Abstract
A-192411.29 is a novel antifungal agent derived from the structural template of the natural product echinocandin. The in vitro activity of A-192411.29 against common pathogenic yeasts was assessed by National Committee for Clinical Laboratory Standards method M27-A. It demonstrated broad-spectrum, fungicidal activity and was active against the most clinically relevant yeasts, such as Candida albicans, Candida tropicalis, and Candida glabrata, as well as less commonly encountered Candida species; in general, its potency on a weight basis was comparable to that of amphotericin B. It maintained potent in vitro activity against Candida strains with reduced susceptibilities to fluconazole and amphotericin B. The in vitro activity of A-192411.29 against Cryptococcus neoformans was comparable to its activity against Candida spp. However, A-192411.29 did not demonstrate complete growth inhibition of Aspergillus fumigatus by the broth microdilution method used. A-192411.29 possesses an antifungal profile comparable to or better than those of fluconazole and amphotericin B against pathogenic yeasts, including strains resistant to fluconazole or amphotericin B, suggesting that it may be a therapeutically useful new antifungal drug.
Serious fungal infections are increasingly recognized as important causes of morbidity and mortality, especially among debilitated patients. Immunosuppression due to malignancy, immunosuppressive and cytotoxic therapies, human immunodeficiency virus infection, and age as well as procedures which cause breaks in skin and mucosal barriers and broad-spectrum antibacterial treatment place patients at risk for fungal infections. In a recent survey of nosocomial bloodstream isolates, Candida species, predominately Candida albicans, were the fourth most prevalent group of pathogens and were isolated from 8.0% of all patients with nosocomial bloodstream infections (7). Candida spp. are also identified as critical pathogens in infections of wounds and sterile body fluids (10). In human immunodeficiency virus-infected patients, mucosal infections such as oropharyngitis, esophagitis, and vaginitis caused by Candida spp. and meningeal, lung, and blood infections caused by Cryptococcus neoformans are very common (3, 12). Aspergillosis and other opportunistic mycoses are also significant infections in immunocompromised patients, although they are less common than infections with yeasts (2, 3).
Options for treatment of serious fungal infections are primarily azole-class compounds and amphotericin B. However, the widespread and prolonged use of azole antifungal agents, especially fluconazole, has increased the prevalence of azole-resistant strains in patients with mucosal candidiasis and fungemia (7, 10, 12, 14). The reduced susceptibility of yeasts to amphotericin B is also identified as a clinical problem for the treatment of fungemia (13, 15). Moreover, current treatment for opportunistic mold infections is limited to amphotericin B and itraconazole (19). Thus, new antifungal agents are needed.
A-192411.29 is a novel antifungal agent derived by total synthesis from the structural template of the natural product echinocandin (Fig. 1) (4; Q. Li, unpublished data). A-192411.29 inhibited C. albicans β-1,3-glucan synthesis by targeting the fungal 1,3-β-d-glucan synthase complex and disrupting the fungal cell wall (D. Frost, unpublished data), a mechanism of action shared by other members of the lipopeptide class of antifungal agents such as MK-0991 (capsofungin) (4), LY-303366 (J. Tang, T. R. Parr, Jr., W. Turner, M. Debono, L. Lagrandeur, F. Burkhardt, M. Rodriguez, M. Zweifel, J. Nissen, and K. Clingerman, Program Abstr. 33rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. 367, p. 186, 1993), and FK-463 (17). A-192411.29 demonstrated in vitro activity equivalent to that of amphotericin B in early studies and effectively treated systemic candidiasis in a mouse infection model (J. Meulbroek, unpublished data). In this report, we describe the antifungal spectrum and potency of A-192411.29.
FIG. 1.
Structure of A-192411.29.
MATERIALS AND METHODS
Strains.
Fungal stains were from the Abbott Laboratories clinical isolate collection or were purchased from the American Type Culture Collection (Manassas, Va.). Quality control strains Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, and C. albicans ATCC 90028 were included in each test as recommended by the National Committee for Clinical Laboratory Standards (NCCLS).
Antifungal agents.
A-192411.29 and fluconazole were synthesized at Abbott Laboratories. Amphotericin B was purchased from Bristol-Meyers Squibb (Wallingford, Conn.). A-192411.29 was dissolved in either dimethyl sulfoxide or distilled H2O, amphotericin B was dissolved in dimethyl sulfoxide, and fluconazole was dissolved in distilled H2O.
Susceptibility tests.
