Activity of the new antifungal triazole, posaconazole, against Cryptococcus neoformans

Francesco Barchiesia,*, Anna Maria Schimizzia, Francesca Casellia, Daniele Gianninib, Valeria Camilettia, Barbara Filenia, Andrea Giacomettia, Luigi Falconi Di Francescoa and Giorgio Scalisea

a Istituto di Malattie Infettive e Medicina Pubblica, Centro di Gestione Presidenza Medicina e Chirurgia, b Università degli Studi di Ancona, Ancona, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The new antifungal derivative posaconazole was tested against three clinical isolates of Cryptococcus neoformans var. neoformans using a broth microdilution procedure performed according to the guidelines established by the NCCLS. Posaconazole MICs were 0.125, 0.25 and 1.0 mg/L for isolates 491, 2337 and 486, respectively. To investigate the in vivo activity of this new compound, we established an experimental model of systemic cryptococcosis in CD1 mice by iv injection of cells of each strain of C. neoformans. Low (3 mg/kg/day) and high (10 mg/kg/day) doses of posaconazole were compared with amphotericin B given at 0.3 mg/kg/day for 10 consecutive days. Survival studies showed that all treatment regimens were effective in prolonging the survival of mice infected with C. neoformans 486 (P < 0.001). Only posaconazole at 10 mg/kg and amphotericin B were effective in prolonging the survival in mice infected with C. neoformans 2337 (P from <0.01 to <0.001), while neither agent was effective in mice infected with C. neoformans 491. Tissue burden experiments performed 24 h after the end of therapy revealed that posaconazole at 10 mg/kg was effective at reducing the fungal burden in both lung and brain tissues of all three strains of C. neoformans. In particular, for C. neoformans 491 and 2337 posaconazole was superior to amphotericin B at reducing the fungal burden in the brain (P < 0.05). The efficacy of posaconazole was also confirmed by determining the capsular antigen serum levels of treated mice versus untreated mice. Our study underlines the excellent activity of posaconazole against this pathogenic yeast.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cryptococcus neoformans is the cause of the most common life-threatening opportunistic fungal infection in patients with AIDS.1 Although the occurrence of cryptococcosis among this group of patients has decreased in the last 3 years due to the introduction of triple HIV therapy, this incidence remains high, particularly in developing countries.2

The ‘gold standard’ therapy for cryptococcosis remains amphotericin B with or without flucytosine.1 For suppression therapy a triazole, such as fluconazole or itraconazole, is the agent of choice.1,3

Recently, the new investigational triazole posaconazole was shown to have potent activity against isolates of C. neoformans in vitro.4–7 Thus far, few studies have been conducted to correlate the in vitro data on posaconazole MICs for this pathogenic yeast with results in vivo.8

Therefore, in the present study we investigated the efficacy of this new triazole in an experimental model of systemic murine cryptococcosis caused by strains of C. neoformans with variable patterns of posaconazole susceptibilty in vitro.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Isolates

Three isolates of C. neoformans, each obtained from an AIDS patient, were used in this study (TableGo). Two strains were obtained from blood (491 and 2337), while C. neoformans 486 was isolated from CSF. All isolates were identified as C. neoformans var. neoformans on the basis of no colour change on canavanine–glycine–bromothymol blue agar.9 All the strains were maintained on Sabouraud dextrose agar (SDA; Difco Laboratories, Detroit, MI, USA) slants at 4°C.


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Table. In vitro and in vivo activities of posaconazole and amphotericin B against three isolates of C. neoformans
 
Antifungal agents

For in vitro studies, a stock solution of posaconazole (Schering Plough Research Institute, Kenilworth, NJ, USA) was prepared in polyethylene glycol (PEG 200; Janssen Chimica, Geel, Belgium), and a stock solution of amphotericin B (Sigma Chemical, Milano, Italy) in dimethyl sulphoxide (Sigma). Further dilutions of both drugs were made in the test medium. The final concentration of the solvent did not exceed 1% in any well.

For in vivo studies, posaconazole was prepared in PEG 200 while amphotericin B was purchased as Fungizone from Bristol-Myers Squibb (S.p.A., Latina, Italy).

Susceptibility testing

Antifungal susceptibility testing was performed using a broth microdilution method, adhering to the recommendations of the NCCLS.10 The test medium was RPMI 1640 (Sigma) buffered to pH 7.0 with 0.165 M MOPS (Gibco Laboratories, Milano, Italy). Final concentrations of both drugs ranged from 0.0078 to 4.0 mg/L. Yeast inocula ranged from 0.5 x 103 to 2.5 x 103 cfu/mL. The microdilution plates were incubated in air at 35°C and read at 72 h. The posaconazole MIC was defined as the lowest concentration of drug at which turbidity in the well was 80% less than that in the control well, while amphotericin B MIC was defined as the lowest concentration of drug at which no fungal growth was detectable.10 Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were routinely tested in parallel with each MIC run.

