusei, C. glabrata,Aspergillus spp., and other filamentous fungi. While both triazoles are generally fungistatic to yeasts, ITR is fungicidal to many strains of aspergilli. Nonetheless, aspergillosis infections treated with ITR fail in 20 to 40% of cases (1, 8).
The widespread use of triazoles in systemic fungal infections is due to their broad spectrum and improved safety profile compared to those of other marketed antifungal drugs. In this study, we report on the in vitro antifungal and fungicidal activities of the new triazole BMS-207147 (BMS). This triazole, also known as ER-30346, has been evaluated previously by Eisai Co. on 90 to 100 strains of yeasts and aspergilli using either SAAM-F (synthetic amino acid medium, fungal agar), SDA (Sabouraud dextrose agar) or the microbroth method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (4, 5). In the current study, 250 fungal strains representing 44 species were tested for their sensitivities to BMS, ITR, FLU, and amphotericin B (AMB) by the NCCLS-approved macrobroth susceptibility testing method for yeasts and modified for filamentous fungi.
MATERIALS AND METHODS
Test organisms.A total of 250 strains from 44 fungal species were used in this study. Almost all (more than 95%) of the strains were clinical isolates; the rest were obtained from the American Type Culture Collection (Rockville, Md.).
Antifungal susceptibility test methods.All isolates (exceptMalassezia furfur) were tested by the reference broth macrodilution method outlined by the NCCLS (6) and modified for antifungal testing of filamentous fungi (2). BMS was obtained from Eisai Co., FLU was from Pfizer, ITR was from Janssen Pharmaceutica, and AMB was from Bristol-Myers Squibb Co.
The interpretative MIC breakpoints for FLU and ITR are obtained from the NCCLS guidelines (6); these breakpoints were meant as interpretative guidelines for Candida spp. The NCCLS-recommended breakpoints for FLU are as follows: ≤8 μg/ml, susceptible; 16 to 32 μg/ml, susceptible-dose dependent (S-DD); and ≥64 μg/ml, resistant. For ITR, the NCCLS-recommended MIC breakpoints as follows: ≤0.13 μg/ml, susceptible; 0.25 to 0.5 μg/ml, S-DD; and ≥1 μg/ml, resistant. At this point, no interpretative MIC breakpoints for BMS have been established. For the purpose of discussion of the MIC results in this report, we will use the ITR interpretative breakpoints for BMS, given that both compounds achieve the same peak levels in plasma in dogs (4). As for AMB, no interpretative MIC breakpoints have been recommended by the NCCLS, though Candida isolates with AMB MICs of >1 μg/ml appear resistant in animal models (8). Thus, AMB resistance will be defined in this study as AMB MICs of ≥2 μg/ml when the NCCLS RPMI 1640 method is used.
Broth macrodilution for yeasts was performed according to the guidelines of the NCCLS (6) and modified for filamentous fungi by the method of Espinel-Ingroff and Kerkering (2). The agar dilution method used for Malassezia furfur was described previously (3).
The MIC endpoints by the broth macrodilution method were determined as recommended by the NCCLS (6). AMB MICs were defined as the lowest drug concentrations which inhibited all visible growth (i.e., 100% inhibition). FLU, ITR, and BMS MICs were defined as the lowest drug concentrations which inhibited 80% of the growth in the growth control tube (as determined by comparison with a 1:5 dilution of the growth control), except with Malassezia furfur, where 100% growth inhibition was the endpoint.
MFCs.Minimum fungicidal concentrations (MFCs) were determined by subculturing 0.1 ml from each tube with no visible growth in the MIC broth macrodilution series onto drug-free SDA plates, as previously described (3). Colony counts were determined, and the MFCs were defined in accordance with the level of decrease in the number of CFU per milliliter, i.e., MFC99 means a 99% reduction in the number of CFU of the final inoculum size per milliliter, MFC95 means a 95% reduction, and MFC90 means a 90% reduction.
