Comparison of In Vitro Antifungal Activities of Free and Liposome-Encapsulated Nystatin with Those of Four Amphotericin B Formulations

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1998, p. 1412 1416 Vol. 42, No. 6 0066-4804/98/$04.00 0 Copyright 1998, American Society for Microbiology Comparison of In Vitro Antifungal Activities of Free and Liposome-Encapsulated Nystatin with Those of Four Amphotericin B Formulations ELIZABETH M. JOHNSON, 1 JOSHUA O. OJWANG, 2 ADRIEN SZEKELY, 1 THOMAS L. WALLACE, 2 AND DAVID W. WARNOCK 1 * Mycology Reference Laboratory, Public Health Laboratory Service, Bristol, United Kingdom, 1 and Aronex Pharmaceuticals Inc., The Woodlands, Texas 77381-1191 2 Received 31 October 1997/Returned for modification 5 January 1998/Accepted 8 April 1998 The in vitro activity of a multilamellar liposomal formulation of nystatin (Nyotran) was compared with those of free nystatin and four pharmaceutical preparations of amphotericin B. MICs for 200 isolates of two Aspergillus spp., seven Candida spp., and Cryptococcus neoformans were determined by a broth microdilution adaptation of the method recommended by the National Committee for Clinical Laboratory Standards. Minimum lethal concentrations (MLCs) of the six antifungal preparations were also determined. Both nystatin formulations possessed fungistatic and fungicidal activities against the 10 species tested. Liposomal nystatin appeared to be as active as free nystatin, with MICs and MLCs that were similar to, or lower than, those of the latter. Neither formulation of nystatin was as active as amphotericin B deoxycholate (Fungizone) or amphotericin B lipid complex (Abelcet), but both were more effective than liposomal amphotericin B (AmBisome). Our results suggest that further evaluation of liposomal nystatin is justified. Nystatin is a polyene antibiotic derived from Streptomyces noursei (12). It is active against a broad spectrum of fungi in vitro and in vivo, including Aspergillus fumigatus, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum (4, 5, 7, 10, 26, 27). However, nystatin is not well absorbed from the gastrointestinal tract, and its parenteral administration results in dose-limiting toxicities and harmful infusion-related reactions (21, 23). For this reason, the clinical application of nystatin has largely been limited to topical use in mucosal and cutaneous forms of candidiasis (25). Like nystatin, amphotericin B is a polyene antibiotic with a broad spectrum of antifungal action. It remains the most effective agent currently available for the treatment of many systemic fungal infections, despite the fact that its clinical use is seriously limited by nephrotoxicity and other side effects (8). To overcome these problems, three new lipid-based parenteral formulations of amphotericin B have been developed (Table 1). These industrial preparations, which differ in their compositions and physicochemical properties, are less toxic than the conventional deoxycholate formulation of amphotericin B, and this has enabled higher doses to be administered to patients with systemic fungal infections (13). The success of the new formulations of amphotericin B has stimulated interest in the development of less toxic parenteral preparations of nystatin. Initial attempts to develop a liposomal formulation of this agent resulted in a preparation which was much less toxic in vivo (19), as well as being active against a range of fungi in vitro and against C. albicans in vivo (18, 19). Nyotran (Aronex Pharmaceuticals Inc.) is a new multilamellar liposomal formulation of nystatin that contains dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) in a 7:3 ratio. It has been found to be effective and well tolerated in the treatment of neutropenic animals * Corresponding author. Mailing address: Mycology Reference Laboratory, Public Health Laboratory, Kingsdown, Bristol BS2 8EL, United Kingdom. Phone: (44) 117-928-5030. Fax: (44) 117-922-6611. E-mail: D.W.Warnock@PHLSBristol.btinternet.com. with disseminated Aspergillus infection (9, 29). It is currently undergoing clinical evaluation in patients with systemic fungal infections and has been reported to be active in some neutropenic individuals for whom amphotericin B treatment failed (2). In this study, we compared the in vitro activity of liposomal nystatin with those of free nystatin and four pharmaceutical preparations of amphotericin B against 200 isolates of Aspergillus spp., Candida spp., and C. neoformans. The in vitro testing method we employed was a microdilution adaptation of the standard broth macrodilution reference method of the National Committee for Clinical Laboratory Standards (20). MATERIALS AND METHODS Test isolates. A