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Pneumocandin L-743,872 enhances the activities of amphotericin B and fluconazole against Cryptococcus neoformans in vitro.

Franzot SP, Casadevall A.

Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Cryptococcus neoformans infections in patients with AIDS are often incurable, despite aggressive antifungal therapy. Combination regimens with additive or synergistic drugs could provide additional options for treating cryptococcal meningitis. We evaluated the efficacy of combination therapies using L-743,872, a pneumocandin antifungal drug, and amphotericin B or fluconazole against 18 strains of C. neoformans, including 11 C. neoformans var. neoformans, 3 C. neoformans var. gattii, and 4 fluconazole-resistant isolates. The combination of subinhibitory concentrations of L-743,872 with amphotericin B significantly enhanced amphotericin B activity against C. neoformans as measured by turbidity (antifungal susceptibility studies using the National Committee of Clinical and Laboratory Standards method), quantitative CFU, and tetrazolium salt reduction assays. Similarly, the addition of subinhibitory concentrations of L-743,872 to fluconazole enhanced fluconazole activity, but the effect was less dramatic than for the pneumocandin-amphotericin B combination. A marked synergism was observed in all combinations of amphotericin B and L-743, 872 (fractional inhibitory concentration index [FIC] of < or = 0.5). Fluconazole-resistant strains showed a susceptibility to amphotericin B and L-743,872 which was comparable to that of susceptible isolates. Combinations of pneumocandin with fluconazole revealed different activities for the various strains, including synergism (FIC < 1.0), additivity (FIC = 1.0), and autonomy (FIC between 1.0 and 2.0). Combination studies with fluconazole and L-743,872 showed additive and autonomous activities against fluconazole-resistant isolates. No antagonistic interactions (FIC < 2.0) were observed for any combination of L-743,872 with either amphotericin B or fluconazole. The results of this study suggest that L-743,872 can enhance the efficacy of fluconazole or amphotericin B in vitro and indicate a potential role for L-743,872 in combination therapy against C. neoformans.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9021188&dopt=Abstract fluconazole Diflucan



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In vivo effects of fluconazole on lymphocyte subpopulations of the thymus and spleen in mice: flow cytometry analysis.

Liu J, Mengel K, Friedberg KD.

Institute of Pharmacology and Toxicology, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany.

Fluconazole is a triazole compound developed for the therapy of fungal infections, especially for systemic fungal infections. In this study 2.5 and 5.0 mg oral doses of fluconazole per kg of body weight were administered to mice for 3-28 days, followed by lymphocyte phenotyping by flow cytometry analysis on a Becton-Dickinson FACScan. The results show that administration of fluconazole neither reduced body and organ weights nor thymocyte and spleen cell count. In the thymus and spleen the distribution of all the lymphocyte subpopulations studied was unchanged. These results suggest that fluconazole has no in vivo effects on the quantity of lymphocyte subpopulations in mice. The results tend to support the use of fluconazole in immunosuppressed patients.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9023584&dopt=Abstract fluconazole Diflucan



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Inhibition of mitochondrial function in isolated rate liver mitochondria by azole antifungals.

Rodriquez RJ, Acosta D Jr.

Department of Pharmacology/Toxicology, University of Texas at Austin 78712-1074, USA.

Ketoconazole is an imidazole oral antifungal agent with a broad spectrum of activity. Ketoconazole has been reported to cause liver damage, but the mechanism is unknown. However, ketoconazole and a related rug, miconazole, have been shown to have inhibitory effects on oxidative phosphorylation in fungi. Fluconazole, another orally administered antifungal azole, has also been reported to cause liver damage despite its supposedly low toxicity profile. The primary objective of this study was to evaluate the metabolic integrity of adult rat liver mitochondria after exposure to ketoconazole, miconazole, fluconazole, and the deacetylated metabolite of ketoconazole by measuring ADP-dependent oxygen uptake polarographically and succinate dehydrogenase activity spectrophotometrically. Ketoconazole, N-deacetyl ketoconazole, and miconazole inhibited glutamate-malate oxidation in a dose-dependent manner such that the 50% inhibitory concentration (I50) was 32,300, and 110 microM, respectively. In addition, the effect of ketoconazole, miconazole, and fluconazole on phosphorylation coupled to the oxidation of pyruvate/malate, ornithine/malate, arginine/malate, and succinate was evaluated. The results demonstrated that ketoconazole and miconazole produced a dose-dependent inhibition of NADH oxidase in which ketoconazole was the most potent inhibitor. Fluconazole had minimal inhibitory effects on NADH oxidase and succinate dehydrogenase, whereas higher concentrations of ketoconazole were required to inhibit the activity of succinate dehydrogenase. N-deacetylated ketoconazole inhibited succinate dehydrogenase with an I50 of 350 microM. In addition, the reduction of ferricyanide by succinate catalyzed by succinate dehydrogenase demonstrated that ketoconazole caused a dose-dependent inhibition of succinate activity (I50 of 74 microM). In summary, ketoconazole appears to be the more potent mitochondrial inhibitor of the azoles studied; complex I of the respiratory chain is the apparent target of the drug's action.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9029271&dopt=Abstract fluconazole Diflucan