RESULTS
Comparative in vitro antifungal spectra of BMS, ITR, and FLU.Of the 116 Candida strains tested, all but 9 strains ofC. tropicalis were susceptible to BMS at MICs of ≤0.5 μg/ml (Table 1). Likewise, eight strains of C. tropicalis and four strains of C. krusei were resistant to ITR (MICs of ≥1 μg/ml) compared to eight C. tropicalis and six C. krusei strains being resistant to FLU (MICs of ≥64 μg/ml). Based on MIC90s, only C. tropicalis strains were considered resistant to BMS, whereas C. tropicalis andC. krusei were resistant to ITR and FLU. Nonetheless, whereas the MIC90s of BMS to C. albicans andCandida parapsilosis were 0.03 to 0.06 μg/ml, the BMS MIC90 for C. krusei was 10-fold higher (at 0.5 μg/ml). This suggests that while BMS was active against C. krusei, it was intrinsically less active against this species.C. krusei is considered intrinsically resistant to FLU. All of the yeast strains tested were susceptible to AMB.
Comparative in vitro activities of triazoles and AMB
Another yeast species often resistant to FLU is C. glabrata. Of the 16 C. glabrata strains tested, 9 (or 56%) have FLU MICs of <64 μg/ml (Table 1). Compared to other yeast species, BMS and ITR were also less active against C. glabrata; 44 and 38% of the C. glabrata strains tested had BMS and ITR MICs of <1 μg/ml, respectively.
All 32 C. neoformans strains tested were susceptible to the three azoles, as were the two Trichosporon beigelii strains (Tables 1 and 2). While one of the twoRhodotorula strains was susceptible to BMS, they were both less susceptible to ITR and FLU than to BMS (Table 2).
MICs of triazoles and AMB for species represented by five or fewer clinical isolates each
Sixteen Aspergillus strains were tested. All but one strain each of Aspergillus niger and Aspergillus fumigatus had MICs of <1 μg/ml to BMS and ITR, respectively (Table 1). In contrast, all 16 strains of Aspergillus were resistant to FLU. BMS and ITR were active against other hyalineHyphomycetes (Acremonium strictum,Paecilomyces variotii, and Penicillium sp.) but inactive (MICs >16 μg/ml) against Fusarium spp. and 4 of the 6 Pseudallescheria boydii strains (Tables 1 and 2). FLU MICs were ≥64 μg/ml for Fusarium spp., one of the fourP. variotii strains, and one of the six P. boydiistrains tested. AMB MICs that were ≥2 μg/ml were observed with one strain of Fusarium spp. and in five of the six strains ofP. boydii.
The 25 dermatophytes were highly susceptible (MICs of ≤0.13 μg/ml) to BMS and ITR. The four dermatophytic strains with FLU MICs of ≥64 μg/ml belonged to the species Microsporum gypseum.
ITR MICs were ≤0.13 μg/ml against all of the dematiaceous fungi tested (Table 2). BMS, on the other hand, was less active than ITR against Alternaria and Curvularia spp., with MICs of 1 to 2 μg/ml. FLU MICs of ≥64 μg/ml were observed with strains of Alternaria spp., Exophiala jeanselmei,Fonsecaea pedrosoi, and Phialophora verrucosa. Cladosporium carrionii, F. pedrosoi, and P. verrucosa strains had AMB MICs of ≥2 μg/ml.
For the most part, BMS and ITR MICs of ≥1 μg/ml were inactive against Sporothrix schenckii and the zygomycetes (Mucor spp., an Absidia strain, and aRhizopus strain) (Table 2). AMB MICs to S. schenckii and the zygomycetes were ≤1 μg/ml.
Fungicidal activities of BMS, ITR, FLU, and AMB.For the five strains of Candida spp. and one C. glabratastrain tested, AMB MFC99s were no more than twofold greater than the MICs (Table 3). The three triazoles were not fungicidal to Candida spp. One strain ofCandida lusitaniae had a FLU MFC99 of 1 μg/ml.
Fungicidal activities of triazoles and AMB for yeast strains
FLU was not fungicidal to Cryptococcus neoformans (Table 3). Even though the MFC99 and MIC95s of BMS and ITR were much higher than the MICs of these drugs, the MFC95values were often <1 μg/ml. Thus, it appears that ITR and BMS were often fungicidal to cryptococci at achievable levels of drug in serum.
BMS and ITR were often fungicidal to aspergilli (Table4). The MFC99s, MFC95s, and MFC90s were usually the same, though these values differed by 4- to 32-fold in four strains. The BMS MFC90s were <1 μg/ml for 7 of 14 Aspergillusstrains compared to 10 of the 14 strains with this level of fungicidal activity with ITR.
Antifungal activities of triazoles and AMB againstAspergillus spp.
The three triazoles were not fungicidal to non-Aspergillusfilamentous fungi (Table 5).