total of 200 isolates were tested. These comprised 10 of Aspergillus flavus, 30ofA. fumigatus, 40ofC. albicans, 20ofCandida glabrata, 10 of Candida kefyr, 20ofCandida krusei, 10ofCandida lusitaniae, 20ofCandida parapsilosis, 20 of Candida tropicalis, and 20 of C. neoformans. Of the 200 isolates, 162 were recent clinical isolates submitted to the Mycology Reference Laboratory, Bristol, United Kingdom, for identification and 38 were obtained from the United Kingdom National Collection of Pathogenic Fungi, held at the Mycology Reference Laboratory. Yeast isolates were identified to the species level by the Auxacolor (Sanofi Diagnostics Pasteur, Paris, France) and API 20C (biomerieux UK Limited, Basingstoke, England) yeast identification systems and by morphology on Oxoid cornmeal agar plates (Unipath Limited, Basingstoke, England). Two reference strains, C. parapsilosis ATCC 90018 and C. krusei ATCC 6258, were included in each batch of broth microdilution tests to ensure quality control. Isolates were retrieved from storage in liquid nitrogen and subcultured twice on plates of Oxoid Sabouraud dextrose agar (supplemented with 0.5% [wt/vol] chloramphenicol) to ensure optimal growth. Prior to testing, subcultures on Sabouraud dextrose agar were incubated at 35 C for 24 h (Candida spp.) or 48 h (C. neoformans). To induce spore formation, the Aspergillus isolates were subcultured on slopes of Oxoid potato dextrose agar and incubated at 35 C for 7 days. Antifungal agents. Pharmaceutical preparations of five antifungal agents were obtained from the respective manufacturers: liposomal nystatin (Nyotran; Aronex Pharmaceuticals Inc.), amphotericin B deoxycholate (Fungizone; Bristol- Myers Squibb Pharmaceuticals Ltd., Hounslow, England), liposomal amphotericin B (AmBisome; Nexstar Pharmaceuticals Ltd., Cambridge, England), amphotericin B colloidal dispersion (ABCD) (Amphocil; Zeneca Ltd., Wilmslow, England), and amphotericin B lipid complex (ABLC) (Abelcet; Liposome Co. Ltd., London, England). Nystatin powder (analytical grade) was obtained from Sigma Chemical Co. (St. Louis, Mo.). 1412

VOL. 42, 1998 ACTIVITIES OF NYSTATIN AND AMPHOTERICIN B 1413 TABLE 1. Structural features of the different parenteral formulations of amphotericin B and nystatin a Formulation Composition Molar ratio Structure Particle size Fungizone Sodium deoxycholate-amphotericin B 4:5 Colloidal dispersion NA AmBisome HSPC-cholesterol-DSPG-amphotericin B 10:5:4:2 Unilamellar liposome 80 nm Amphocil (ABCD) Sodium cholesteryl sulfate-amphotericin B 1:1 Lipid disc 120 nm Abelcet (ABLC) DMPC-DMPG-amphotericin B 7:3:10 Lipid ribbon 2 5 m Nyotran DMPC-DMPG-nystatin 7:3:1 Multilamellar liposome 0.1 3 m a HSPC, hydrogenated soy phosphatidylcholine; DSPG, distearoylphosphatidylglycerol; NA, not applicable. The pharmaceutical preparations were reconstituted according to the manufacturers instructions. Further dilutions were made with RPMI 1640 medium (with L-glutamine and without bicarbonate) (Sigma), supplemented with glucose (2%), and buffered to ph 7.0 with 0.165 M morpholinopropanesulfonic acid (MOPS; Sigma). Nystatin was dissolved in dimethyl sulfoxide; the solution was diluted with dimethyl sulfoxide and then with RPMI 1640 medium (20). Antifungal susceptibility testing. Broth microdilution MICs were determined according to National Committee for Clinical Laboratory Standards recommendations (20). The antifungal agents were tested over a final concentration range of 0.015 to 8 g/ml. Testing was performed in 96-well round-bottom microtiter plates. Cell suspensions of Candida spp. and C. neoformans were prepared in RPMI 1640 medium and adjusted to give a final inoculum concentration of 0.5 10 3 to 2.5 10 3 cells/ml. Spore suspensions of Aspergillus spp. were prepared in RPMI 1640 medium and adjusted to a final concentration of 0.4 10 4 to 5 10 4 spores/ml. The plates were incubated at 35 C and read after 48 h. The MIC was defined as the lowest concentration at which there was complete inhibition of growth. Determination of MLC. The minimum lethal concentration (MLC) was determined after 48 h of incubation by removing 10 l of the contents from all wells showing no visible growth and spreading them onto Sabouraud dextrose agar plates. The plates were incubated at 35 C for 48 h (Aspergillus and Candida spp.) or 72 h (C. neoformans). The MLC was defined as the lowest concentration at which 95% of the inoculum was killed. RESULTS Table 2 summarizes the in vitro susceptibilities of the 200 isolates of Aspergillus spp., Candida spp., and C. neoformans to the six different formulations of nystatin and amphotericin B. The data are reported as MIC and MLC ranges and the MICs and MLCs at which 50 and 90% of