a Department of Microbiology, Asesoría Científica y de Investigación Aplicada, E-08031 Barcelona b Department of Microbiology and Parasitology, School of Medicine, University of the Pais Vasco, Bilbao c Service of Microbiology, Hospital Vall dóHebrón, Barcelona, Spain d Pre-Clinical Research and Development, Aronex Pharmaceuticals Incorporated, The Woodlands, TX, USA
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Abstract |
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Introduction |
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Liposomal nystatin is a multilamellar liposomal formulation of nystatin, which contains nystatin, dimyristoyl phosphatidyl choline and dimyristoyl phosphatidyl glycerol in a ratio (by weight) of 1:7:3. López-Berestein and co-workers formulated nystatin into liposomes. Different studies have demonstrated good antifungal activity and reduced toxicity following iv administration.3 ,4 ,5 ,6 ,7 ,8
The purpose of this study was to compare the in-vitro activity of liposomal nystatin with those of other commercially available antifungal drugs. The comparator drugs were nystatin, amphotericin B (desoxycholate), amphotericin B cholesteryl sulphate (ABCD), liposomal amphotericin B (LAB), amphotericin B lipid complex (ABLC), fluconazole and itraconazole.
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Materials and methods |
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Assay methodology was based on that described by Anaissie et al.10 and NCCLS.9 Rosewell Park Memorial Institute (RPMI) 1640 (with glutamine and without bicarbonate) (Sigma-Aldrich, St Louis, MO, USA) was used as the culture medium; this contained 0.165 MOPS (0.165 Molar (3-(N-morpholino)-propanesulphonic acid)) (Sigma-Aldrich), pH 7.0. Incubation was carried out at at 25°C. The medium was sterilized by filtration using a 0.22 µM filtration unit (Steritop-GP, Millipore Co., Bedford, MA, USA). Liposomal nystatin and nystatin were supplied by Aronex Pharmaceuticals, Inc. (The Woodlands, TX, USA). ABLC was obtained from The Liposome Company, Inc. (Princeton, NJ, USA), LAB from Vestar Deutschland Gmbh (Braunsleweig, Germany), ABCD from Sequus Pharmaceuticals, Inc. (Menlo Park, CA, USA), fluconazole from Pfizer (Sandwich, UK) and itraconazole from Jansen Pharmaceutica (Beerse, Belgium). The lyophilized drugs were reconstituted following manufacturer's instructions, liposomal nystatin being dissolved in sterile saline (1000 mg/L), and LAB, ABCD and amphotericin B in sterile water (2000 mg/L); ABLC was diluted (1:250) with RPMI 1640 (2000 mg/L). Nystatin and itraconazole were prepared in dimethylsulphoxide (2000 mg/L). Fluconazole was diluted in sterile water and later in RPMI 1640 (128 mg/L). Yeast inocula were prepared by choosing five colonies > 1 mm in diameter, from SDA plates and suspending the material in 5 mL of 0.85% saline (Grifols, Barcelona, Spain). Suspensions were mixed for 15 s and adjusted to 0.5 McFarland Standard (15 x 106 cfu/mL) and later diluted 1:1000 in RPMI 1640 to give a final inoculum of 0.52.5 x 103 cfu/mL.
The microtitre cells were examined using an inverted mirror, each well being compared with the control (drug-free) well. MICs for liposomal nystatin, ABLC, LAB, ABCD, amphotericin B and nystatin were taken as the lowest concentration at which clear wells were observed. Those for fluconazole and itraconazole were taken to be the lowest concentration at which a definite decrease in turbidity was observed. MICs were read at 24/48 h for Candida spp., and 4872 h for Cryptococcusspp., R. rubra and Trichosporon spp., or until visible growth was detected in the control. Minimal fungicidal concentrations (MFCs) were calculated by subculturing 10 µL from each well onto SDA. The MFC was taken as the concentration at which less than three colonies were detected after incubation. Assays were performed in duplicate. SPSS PCsoftware (SPSS Inc., Chicago, IL, USA) was used to determine MIC50 and MIC90 values. MIC50 was calculated when there were at least five isolates per fungal strain; MIC90 was calculated when there were at least 10 isolates per fungal strain. The criterion for statistical significance was P < 0.05.
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Results and discussion |
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The results of the MIC determinations are shown in the Table (MIC50, MIC90 and mean MIC). Species of which less than six isolates were examined have been included in the `all strains' table only. Inhibitory concentrations were remarkably similar for polyenes, with values for MIC50s ranging from 0.31 to 0.62 mg/L, MIC90s ranging from 0.62 to 1.25 mg/L and mean MICs ranging from 0.54 to 1.07 mg/L. No statistically significant differences were found between the in-vitro antifungal activities of polyenes. Polyene MFCs paralleled MIC results, and values were one to two dilutions higher than MIC values.
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Although there was no overall statistical significance in the differences between the in-vitro antifungal activity of liposomal nystatin, nystatin, amphotericin B, LAB, ABLC and ABCD, there were differences in the activity of these drugs for individual isolates. Amphotericin B and ABLC had high MIC values (3.31 and 10 mg/L, respectively) for an isolate of C. laurentii, but liposomal nystatin, nystatin, LAB, ABCD and itraconazole had relatively low MICs (0.62, 0.31, 1.25, 0.31 and 0.019 mg/L, respectively) for the same isolate. LAB, ABLC and ABCD had higher mean MIC values (5.16, 6.33 and 3.28 mg/L, respectively) against Trichosporon spp. than liposomal nystatin, nystatin, amphotericin B and itraconazole (0.94, 0.51, 0.74 and 0.23 mg/L, respectively). Moreover, LAB and ABCD had high MIC90 values for C. glabrata.