Fungicidal activities of triazoles and AMB against filamentous fungi other than Aspergillus spp.
Susceptibility testing of BMS.Since test factors can influence the MICs to azoles, we examined a number of test factors (temperature, inoculum size, pH, duration of incubation, and human serum) on three yeast strains (data not shown). The MICs of BMS, ITR, and FLU were not affected by incubation temperature (30, 35, or 37°C) or pH (3, 4, 5, 6, or 7). The MICs increased no more than fourfold with an additional 24 h of incubation. Increasing the inocula from 102 to 105 CFU/ml affected BMS MIC the least (up to 2-fold increase), while up to 16- and 8-fold increases were observed in ITR MICs and FLU MICs, respectively. In the presence of 50% human serum, the MICs of the three azoles remained essentially unchanged (≤4-fold increase) against two strains of C. albicans. Interestingly, with the C. tropicalis strain, the MICs of the azoles decreased by 8- to 16-fold in the presence of human serum.
The MIC distribution of BMS and ITR are listed in Table6. Only 1 of the 24 ITR MICs withC. parapsilosis ATCC 22019 was outside the acceptable quality control range recommended by the NCCLS. The ITR MICs forC. krusei ATCC 22492 was within the NCCLS-recommended, acceptable quality control range. The recommended acceptable quality control MIC ranges for BMS are 0.015 to 0.06 μg/ml for C. parapsilosis ATCC 22019 and 0.13 to 0.5 μg/ml for C. krusei ATCC 22492.
MIC distribution of BMS and ITR strains to NCCLS-recommended quality control
DISCUSSION
BMS appears to have a broader anticandidal spectrum than ITR and FLU do. Based on their MIC90s, ITR and FLU were inactive against some strains of C. krusei and C. tropicalis, compared to BMS, which was active against all of theC. krusei strains tested but was inactive against some strains of C. tropicalis. Only 40 to 60% of the C. glabrata strains were susceptible or S-DD to the three triazoles. With the exception of the MIC90s reported by Hata et al. (4, 5) against C. tropicalis and C. glabrata, our results confirmed their findings. Hata et al. reported MIC90s in the 0.03- to 0.4-μg/ml range for the three triazoles against C. tropicalis when SAAM-F medium was used (5) but in the 12.5- to >100-μg/ml range when RPMI 1640 medium was used (4). In both studies by Hata et al. (4, 5), the BMS MIC90s to C. glabratawere 0.4 μg/ml versus the >16 μg/ml result obtained in this study.
The three triazoles were active against C. neoformans, though only BMS and ITR were fungicidal to this yeast species. In this study and the Hata et al. studies (4, 5), BMS was 2- to 4-fold more active than ITR and 40-fold more active than FLU against yeast species.
In this study, BMS and ITR were inhibitory at 1 μg/ml to all but one of the 16 strains of Aspergillus spp. Similarly, Hata et al. observed the consistent activity of BMS and ITR againstAspergillus spp. (4, 5). The antiaspergillus potencies of BMS and ITR are comparable. FLU was inactive against aspergilli. BMS and ITR were also fungicidal to 50 to 74% of theAspergillus strains tested.
The activities of BMS and ITR against other filamentous fungi are variable compared to FLU, which was inactive against most filamentous fungi. ITR and BMS were uniformly active against dermatophytes, while FLU was less active against Microsporum gypseum. Acremonium strictum, Paecilomyces variotii, andPenicillium sp. were susceptible to BMS and ITR. Though both ITR and BMS were active against most dematiaceous fungi, ITR appeared to be somewhat more active than BMS. BMS and ITR were less active against most strains of Pseudallescheria boydii,Sporothrix schenckii, and the zygomycetes, and both were generally inactive against Fusarium spp. UnlikeAspergillus spp., BMS and ITR were not fungicidal to the other filamentous fungi.
In summary, BMS is a new triazole that is two- to fourfold more potent than ITR and up to 40-fold more active than FLU against many species of fungi. Its spectrum includes some yeast strains that are resistant to FLU. BMS is like ITR in that it is fungicidal to cryptococci and many strains of aspergilli. The in vitro profile of BMS warrants its development as a therapeutic agent in humans.
FOOTNOTES
- Received 25 July 1997.
- Returned for modification 10 September 1997.
- Accepted 17 November 1997.
- Copyright © 1998 American Society for Microbiology