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Itraconazole cyclodextrin solution: the role of in vitro susceptibility testing in predicting successful treatment of HIV-related fluconazole-resistant and fluconazole-susceptible oral candidosis.

Cartledge JD, Midgley J, Gazzard BG.

Department of HIV & Genito-Urinary Medicine, St Stephen's Centre, Chelsea & Westminster Healthcare NHS Trust, London, UK.

OBJECTIVES: This study assessed the ability of in vitro susceptibility testing of clinical Candida isolates to predict in vivo response to itraconazole cyclodextrin solution. METHODS: One hundred specimens were obtained from HIV-positive patients with oral thrush, of which 72 speciments were from patients who were clinically unresponsive to fluconazole at standard doses and had fluconazole-resistant isolates in vitro. Susceptibility to itraconazole was assessed by measuring the relative growth of an isolate in liquid medium containing a single concentration of itraconazole and then expressing growth in itraconazole as a percentage of growth in antifungal-free medium. RESULTS AND CONCLUSIONS: Where specimens yielded only one isolate, a cut-off relative growth in itraconazole of 68% discriminated between isolates from patients failing to respond clinically to itraconazole solution and those from patients successfully treated with the preparation (specificity 100%; sensitivity 88%). The presence of mixed infection reduced the predictive accuracy of the test. Only 30% of fluconazole-resistant isolates were cross-resistant to itraconazole. No isolates were resistant to itraconazole but susceptible to fluconazole. Non-response to itraconazole solution was attributed to resistant yeast infection in the majority of cases, and this susceptibility method accurately identified specimens from patients unlikely to respond to the drug.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9030362&dopt=Abstract fluconazole Diflucan



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The in-vivo activity of an antifungal antibiotic, benanomicin A, in comparison with amphotericin B and fluconazole.

Ohtsuka K, Watanabe M, Orikasa Y, Inouye S, Uchida K, Yamaguchi H, Kondo S, Takeuchi T.

Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd, Yokohama, Japan.

The in-vivo antifungal activity of benanomicin A administered intravenously or subcutaneously was compared with that of amphotericin B and fluconazole using animal models of systemic infections with Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. The efficacy of benanomicin A in C. albicans infection was more pronounced when administered in multiple doses than in a single dose. This was also true of fluconazole, but not of amphotericin B, which showed no difference between single and multiple dosings. Benanomcin A eradicated C. albicans cells from the kidneys of infected mice in a manner comparable to that of amphotericin B, but more effectively than fluconazole. The histopathological findings obtained from the kidneys of the C. albicans-infected mice confirmed the therapeutic efficacy of benanomicin A. The subcutaneous ED50 values of benanomicin A were 1.30 mg/kg/day (C. albicans) and 19.0 mg/kg/day (A. fumigatus) which were intermediate between those of amphotericin B and fluconazole in the two models. The subcutaneous ED50 value of benanomicin A for C. neoformans was 21.5 mg/kg/day, which was higher than that of amphotericin B.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9044030&dopt=Abstract fluconazole Diflucan



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Reversible fluconazole resistance in Candida albicans: a potential in vitro model.

Calvet HM, Yeaman MR, Filler SG.

Department of Medicine, Harbor-UCLA Research and Education Institute, St. John's Cardiovascular Research Center, Torrance 90509, USA.

To study the development and potential mechanisms of antifungal resistance in relation to antifungal exposure, reversible fluconazole resistance was examined in vitro. Candida albicans ATCC 36082 blastospores were passed in liquid yeast nitrogen base medium containing either 4, 8, 16, or 128 micrograms of fluconazole per ml, and susceptibility testing was performed after each passage. High-level fluconazole resistance (50% inhibitory concentration, > 256 micrograms/ml) developed in the isolates after serial passage in medium containing 8, 16, or 128 micrograms of fluconazole per ml, but not in isolates passed in 4 micrograms of fluconazole per ml. Reduced susceptibility was noted within four to seven passages, which was equivalent to 14 to 19 days of exposure to the drug. However, all isolates returned to the susceptible phenotype after 8 to 15 passages in medium lacking the drug; thus, fluconazole resistance was reversible in vitro. In vivo, organisms retained the resistant phenotype after a single passage in the rabbit model of infective endocarditis. Restriction digest profiles and karyotypic analysis of the parent strain and selected fluconazole-resistant and -susceptible isolates from each group were identical. Investigations into the molecular mechanisms of this reversible resistance failed to reveal increased accumulation of mRNA for 14 alpha-demethylase, the target enzyme for fluconazole, or for the candidal multidrug transporters CDR1 and BENr. This process of continuous in vitro exposure to antifungal drug may be useful as a model for studying the effects of different antifungal agents and dosing regimens on the development of resistance and for defining the mechanism(s) of reversible resistance.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9055988&dopt=Abstract fluconazole Diflucan