Susceptibility determinations for yeasts were made by the broth microdilution method as described in NCCLS document M27-A (8). Yeasts used as inocula for the tests were grown overnight on Sabouraud dextrose agar (Becton Dickinson Microbiology Systems, Cockeysville, Md.) at 35°C; C. neoformans, however, was grown for 48 h. Tests were performed in RPMI 1640 (Gibco BRL, Gaithersburg, Md.) buffered to pH 7.0 with 0.165 mol of morpholinepropanesulfonic acid (MOPS; Sigma Chemical Co., St. Louis, Mo.) per liter. A-192411.29 and amphotericin B MICs were determined to be the lowest drug concentration that inhibited visible fungal growth. Fluconazole MICs were determined to be the lowest drug concentration that inhibited fungal growth by at least 80% compared with the growth in drug-free medium. For determination of medium effects, buffered RPMI 1640 supplemented with 20 g of glucose per liter, yeast nitrogen broth (pH 7.0; Difco Laboratories, Detroit, Mich.) supplemented with 5 g of glucose per liter, antibiotic medium 3 (Becton Dickinson Microbiology Systems), and Sabouraud dextrose broth (Difco Laboratories) were substituted for the buffered RPMI 1640 recommended by NCCLS in document M27-A (6). For determination of inoculum effects, NCCLS document M27-A (6) was followed, except that strains were suspended to a turbidity equivalent to that of a 0.5 McFarland standard in 0.9% (wt/vol) NaCl and were further diluted in 0.9% NaCl to achieve the desired inoculum levels. Inoculum densities were verified by determining the number of viable colonies per milliliter on the Sabouraud dextrose agar after serial dilutions in 0.9% NaCl.
For C. albicans, fluconazole MICs were determined by the Etest method on RPMI 1640 buffered to pH 7.0 with 0.165 mol of MOPS per liter and supplemented with 20 g of glucose per liter and 15 g of Bacto Agar (Difco Laboratories) per liter by following the manufacturer's directions (Antifungal susceptibility testing of yeasts, technical guide 4, AB Biodisk, Solna, Sweden, 1997; Media for antifungal susceptibility testing of yeasts and moulds, Etest customer information sheet no. 5, AB Biodisk, Solna, Sweden). Fluconazole Etest strips were purchased from AB Biodisk (Piscataway, N.J.). The tests were incubated at 35°C for 24 to 48 h. MICs were determined at the intersection of the ellipse that demarcates significantly reduced growth by use of the printed scale (11; Antifungal susceptibility testing of yeasts, technical guide 4, AB Biodisk, Solna, Sweden).
The susceptibilities of the A. fumigatus isolates were determined by the broth microdilution method. Cultures were grown on Sabouraud dextrose agar at 35°C until sporulation occurred, typically 72 h. Stock spore suspensions were harvested with yeast nitrogen broth (pH 7.0) supplemented with 5 g of glucose per liter and 25% (vol/vol) glycerol and were stored at 4°C until use. The numbers of CFU per milliliter were determined by plating serial dilutions of the stock suspension on Sabouraud dextrose agar. Before inoculation for the susceptibility tests, the spore suspensions were diluted to achieve 2 × 104 to 2 × 105 CFU/ml in yeast nitrogen broth plus 0.5% glucose (pH 7.0) and were incubated for 24 h at 35°C to germinate the spores. Serial twofold dilutions of antibiotics were made in yeast nitrogen broth plus 0.5% glucose in 100-μl volumes and were inoculated with 100 μl of the germinated spore suspensions. Incubation was for 72 h at 35°C. MICs were determined as the lowest concentration that inhibited visible fungal growth.
Time-kill analysis.
C. albicans CCH442 was grown overnight at 35°C on Sabouraud dextrose agar. Isolated colonies were selected and suspended in 0.9% NaCl to a turbidity equivalent to that of a 0.5 McFarland standard. Flasks that contained RPMI 1640 buffered with 0.165 mol of MOPS per liter to pH 7.0 plus test antibiotics at four times the MIC or no antibiotic (growth control) were prepared. The flasks were inoculated with the yeast suspension to a final concentration of approximately 105 CFU/ml. The cultures were incubated at 35°C with shaking for up to 24 h. At the indicated times, aliquots were removed and the numbers of viable colonies per milliliter were determined on Sabouraud dextrose agar after serial dilutions in 0.9% NaCl.
RESULTS AND DISCUSSION
In vitro activity of A-192411.29.