the isolates were inhibited or killed. In each batch of tests, the MICs for the quality control strains were within the accepted limits. The two preparations of nystatin were more active in vitro than liposomal amphotericin B against the 10 species tested but less active than ABLC or amphotericin B deoxycholate against 9 of the species tested. In the case of A. flavus, both forms of nystatin were as active as ABLC but less active than amphotericin B deoxycholate. Liposomal nystatin was more active than free nystatin: in most cases, the MIC 50 s, MIC 90 s, MLC 50 s, and MLC 90 s were 1 to 2 doubling dilutions lower than those of free nystatin. Of the four different amphotericin B preparations tested, ABLC appeared to be the most active in vitro, and liposomal amphotericin B appeared to be the least active in vitro. In most cases, the MIC 50 s, MIC 90 s, MLC 50 s, and MLC 90 s of ABLC were identical to, or lower than, those of amphotericin B deoxycholate for 9 of the 10 species tested (the exception being A. flavus), while those of ABCD and liposomal amphotericin B were 2 to 4 doubling dilutions higher than those of amphotericin B deoxycholate. DISCUSSION Nystatin has been a useful antifungal agent since the 1950s. However, toxic side effects following parenteral administration have limited its clinical application to the topical treatment of otomycosis and mucosal and cutaneous forms of candidiasis (25). The results of this investigation confirm and extend those of earlier reports which indicated that nystatin is a broadspectrum antifungal agent which is active in vitro and in vivo against Aspergillus spp. (9, 27, 29), Candida spp. (4, 18, 19), and C. neoformans (26). Other reports have demonstrated that nystatin is effective against H. capsulatum in vitro and in vivo (5, 7) and against C. immitis in vivo (10). Nyotran is a new multilamellar liposomal formulation of nystatin in which the phospholipid component consists of DMPC and DMPG at a molar ratio of 7:3 (Table 1). The nystatin content of this formulation is 7 mol%. The phospholipid component of Nyotran is identical to that of the earlier multilamellar formulation of amphotericin B developed by Lopez- Berestein et al. (17), which contained amphotericin B at a concentration of 5 to 10 mol%. Increasing the amphotericin B content of that formulation to 25 to 50 mol% resulted in the complete loss of defined liposomal structures and their replacement with ribbon-like structures (15). This form of lipidbased amphotericin B is now marketed as ABLC (Abelcet). Our results indicate that liposomal nystatin is more active in vitro than free nystatin against Aspergillus spp., Candida spp., and C. neoformans, with MICs and MLCs that were identical to, or lower than, those of the latter. This contrasts with several reports which indicated that multilamellar liposomal formulations of amphotericin B (in which the phospholipid component consists of DMPC and DMPG in a 7:3 molar ratio) are less active in vitro than free amphotericin B (14, 24). Time-kill studies demonstrated consistent differences between the two formulations, the lower rate of killing with the liposomal amphotericin B preparation suggesting that the antibiotic must dissociate from the phospholipid carrier before producing a fungicidal effect (24). This investigation is the first to have compared the in vitro fungistatic and fungicidal activities of the four parenteral formulations of amphotericin B that are now available for clinical use. Our results confirm those of several previous investigations which found that liposomal amphotericin B is less active in vitro against C. albicans than is amphotericin B deoxycholate (22, 28). However, an earlier investigation showed that liposomal amphotericin B and free amphotericin B had comparable in vitro fungistatic and fungicidal activities against Aspergillus spp., Candida spp., Fusarium spp., and C. neoformans (1). Our results also support those of a previous report which found that the concentrations of ABCD required to inhibit the growth of Aspergillus spp., Candida spp., and C. neoformans in vitro are sometimes higher than those of amphotericin B deoxycholate (11). Legrand et al. (16) have demonstrated that differences in structure and lipid composition affect the rate at which amphotericin B is released from lipid-based preparations of this agent. After 1hofincubation in aqueous solution, the proportion of amphotericin B released from preparations containing 0.05 g/ml ranged from 20% for liposomal amphotericin B to 75% for ABLC. In other tests, Legrand et al. (16) measured potassium ion release from C. albicans cells during short-term (1 h) incubation with different amphotericin B preparations.