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In vitro activities of voriconazole (UK-109,496) against fluconazole-susceptible and -resistant Candida albicans isolates from oral cavities of patients with human immunodeficiency virus infection.

Ruhnke M, Schmidt-Westhausen A, Trautmann M.

Abteilung Innere Medizin und Poliklinik, Virchow-Klinikum der Humboldt-Universitat, Berlin, Germany. mruhnke ukrv.de

The susceptibility of Candida albicans to a new antifungal triazole, voriconazole (UK-109,496), was investigated in 105 isolates obtained from the oral cavities of patients with human immunodeficiency virus (HIV) infection to study this drug's activity against fluconazole-susceptible and -resistant isolates. MICs were determined by a broth microdilution technique according to document M27-T from the National Committee for Clinical Laboratory Standards and by using a broth microdilution technique and a synthetic high-resolution medium. These antifungal susceptibility testing methods showed high levels of agreement (93% for fluconazole and 86% for voriconazole). Data from in vitro studies showed that voriconazole has good activity against fluconazole-susceptible and -resistant C. albicans isolates; the MICs at which 90% of all isolates were inhibited were 0.19 to 0.39 microgram/ml. We found that for isolates for which fluconazole MICs were high, voriconazole MICs were proportionally higher than those for fluconazole-susceptible C.albicans (P < 0.001). Pretreatment isolates from six patients with fluconazole-refractory esophageal candidiasis were included in the study. For these isolates the MICs were < or = 0.39 microgram/ml, and all patients responded to voriconazole. These results suggest that voriconazole is effective even in the treatment of fluconazole-refractory esophageal candidiasis and should be studied further to determine its clinical relevance in patients with HIV infection.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9055995&dopt=Abstract fluconazole Diflucan



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Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro.

Moran GP, Sullivan DJ, Henman MC, McCreary CE, Harrington BJ, Shanley DB, Coleman DC.

Department of Oral Medicine and Pathology, School of Dental Science, Trinity College, University of Dublin, Republic of Ireland.

Candida dubliniensis is a recently described species of Candida associated with oral candidiasis in human immunodeficiency virus (HIV)-infected individuals. Nineteen oral isolates of C. dubliniensis recovered from 10 HIV-positive and 4 HIV-negative individuals and one vaginal isolate from an additional HIV-negative subject were assessed for fluconazole susceptibility by broth microdilution (BMD), hyphal elongation assessment, and Etest. The susceptibilities of these 20 isolates to itraconazole and amphotericin B and of 10 isolates to ketoconazole were also determined by BMD only. Sixteen of the C. dubliniensis isolates were susceptible to fluconazole (MIC range, 0.125 to 1.0 microgram ml-1), and four (recovered from two AIDS patients) were fluconazole resistant (MIC range, 8 to 32 micrograms ml-1). Fluconazole susceptibility data obtained by hyphal elongation assessment correlated well with results obtained by BMD, but the corresponding Etest MIC results were one to four times higher. All of the isolates tested were found to be sensitive to itraconazole, ketoconazole, and amphotericin B. Sequential exposure of two fluconazole-sensitive (MIC, 0.5 microgram ml-1) C. dubliniensis isolates to increasing concentrations of fluconazole in agar medium resulted in the recovery of derivatives which expressed a stable fluconazole-resistant phenotype (BMD-determined MIC range, 16 to 64 micrograms ml-1), even after a minimum of 10 consecutive subcultures on drug-free medium and following prolonged storage at -70 degrees C. The clonal relationship between the parental isolates and their respective fluconazole-resistant derivatives was confirmed by genomic DNA fingerprinting and karyotype analysis. The results of this study demonstrate that C. dubliniensis is inherently susceptible to commonly used antifungal drugs, that fluconazole resistance does occur in clinical isolates, and that stable fluconazole resistance can be readily induced in vitro following exposure to the drug.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9056003&dopt=Abstract fluconazole Diflucan