Our results for the activity of liposomal nystatin in vitro are similar to those recently described by Johnson et al.10 for C. albicans, C. tropicalis, C. parapsilosis and C. krusei, but it was shown to be slightly more active in this study against C. neoformans and C. glabrata. Liposomal nystatin has previously been reported to be active in vitro against LAB-resistant yeasts11 and to have been effective in some patients who have failed therapy with amphotericin B and ABLC.5 ,6 ,78 It has also been reported to be effective in at least 60% of episodes of refractory candidaemia, including those treated with amphotericin B, fluconazole and/or 5-fluorocytosine. 1 ,6 ,7 A 67% success rate has been reported in the treatment of 43 patients with candidaemia, causing renal toxicity in only 14% of them compared with 37% of the patients in an AMB control. 1 ,4 ,5 ,6 ,7 These results are encouraging and liposomal nystatin may be useful for the treatment of systemic fungal infections, particularly for those where other antifungal treatments have failed.
MIC and MFC values compared favourably with the blood concentrations achieved following iv administration of liposomal nystatin to patients. 1 ,6 ,7 ,8 At the therapeutic dosages of 2 and 4 mg/kg currently being used in liposomal nystatin clinical trials, the concentration of nystatin in the blood has been found to be 4.8 ± 4.0 mg/L and 24.3 ± 2.8 mg/L.12 The overall MIC90 and MFC90 values of liposomal nystatin were 1.25 and 5.0 mg/L. These results suggest that 2 or 4 mg/kg dosages to humans can achieve blood concentrations that should be sufficient for antifungal activity against most, if not all, yeasts likely to be encountered in a clinical context. In conclusion, the study shows that liposomal nystatin was active against all yeasts, showed as a fungicidal activity in vitro, and should find a place in the treatment of human systemic fungal infections.
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Acknowledgments |
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Notes |
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References |
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2 . 2.Gordon, L. E. (1995). Nystatin therapy in experimental coccichomycosis. American Review of Tuberculosis 72, 6770.
3 . 3.Wallace, T. L., Paetznick, V., Cossum, P. A., López-Berestein, G., Rex, J. H. & Anaissie, E. (1997). Activity of liposomal nystatin against disseminated Aspergillus fumigatus in neutropenic mice. Antimicrobial Agents and Chemotherapy 41, 223843.[Abstract]
4 . 4.López-Berestein, G., Fainstein, V., Hopfer, R. L., Mehta, K., Sullivan, M. P., Keating, M. et al. (1985). Liposomal amphotericin-B for the treatment of systemic fungal infections in patients with cancer: a preliminary study. Journal of Infectious Diseases 151, 70410.[ISI][Medline]
5 . 5.Gonzalez, C. E., Giri, N., Shetty, D., Klighys, K., Love, W., Sein, T. et al. (1996). Efficacy of a lipid formulation of nystatin against invasive pulmonary Aspergillosis. In Program and Abstracts of the Thirty-Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA. Abstract B54. p. 31. American Society for Microbiology, Washington, DC.
6 . 6.Boutati, E., Maltezou, H. C., López-Berestein, G., Vartivarian, S. E. & Anaissie, E. J. (1995) Phase I study of maximum tolerated dose of intravenous liposomal nystatin for the treatment of refractory febrile neutropenia (RFN) in patients with haematological malignancies. In Program and Abstracts of the Thirty-Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Washington, DC.
7 . 7.Roston, K., Baird, I., Graham, D. R. & Jauregui, L. (1998) Treatment of refractory candidemia in non-neutropenic patients with liposomal nystatin (NYOTRANTM). In Program and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. Abstract, p. 31. American Society for Microbiology, Washington, DC.
8 . 8.Cossum, P. A., Wyse, J., Simmons, V., Wallace, T. L. & Rios, A (1996). Pharmacokinetics of NYOTRANTM (liposomal nystatin) in human patients. In Program and Abstracts of the Thirty-Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA. Abstract A88. p. 17. American Society for Microbiology, Washington, DC.
9 . 9.National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: M27-A. NCCLS, Villanova, PA.
10 . 10.Anaissie, E., Paetznick, V., Proffit, R., Adler-Moore, J. & Bodey, G. P. (1991). Comparison of the in-vitro antifungal activity of free and liposome-encapsulated amphotericin B. European Journal of Clinical Microbiology and Infectious Disease 10, 6658.[ISI][Medline]
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11.Johnson, E. M., Ojwang, J. O., Szekely, A., Wallace,
T. L. & Warnock, D. W. (1998) Comparison of in-vitro antifungal activities of
free and liposome-encapsulated nystatin with those of four amphotericin B formulations. Antimicrobial Agents and Chemotherapy 42, 1412
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12 . 12.Wallace, T. L. & Lopez-Berenstein, G. (1998). Nystatin and liposomal nystatin. In Antimicrobial Chemotherapy and Vaccines (Yu, V. L., Merigan, T. C., Barriere, S. & White, N. J., Eds), pp. 118591. Williams & Wilkins, Baltimore, MD.
Received 2 November 1998; returned 5 February 1999; revised 16 March 1999; accepted 29 April 1999