1414 JOHNSON ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. In vitro susceptibilities of 200 isolates to four amphotericin B and two nystatin formulations Species (no. of isolates) Antifungal agent MIC ( g/ml) MLC ( g/ml) Range MIC 50 MIC 90 Range MLC 50 MLC 90 A. flavus (10) ABLC 2 8 8 8 2 8 8 8 Amphotericin B deoxycholate 0.5 2 1 1 0.5 2 1 1 ABCD 8 8 8 8 8 8 8 8 Liposomal nystatin 2 4 4 4 4 8 4 8 Nystatin 4 8 8 8 4 8 8 8 A. fumigatus (30) ABLC 0.12 0.25 0.25 0.5 0.12 4 0.5 2 Amphotericin B deoxycholate 0.5 2 1 1 0.5 8 1 4 ABCD 4 8 8 8 4 8 8 8 Liposomal amphotericin B 8 8 8 8 8 8 8 Liposomal nystatin 1 4 2 4 2 8 4 8 Nystatin 2 8 8 8 4 8 8 8 C. albicans (40) ABLC 0.06 0.25 0.12 0.12 0.12 0.5 0.25 0.5 Amphotericin B deoxycholate 0.25 0.5 0.25 0.25 0.25 1 0.5 0.5 ABCD 1 2 1 1 1 8 2 4 Liposomal amphotericin B 2 8 4 4 8 8 8 8 Liposomal nystatin 1 2 1 1 1 2 1 2 Nystatin 1 2 2 2 1 2 2 2 C. glabrata (20) ABLC 0.12 0.5 0.25 0.25 0.25 2 0.5 2 Amphotericin B deoxycholate 0.25 0.5 0.25 0.5 0.25 0.5 0.5 0.5 ABCD 1 4 2 2 1 4 2 4 Liposomal amphotericin B 4 8 8 8 8 8 8 8 Liposomal nystatin 1 4 2 2 2 8 2 4 Nystatin 2 8 2 4 2 8 4 4 C. kefyr (10) ABLC 0.12 1 0.25 1 0.25 1 0.5 1 Amphotericin B deoxycholate 1 1 1 1 2 1 2 ABCD 4 8 8 8 8 8 8 8 Liposomal nystatin 1 4 1 4 1 4 1 4 Nystatin 2 8 4 8 4 8 4 8 C. krusei (20) ABLC 0.06 2 0.5 1 0.25 8 4 8 Amphotericin B deoxycholate 0.12 1 0.5 1 0.25 2 0.5 1 ABCD 2 8 8 8 4 8 8 8 Liposomal nystatin 1 4 2 2 2 4 4 4 Nystatin 2 8 4 8 4 8 8 8 C. lusitaniae (10) ABLC 0.06 0.25 0.12 0.25 0.12 2 0.25 1 Amphotericin B deoxycholate 0.5 2 0.5 1 0.5 2 1 2 ABCD 4 8 4 8 4 8 8 8 Liposomal amphotericin B 4 8 8 8 8 8 8 8 Liposomal nystatin 1 2 1 2 1 4 2 4 Nystatin 2 8 4 8 4 8 4 8 C. parapsilosis (20) ABLC 0.06 0.25 0.12 0.25 0.5 4 1 2 Amphotericin B deoxycholate 0.25 0.5 0.25 0.5 0.5 2 0.5 1 ABCD 1 2 2 2 2 8 4 8 Liposomal amphotericin B 2 8 8 8 8 8 8 8 Liposomal nystatin 1 2 1 2 2 8 4 8 Nystatin 2 4 2 4 4 8 4 8 C. tropicalis (20) ABLC 0.12 1 0.25 1 0.25 2 1 1 Amphotericin B deoxycholate 0.25 0.5 0.5 0.5 0.25 8 0.5 0.5 ABCD 2 8 4 8 4 8 8 8 Liposomal nystatin 1 4 2 4 2 8 2 4 Nystatin 2 4 4 4 2 8 4 8 C. neoformans (20) ABLC 0.03 0.25 0.06 0.12 0.06 0.5 0.12 0.25 Amphotericin B deoxycholate 0.12 0.5 0.25 0.5 0.25 2 0.5 0.5 ABCD 1 4 2 4 2 8 4 8 Liposomal amphotericin B 4 8 8 8 8 8 8 Liposomal nystatin 1 4 1 4 1 8 2 4 Nystatin 1 8 1 8 2 8 2 8

VOL. 42, 1998 ACTIVITIES OF NYSTATIN AND AMPHOTERICIN B 1415 Liposomal amphotericin B was about ninefold less active than free amphotericin B against the one strain tested, while ABLC was almost as active as free amphotericin B. In longer-term experiments (24 h of incubation) with the same C. albicans strain, the differences between the preparations were less marked, but liposomal amphotericin B was still twofold less active than free amphotericin B (16). It seems that differences in the rates of release of the agent from the phospholipid carrier could well account for the marked differences in fungistatic and fungicidal activities that we and others have observed among the four amphotericin B preparations. Comparison of the MICs of nystatin and amphotericin B deoxycholate for the different organisms studied in this work suggests an almost constant relationship between them. For