The in vitro activity of A-192411.29 was assessed against 124 strains of yeast (Table 1). A-192411.29 was as active as amphotericin B against 41 strains of C. albicans; all strains were inhibited by ≤1 μg/ml. A-192411.29 was also as active as amphotericin B against Candida glabrata, Candida tropicalis, C. krusei, Candida lusitaniae, and Candida stellatoidea; all 64 isolates were inhibited by ≤4 μg/ml. C. parapsilosis, Candida kefyr, and Candida guillermondii were also inhibited by A-192411.29 at ≤4 μg/ml, although amphotericin B was slightly more active than A-192411.29 against these species. Although there was variation in susceptibility to A-192411.29 in the group of strains tested, with MICs ranging from 0.25 to 4 μg/ml, no highly resistant strains were detected. Therefore, A-192411.29 demonstrates broad-spectrum and potent antifungal activity against pathogenic Candida spp.
TABLE 1.
Comparative in vitro activities of A-192411.29, amphotericin B, and fluconazole against yeasts
| Species | No. of isolates | Antifungal agent | MIC (μg/ml)
|
||
|---|---|---|---|---|---|
| Range | 50% | 90% | |||
| Candida albicans | 41 | A-192411.29 | 0.25–1 | 0.5 | 1 |
| Amphotericin B | 0.5–1 | 0.5 | 1 | ||
| Fluconazolea | 0.19–>256 | 0.25 | >256 | ||
| Candida albicans | 29 | A-192411.29 | 0.25–1 | 0.5 | 0.5 |
| Fluconazole susceptible | Amphotericin B | 0.5–1 | 0.5 | 1 | |
| Fluconazolea | 0.19–3 | 0.19 | 0.38 | ||
| 12 | A-192411.29 | 0.25–1 | 0.5 | 1 | |
| Fluconazole resistant | Amphotericin B | 0.5–8 | 0.5 | 0.5 | |
| Fluconazolea | 6–>256 | 64 | >256 | ||
| Candida glabrata | 20 | A-192411.29 | 1–2 | 2 | 2 |
| Amphotericin B | 0.25–1 | 0.5 | 1 | ||
| Fluconazole | 16–>128 | 64 | >128 | ||
| Candida tropicalis | 14 | A-192411.29 | 0.25–1 | 1 | 1 |
| Amphotericin B | 0.5–1 | 0.5 | 0.5 | ||
| Fluconazole | 0.25–2 | 0.5 | 2 | ||
| Candida krusei | 21 | A-192411.29 | 1–4 | 2 | 2 |
| Amphotericin B | 1 | 1 | 1 | ||
| Fluconazole | >32 | >32 | >32 | ||
| Candida parapsilosis | 7 | A-192411.29 | 2–4 | ||
| Amphotericin B | 0.5–1 | ||||
| Fluconazole | 0.5–4 | ||||
| Candida kefyr | 7 | A-192411.29 | 1–4 | ||
| Amphotericin B | 0.5–1 | ||||
| Fluconazole | 1–2 | ||||
| Candida lusitaniae | 7 | A-192411.29 | 1–2 | ||
| Amphotericin B | 0.5–1 | ||||
| Fluconazole | 0.5–4 | ||||
| Candida guillermondii | 2 | A-192411.29 | 4 | ||
| Amphotericin B | 0.5 | ||||
| Fluconazole | 4 | ||||
| Candida stellatoidea | 2 | A-192411.29 | 0.25 | ||
| Amphotericin B | 0.5 | ||||
| Fluconazole | >32 | ||||
| Cryptococcus neoformans | 3 | A-192411.29 | 4 | ||
| Amphotericin B | 0.25 | ||||
| Fluconazole | 2–16 | ||||
Fluconazole MICs were determined by the Etest for C. albicans.
A-192411.29 was highly active against fluconazole-resistant Candida spp. A-192411.29 was as potent against fluconazole-resistant C. albicans strains as it was against fluconazole-susceptible strains. The MICs at which 90% of strains are inhibited (MIC90s) for A-192411.29, as determined by the broth microdilution method, were 0.5 and 1 μg/ml for fluconazole-resistant and -susceptible strains, respectively, with corresponding fluconazole MIC90s of >256 and 0.38 μg/ml, respectively, as determined by the Etest method. Similarly, C. glabrata and C. krusei strains that demonstrated reduced fluconazole susceptibilities were highly susceptible to A-192411.29. As determined by the broth microdilution methods, the MIC90s of A-192411.29 were 2 μg/ml for both species, while the fluconazole MIC90s were >128 μg/ml for C. glabrata and >32 μg/ml for C. krusei.
