Abstract
The in vitro activity of a new water-soluble polyene, SPA-S-843, was evaluated against 116 strains of Candida, Cryptococcus, and Saccharomyces spp. and compared with that of amphotericin B. SPA-S-843 demonstrated better inhibitory activity against all of the yeasts examined and better fungicidal activity against Candida albicans, Candida glabrata, Candida krusei, and Candida tropicalis than did amphotericin B.
The incidence of systemic fungal infections has increased (1, 6, 10, 13, 19) over the last 15 years. Amphotericin B is the most commonly used drug for systemic fungal infection, but it is somewhat toxic (16). While the azoles are much less toxic than amphotericin B, therapeutic failure and the emergence of azole resistance have limited their utility (3, 7, 14). SPA-S-843 (N-dimethylaminoacetyl-partricin A 2-dimethylaminoethylamide diascorbate), a new water-soluble partricin A derivative, is currently under development by Società Prodotti Antibiotici (Milan, Italy) for antifungal potential and low toxicity.
In this study, a group of 116 yeasts comprising 48 strains of Candida albicans, 11 of Candida glabrata, 10 of Candida krusei, 4 of Candida parapsilosis, 12 of Candida tropicalis, 21 of Cryptococcus neoformans, and 10 of Saccharomyces cerevisiae were compared for susceptibilities to amphotericin B and SPA-S-843. Two organisms, C. krusei ATCC 6258 and C. parapsilosis ATCC 22019, were included as quality control strains (11, 12). The MICs of SPA-S-843 and of amphotericin B were determined by the National Committee for Clinical Laboratory Standards (NCCLS) reference method (9). The medium, antibiotic medium 3 (AM3) (5, 8, 9, 15, 18), supplemented with 2% glucose, was selected as suggested by NCCLS document M27-A because in tests against Candida spp. it may enhance detection of resistance. In selected tests with the quality control strains, two additional media were used: yeast nitrogen base (YNB), supplemented with 1% glucose and 0.15% asparagine, and RPMI 1640, supplemented with 0.03% l-glutamine; all media were buffered to pH 7.0 with 0.165 M morpholinopropanesulfonic acid. A standard inoculum was diluted to a final concentration of 0.5 × 103 cells/ml. The MIC was defined as the lowest drug concentration that completely inhibited growth after 48 h of incubation.
Minimum fungicidal concentrations (MFCs) were determined by subculturing 0.1 ml from all tubes with no visible growth after 48 h onto drug-free Sabouraud dextrose agar plates (4, 17). The MFC99 was defined as the lowest drug concentration allowing the final inoculum to grow by 1%.
To evaluate differences between effects, Student’s paired t test (two-tailed) was applied; P values of <0.05 were considered significant.
Table 1 shows the MIC ranges, the MICs at which 50% of the isolates were inhibited (MIC50s), the MIC90s, and the geometric means (GM) of the MICs, as well as the ranges and GM of the MFC99s, of SPA-S-843 and amphotericin B for the strains tested. SPA-S-843 had significantly lower MIC50s, MIC90s, and MIC ranges against S. cerevisiae, C. tropicalis, C. neoformans, and C. glabrata than did amphotericin B (P < 0.001).
TABLE 1.