instance, the MIC 50 s of the two agents for C. albicans, C. glabrata, and C. parapsilosis were 0.25 and 2 g/ml, while the corresponding values for C. krusei, C. lusitaniae, and C. tropicalis were 0.5 and 4 g/ml (Table 2). Likewise, the MIC 50 sof the two agents for A. flavus and A. fumigatus were 1 and 8 g/ ml. This relationship between the susceptibilities of different organisms to amphotericin B and nystatin is not unexpected, given their similar mechanisms of action. Both drugs are believed to bind to ergosterol in fungal cell membranes, causing impairment of membrane barrier function (25). Our results suggest that organisms which are resistant to one agent might be cross-resistant to the other. There are published reports of C. tropicalis strains which were resistant to both nystatin and amphotericin B (30). However, there are also reports of amphotericin B-resistant strains of C. albicans that were not cross-resistant to nystatin (3). An ergosterol-deficient, nystatin-resistant mutant of C. albicans was shown to be cross-resistant to amphotericin B, but in contrast, an ergosterol-producing, amphotericin B-resistant mutant of the same fungus was not cross-resistant to nystatin (3). These data suggest that the mechanisms of action of the two agents are different in some fungal strains. Incorporation of antimicrobial drugs into phospholipid carriers has proved most useful for agents that have dose-limiting toxicities. Two such drugs are amphotericin B and nystatin. Lipid-based parenteral formulations of amphotericin B are less toxic than the conventional preparation, and this has enabled higher doses to be used in the treatment of human fungal infections (13). Likewise, liposomal nystatin is much less toxic to animals than is nystatin (19). Initial data from healthy human subjects indicates that parenteral administration of liposomal nystatin, at doses of 2 to 5 mg/kg of body weight, is well tolerated and results in concentrations in serum of 4.8 to 24.1 mg/liter at the end of the infusion (6). Although these levels are higher than the minimal fungicidal concentrations of liposomal nystatin for most of the fungal strains tested in this work, there is a limit to how far the results of in vitro studies can be used to predict drug behavior in human infections. In conclusion, our work indicates that, although nystatin is less active in vitro than amphotericin B, it does possess useful fungistatic and fungicidal activities against Aspergillus spp., Candida spp., and C. neoformans. Whether the new liposomal formulation of nystatin will have a clinical advantage over amphotericin B cannot be predicted from this in vitro evidence, because there are a large number of other factors, such as concentrations in tissue of the agent at the site of infection, that contribute to clinical outcome. However, initial results from animal models suggest that liposomal nystatin is effective in disseminated Aspergillus infection (9, 29). Liposomal nystatin has also been reported to be active in some human patients who failed to respond to amphotericin B (2). Our findings suggest that further evaluation of liposomal nystatin is justified. ACKNOWLEDGMENT This study was partially supported by a grant from Aronex Pharmaceuticals Inc. REFERENCES 1. Anaissie, E., V. Paetznick, R. Proffitt, J. Adler-Moore, and G. P. Bodey. 