As the current NCCLS method is unable to adequately identify amphotericin B resistance, we independently determined the susceptibility of C. albicans to amphotericin B by testing in antibiotic medium 3 (15). The amphotericin B MIC for one resistant strain in our collection was 64 μg/ml in antibiotic medium 3, while the MIC range for 31 other strains was 0.12 to 0.25 μg/ml. The MIC of A-192411.29 for this amphotericin B-resistant strain of C. albicans by the NCCLS methodology was 1 μg/ml and was indistinguishable from the MICs of A-192411.29 for the amphotericin B-susceptible strains, which ranged from 0.25 to 1 μg/ml.
The in vitro anti-Candida activities of three other echinocandin-class antifungal compounds have been evaluated by the NCCLS method described in document M27-A (1, 9, 17, 18, 20). On the basis of a comparison of the results for M-0991, LY-303366, and FK-463 reported in the literature and the results for A-192411.29 presented in this report, A-192411.29 appears to be slightly less active in vitro (two-to fourfold) than M-0991 and LY-303366 (1, 9, 18, 20), while FK-463 appears to be more active than M-0991 and LY-303366 (17).
The three isolates of C. neoformans tested were susceptible to A-192411.29 at 4 μg/ml (Table 1). In contrast, MK-0991, LY-303366, and FK-463 have little activity against C. neoformans in vitro. The reported MIC90s for C. neoformans are 32 μg/ml for MK-0991 (1), >10.24 μg/ml for LY-303366 (20), and >64 μg/ml for FK-463 (17). MK-0991, LY303366, and FK-463 are possibly poorer inhibitors of the synthesis of the glucans present in the C. neoformans cell wall such as α-(1,3)-d-glucan, α-(1-6)-glucan, or β-(1,6)-glucan, while they are effective inhibitors of the synthesis of β-(1,3)-d-glucan found in the cell walls of Candida spp. (1, 4, 17, 20). The similar in vitro activity of A-192411.29 against C. neoformans and Candida spp. suggests that the compound may effectively inhibit the synthesis of C. neoformans cell wall glucans.
A-192411.29 was fungicidal against clinical isolate C. albicans CCH442 by time-kill analysis (Fig. 2). The MICs obtained with the larger inoculum required to detect fungicidal activity were 0.12 μg/ml for A-192411.29 and 0.25 μg/ml for amphotericin B. When the two compounds were tested at four times their respective MICs, A-192411.29 caused a 99.2 to 99.8% loss of yeast viability after 4 h of incubation, while amphotericin B caused a >99.9% loss of viability after 1 h of incubation. Fungicidal activity is an advantage of fungal cell wall synthesis inhibitors of the lipopeptide class like A-192411.29, MK-0991, LY-303366, and FK-463 (1, 17, 19, 20). Cell lysis and subsequent cell death result from an inability of the weakened cell wall to maintain protoplast integrity under the stress between high internal and low external osmotic pressures (4).
FIG. 2.
Time-kill analysis of A-192411.29 and amphotericin B at antibiotic concentrations equal to four times the MIC for C. albicans CCH 442. ■, drug-free control; ▴, A-192411.29, 0.5 μg/ml; ●, amphotericin B, 1 μg/ml.
A-192411.29 did not have detectable activity against the seven strains of A. fumigatus tested by use of pregerminated spores in yeast nitrogen broth supplemented with glucose. The MICs, defined as complete inhibition of mycelial growth, for the seven strains were all >100 μg/ml, while the MICs of amphotericin B ranged from 0.39 to 0.78 μg/ml. Substantially inhibited growth at sub-MICs of A-192411.29 was not detected visually for any strain tested by this method. Significant growth inhibition is reported for MK-0991 (1, 5, 8), LY-303366 (8, 20), and FK-463 (17) at sub-MICs by other test methods and is suggested to be therapeutically relevant on the basis of data obtained with animal models. Our method for susceptibility testing might result in the lack of detectable in vitro activity for A-192411.29 against A. fumigatus. Alternatively, A-192411.29 might be inherently less active against A. fumigatus than MK-0991, LY-303366, and FK-463. Concurrent testing of the three lipopeptides by the same in vitro method, demonstration of similar microscopic changes in mycelial morphology as a result of treatment with the three compounds (5), or evidence of the efficacy of A-192411.29 in an animal model of invasive aspergillosis is needed to resolve this point.