In vitro activities of SPA-S-843 and amphotericin B against yeast strains
| Organism (no. of strains) | Druga | MIC (μg/ml)
|
MFC99 (μg/ml)
|
||||
|---|---|---|---|---|---|---|---|
| Range | 50% | 90% | GM | Range | GM | ||
| C. albicans (48) | SPA-S-843 | 0.009–0.15 | 0.075 | 0.15 | 0.054 | 0.009–2.4 | 0.094 |
| AmB | 0.075–0.3 | 0.15 | 0.15 | 0.124 | 0.075–0.3 | 0.181 | |
| C. glabrata (11) | SPA-S-843 | 0.019–0.075 | 0.038 | 0.075 | 0.042 | 0.15–2.4 | 0.386 |
| AmB | 0.15–0.6 | 0.3 | 0.3 | 0.264 | 0.3–0.6 | 0.529 | |
| C. krusei (10) | SPA-S-843 | 0.019–0.3 | 0.075 | 0.3 | 0.08 | 0.019–0.6 | 0.131 |
| AmB | 0.15–0.3 | 0.3 | 0.3 | 0.227 | 0.15–1.2 | 0.345 | |
| C. parapsilosis (4) | SPA-S-843 | 0.038–0.075 | 0.3–4.8 | ||||
| AmB | 0.075–0.3 | 0.3–0.6 | |||||
| C. tropicalis (12) | SPA-S-843 | 0.019–0.075 | 0.038 | 0.075 | 0.042 | 0.038–0.3 | 0.089 |
| AmB | 0.075–0.3 | 0.15 | 0.3 | 0.15 | 0.15–0.3 | 0.225 | |
| C. neoformans (21) | SPA-S-843 | 0.019–0.075 | 0.038 | 0.075 | 0.035 | 0.038–1.2 | 0.461 |
| AmB | 0.038–0.3 | 0.15 | 0.3 | 0.15 | 0.075–1.2 | 0.263 | |
| S. cerevisiae (10) | SPA-S-843 | 0.002–0.019 | 0.009 | 0.019 | 0.009 | 0.019–0.6 | 0.15 |
| AmB | 0.018–0.15 | 0.075 | 0.075 | 0.065 | 0.038–0.15 | 0.08 | |
AmB, amphotericin B.
SPA-S-843 was most fungicidal against C. krusei, C. tropicalis, and C. albicans, showing MFC99s twice as high as the MICs; the differences between the values of SPA-S-843 and amphotericin B against the first two species were significant (P ≤ 0.01). The GM of the MFC99 of SPA-S-843 against C. glabrata was about eightfold higher than the GM of the MIC but lower again than the GM of the MFC99 of amphotericin B (difference not significant). The MFC99s of SPA-S-843 against S. cerevisiae and C. neoformans were twice as high as those of amphotericin B (P of <0.05 and <0.001, respectively). Reproducibility was assessed by retesting a random sample of organisms and evaluating the results by analysis of variance; the tests were reproducible, presenting the same results or results within one dilution of the original values.
Since an important difference between SPA-S-843 and amphotericin B is the water solubility of SPA-S-843, some in vitro assays were performed by dissolving SPA-S-843 in water or in dimethyl sulfoxide. The drug retained the same activities in either case.
Table 2 summarizes the MICs obtained with the quality control strains; the results show that the MICs of amphotericin B obtained in AM3 are within the range established by the NCCLS and lower that those obtained in YNB and RPMI 1640.
TABLE 2.
Quality control strains for antifungal susceptibility testing of yeasts
| Organism | Druga | MIC (μg/ml) in:
|
||
|---|---|---|---|---|
| AM3 | YNB | RPMI 1640 | ||
| C. parapsilosis ATCC 22019 | SPA-S-843 | 0.075 | 0.6 | 0.3 |
| AmB | 0.3 | 1.2 | 0.6 | |
| C. krusei ATCC 6258 | SPA-S-843 | 0.075 | 0.6 | 0.6 |
| AmB | 0.6 | 0.6 | 1.2 | |
AmB, amphotericin B.
In AM3 and RPMI 1640, SPA-S-843 showed better activity than amphotericin B against the quality control strains. In YNB, SPA-S-843 showed better activity than amphotericin B against C. parapsilosis ATCC 22019 and the same activity against C. krusei ATCC 6258.
Activity of SPA-S-843 was influenced by the composition of the medium, while the MICs of amphotericin B did not substantially change in different media (±1 dilution). The MICs of SPA-S-843 showed great differences, up to eight times, depending on the medium used. SPA-S-843 is considerably more active in AM3. Different batches of AM3 did not influence the determinations of the MICs of SPA-S-843. Compared with other test media, RPMI 1640 may generate higher SPA-S-843 MICs against yeasts.
These in vitro data were confirmed by recently conducted in vivo tests (2) that showed that among mice intravenously infected with C. albicans and C. neoformans, the percentages of surviving animals treated with SPA-S-843 were higher than those of animals treated with amphotericin B.
Thus, SPA-S-843 is a possible alternative to amphotericin B for the treatment of systemic yeast infections.
Acknowledgments
We thank Sara Aldrovandi and Roberto Grisetti of the Società Prodotti Antibiotici Microbiology Research Laboratory for technical collaboration.
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