1991. Comparison of the in vitro antifungal activity of free and liposome-encapsulated amphotericin B. Eur. J. Clin. Microbiol. Infect. Dis. 10:665 668. 2. Boutati, E. I., H. C. Maltezou, G. Lopez-Berestein, S. E. Vartivarian, and E. J. Anaissie. 1995. Phase I study of maximum tolerated dose of intravenous liposomal nystatin (L-NYST) for the treatment of refractory febrile neutropenia (RFN) in patients with hematological malignancies, abstr. LM22, p. 330. In Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 3. Broughton, M. C., M. Bard, and N. D. Lees. 1991. Polyene resistance in ergosterol producing strains of Candida albicans. Mycoses 34:75 83. 4. Brown, R., E. L. Hazen, and A. Mason. 1953. Effect of fungicidin (nystatin) in mice injected with lethal mixtures of aureomycin and Candida albicans. Science 117:609 610. 5. Campbell, C. C., E. T. O Dell, and G. B. Hill. 1955. Therapeutic activity of nystatin in experimental systemic mycotic infections. Antibiot. Annu. 1955: 858 862. 6. Cossum, P. A., J. Wyse, V. Simmons, T. L. Wallace, and A. Rios. 1996. Pharmacokinetics of Nyotran (liposomal nystatin) in human patients, abstr. A88, p. 17. In Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 7. Drouchet, E., J. Schwarz, and E. Bingham. 1956. Evaluation of the action of nystatin on Histoplasma capsulatum in vitro and in hamsters and mice. Antibiot. Chemother. (Washington, D.C.) 6:23 35. 8. Gallis, H. A., R. H. Drew, and W. W. Pickard. 1990. Amphotericin B: 30 years of clinical experience. Rev. Infect. Dis. 12:308 329. 9. Gonzalez, C. E., N. Giri, D. Shetty, K. Kligys, W. Love, T. Sein, R. Schaufele, C. Lyman, J. Bacher, and T. J. Walsh. 1996. Efficacy of a lipid formulation of nystatin against invasive pulmonary aspergillosis, abstr. B54, p. 31. In Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 10. Gordon, L. E., C. E. Smith, and D. S. Wedin. 1955. Nystatin (Mycostatin) therapy in experimental coccidioidomycosis. Am. Rev. Tuberc. Pulm. Dis. 72:64 70. 11. Hanson, L. H., and D. A. Stevens. 1992. Comparison of antifungal activity of amphotericin B deoxycholate suspension with that of amphotericin B cholesteryl sulfate colloidal dispersion. Antimicrob. Agents Chemother. 36:486 488. 12. Hazen, E. L., and R. Brown. 1951. Fungicidin, an antibiotic produced by a soil actinomycete. Proc. Soc. Exp. Biol. Med. 76:93 97. 13. Hiemenz, J. W., and T. J. Walsh. 1996. Lipid formulations of amphotericin B: recent progress and future directions. Clin. Infect. Dis. 22(Suppl. 2): S133 S144. 14. Hopfer, R. L., K. Mills, R. Mehta, G. Lopez-Berestein, V. Fainstein, and G. P. Bodey. 1984. In vitro antifungal activities of amphotericin B and liposome-encapsulated amphotericin B. Antimicrob. Agents Chemother. 25: 387 389. 15. Janoff, A. S., W. R. Perkins, S. L. Saletan, and C. E. Swenson. 1993. Amphotericin B lipid complex (ABLC): a molecular rationale for the attenuation of amphotericin B related toxicities. J. Liposome Res. 3:451 471. 16. Legrand, P., M. Cheron, L. Leroy, and J. Bolard. 1997. Release of amphotericin B from delivery systems and its action against fungal and mammalian cells. J. Drug Targeting 4:311 319. 