Effect of test method on in vitro activity of A-192411.29.
The test method can affect the determination of antifungal activity (15, 16). NCCLS recommends the use of RPMI 1640 buffered to pH 7.0 with MOPS as the test medium, and this may be supplemented with 20 g of glucose per liter (6). We compared the in vitro activity of A-192411.29 against seven strains of Candida in RPMI 1640 supplemented with glucose, yeast nitrogen broth supplemented with glucose, antibiotic medium 3, and Sabouraud dextrose broth with its activity in RPMI 1640 (Table 2). The addition of glucose to RPMI 1640 did not alter the activity of A-192411.29. The MICs of A-192411.29 were approximately fourfold higher in yeast nitrogen broth than in RPMI 1640. In contrast, the MICs of A-192411.29 were about eightfold lower in antibiotic medium 3 and Sabouraud dextrose broth than in RPMI 1640. Similar results are reported for the lipopeptide LY-303366, which is also more active in antibiotic medium 3 than in RPMI 1640 (9). Amphotericin B was used as a control, and our results (data not shown) are similar to those reported previously (15); namely, amphotericin B was most active in antibiotic medium 3 and Sabouraud dextrose broth and the amphotericin B-resistant strain was clearly distinguished from the amphotericin B-susceptible strains in antibiotic medium 3 and Sabouraud dextrose broth but not in RPMI 1640-based medium.
TABLE 2.
Effect of different test media on in vitro activity of A-192411.29
| Strain | MIC (μg/ml)
|
||||
|---|---|---|---|---|---|
| RPMI 1640 | RPMI 1640 plus 2% glucose | YNBa plus 0.5% glucose | Antibiotic medium 3 | Sabouraud dextrose broth | |
| C. albicans CCH442 | 0.12 | 0.12 | 0.5 | ≤0.015 | 0.03 |
| C. albicans CAF2 | 0.12 | 0.12 | 0.5 | ≤0.015 | ≤0.015 |
| C. albicans ATCC 38247 | 0.5 | 0.25 | 1 | 0.06 | ≤0.015 |
| C. albicans 5765 | 0.12 | NDb | 0.5 | 0.06 | 0.03 |
| C. albicans ATCC 90028 | 0.12 | 0.25 | 0.5 | 0.03 | 0.03 |
| C. parapsilosis ATCC 22019 | 2 | 4 | 8 | 8 | 0.12 |
| C. krusei ATCC 6258 | 1 | 1 | 1 | 0.12 | ≤0.015 |
YNB, yeast nitrogen broth.
ND, not determined.
Overall, the density of the inoculum did not significantly affect the in vitro activity of A-192411.29 against two species of Candida. The MICs of A-192411.29 for two strains of C. albicans ranged from 0.06 to 0.25 μg/ml, with no relationship between the MIC and the inoculum density for inocula of 103, 104, and 105 CFU/ml. For a single strain of C. krusei, the MICs were 0.5 μg/ml for all three inoculum densities. For C. parapsilosis, there was a trend to higher MICs with an increased inoculum density; the MICs were 1, 2, and 4 μg/ml for inocula of 103, 104, and 105 CFU/ml, respectively, for a single strain. The activity of amphotericin B in our study was not affected by the inoculum density for any strain.
New antifungal agents are needed because of the importance of fungal infections in compromised patients, the limitations of currently available antifungal agents regarding their spectra of activity and toxicities, and the increasing prevalence of pathogens resistant to the current antifungal agents. A-192411.29 is a novel lipopeptide which acts by inhibiting cell wall synthesis in fungi. We have demonstrated that it has broad-spectrum, fungicidal activity and is active against the most clinically relevant yeasts, such as C. albicans, C. tropicalis, and C. glabrata, as well as less commonly encountered Candida species. It was also highly active against Candida strains resistant to fluconazole and amphotericin B. In contrast to other lipopeptide antifungal compounds like MK-0991 (capsofungin) and LY-303366 (1, 20) and FK-463 (17), A-192411.29 was active in vitro against C. neoformans. However, unlike the other echinocandins in clinical development, A-192411.29 did not demonstrate activity against A. fumigatus in vitro, although this may be due to differences in the susceptibility test methods used. Since A-192411.29 has been effective as treatment for systemic and renal infections caused by C. albicans in animal models (J. Meulbroek, unpublished data), it may be a therapeutically useful new antifungal agent.
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