17. Lopez-Berestein, G., R. Mehta, R. L. Hopfer, K. Mills, L. Kasi, K. Mehta, V. Fainstein, M. Luna, E. M. Hersh, and R. Juliano. 1983. Treatment and prophylaxis of disseminated Candida albicans infection in mice with liposomal encapsulated amphotericin B. J. Infect. Dis. 147:939 945. 18. Mehta, R. T., R. L. Hopfer, L. A. Gunner, R. L. Juliano, and G. Lopez- Berestein. 1987. Formulation, toxicity, and antifungal activity in vitro of liposome-encapsulated nystatin as therapeutic agent for systemic candidiasis. Antimicrob. Agents Chemother. 31:1897 1900. 19. Mehta, R. T., R. L. Hopfer, T. McQueen, R. L. Juliano, and G. Lopez- Berestein. 1987. Toxicity and therapeutic effects in mice of liposome-encapsulated nystatin for systemic fungal infections. Antimicrob. Agents Chemother. 31:1901 1903. 20. National Committee for Clinical Laboratory Standards. 1997. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard. Document M27-A. National Committee for Clinical Laboratory Standards, Wayne, Pa. 21. Newcomer, V. D., E. T. Wright, T. H. Sternberg, J. H. Graham, R. H. Wier,

1416 JOHNSON ET AL. ANTIMICROB. AGENTS CHEMOTHER. and R. O. Egeberg. 1955. Evaluation of nystatin in the treatment of coccidioidomycosis in man, p. 260 267. In T. H. Sternberg and V. D. Newcomer (ed.), Therapy of fungus diseases. Little, Brown and Co., Boston, Mass. 22. Pahls, S., and A. Schaffner. 1994. Comparison of the activity of free and liposomal amphotericin B in vitro and in a model of systemic and localized murine candidiasis. J. Infect. Dis. 169:1057 1061. 23. Procknow, J. J., and C. G. Loosli. 1958. Treatment of deep mycoses. Arch. Intern. Med. 101:765 802. 24. Ralph, E. D., A. M. Khazindar, K. R. Barber, and C. W. M. Grant. 1991. Comparative in vitro effects of liposomal amphotericin B, amphotericin B deoxycholate, and free amphotericin B against fungal strains determined by using MIC and minimum lethal concentration susceptibility studies and time-kill curves. Antimicrob. Agents Chemother. 35:188 191. 25. Richardson, M. D., and D. W. Warnock. 1997. Fungal infection: diagnosis and management, 2nd ed. Blackwell Scientific Publications, Oxford, United Kingdom. 26. Solotorovsky, M., G. Quabeck, and S. Winsten. 1958. Antifungal activity of candidin, nystatin, euclin, and stilbamidine against experimental infections in the mouse. Antibiot. Chemother. (Washington, D.C.) 8:364 371. 27. Stanley, V. C., and M. P. English. 1965. Some effects of nystatin on the growth of four Aspergillus species. J. Gen. Microbiol. 40:107 118. 28. van Etten, E. W. M., M. T. Ten Kate, L. E. T. Stearne, and I. A. J. M. Bakker-Woudenberg. 1995. Amphotericin B liposomes with prolonged circulation in blood: in vitro antifungal activity, toxicity, and efficacy in systemic candidiasis in leukopenic mice. Antimicrob. Agents Chemother. 39:1954 1958. 29. Wallace, T. L., V. Paetznick, P. A. Cossum, G. Lopez-Berestein, J. H. Rex, and E. Anaissie. 1997. Activity of liposomal nystatin against disseminated Aspergillus fumigatus infection in neutropenic mice. Antimicrob. Agents Chemother. 41:2238 2243. 30. Woods, R. A., M. Bard, I. E. Jackson, and D. J. Drutz. 1974. Resistance to polyene antibiotics and correlated sterol changes in two isolates of Candida tropicalis from a patient with amphotericin B-resistant funguria. J. Infect. Dis. 129:53 58.