Animal studies

A murine model of systemic cryptococcosis was established in CD1 male mice (weight 30 g; Charles River Laboratories, Calco, Italy) by injection via the lateral tail vein of viable yeast cells grown overnight in brain–heart infusion broth. Animal experiments were conducted with the approval of the University of Ancona ethics committee. Posaconazole was administered by oral gavage at concentrations of 3 and 10 mg/kg/day, while amphotericin B was given ip at 0.3 mg/kg/day. Therapy was started 24 h after the infection and continued for 10 consecutive days. In survival studies the mice were observed through days 40 or 60 and deaths were recorded daily. Moribund mice were killed, and their deaths were recorded as occurring on the next day. In tissue burden studies the mice were killed 24 h after the end of therapy, and the number of viable cfu per gram of brain and lungs of each animal was determined by quantitative plating of organ homogenates onto SDA plates. There were 10–13 mice per group in survival studies, and seven mice per group in tissue burden studies. Three additional mice per group were killed 24 h after the end of therapy and were used to study the effects of drugs on the clearance of cryptococcal capsular polysaccharidic antigen (PA) in serum. Testing was performed by a commercially available agglutination assay (Crypto-La Test, Bouty, S.p.A., Milano, Italy).

Statistical analysis

The log rank test was used to determine the difference between survival groups, and the Mann–Whitney U-test was used to determine the significance in tissue burden studies. The results of PA serum levels were determined by the analysis of variance followed by Bonferroni t-test. Differences were considered significant when P was <0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Antifungal susceptibility testing of both drugs showed identical results in five independent experiments. Posaconazole MICs were 0.125, 0.25 and 1.0 mg/L for isolates 491, 2337 and 486, respectively, and the MIC of amphotericin B was 0.5 mg/L for all isolates (TableGo).

Survival results are given in the Table. Mice were infected with 2.0 x 105, 4.5 x 105 and 4.8 x 105 cfu/mouse in experiments with C. neoformans 486, 491 and 2337, respectively. For C. neoformans 486, all treatment regimens were effective in prolonging survival against the controls (P < 0.001). The effectiveness of posaconazole was shown to be dose dependent, with posaconazole at 10 mg/kg/day being more effective than posacoanzole at 3 mg/kg/day (P < 0.05). For C. neoformans 491, neither posaconazole nor amphotericin B was effective in prolonging survival. For C. neoformans 2337, only posaconazole at 10 mg/kg/day (P < 0.01) and amphotericin B (P < 0.001) were effective in prolonging survival. For this strain, amphotericin B was significantly more active than both triazole dosing regimens (P < 0.001).

The second set of experiments consisted of three tissue burden studies. Mice were infected with 1.8 x 105, 2.0 x 105 and 2.1 x 105 cfu/mouse in experiments with C. neoformans 486, 491 and 2337, respectively. Mice were treated for 10 days, and killed 1 day later. In these studies, posaconazole was only given at 10 mg/kg/day. The results are reported in the Table. For C. neoformans 486, both treatments were equally effective at reducing fungal burdens in the brain, whereas only the triazole was effective at reducing fungal burdens in the lung. Posaconazole was also more effective than amphotericin B (P < 0.05). Similar results were obtained for C. neoformans 491. In particular, for this strain posaconazole was more effective than amphotericin B at reducing fungal burdens in both organs (P < 0.05). For C. neoformans 2337, both posaconazole and amphotericin B were effective at reducing fungal burdens in both organs, with the triazole more effective than the polyene in the brain tissue (P < 0.05).

Antigen titres are reported in the Table. Both drugs were significantly effective at reducing PA serum levels for all three strains (P <0.05 to <0.001). In addition, posaconazole was superior to amphotericin B against C. neoformans 486 (P < 0.01).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, we correlated the in vitro activity of posaconazole with its efficacy in vivo against C. neoformans. Three clinical isolates of C. neoformans showing variable degrees of posaconazole susceptibility in vitro were used in the study. Our data on survival did not correlate with the in vitro data which showed that the most susceptible isolate (491) was resistant to treatment with either low or high doses of posaconazole, as it was with amphotericin B. On the other hand, the least susceptible strain (486) proved to be the most susceptible in vivo, with both posaconazole doses being effective at prolonging the survival compared with controls. These findings underline the lack of complete correlation between in vitro and in vivo results in testing this triazole against C. neoformans. It must be noted, however, that the posaconazole MICs only encompassed a three dilution difference between the most and the least susceptible isolate (0.125–1.0 mg/L), and that we did not use any highly resistant strain.

Unlike antibacterial agents, for which standardized susceptibility testing procedures and interpretive breakpoints are well established, reproducible methods and tentative breakpoints for antifungal agents have only recently been introduced.11 So far, most of the studies investigating the relationship between in vitro and in vivo results of antifungal efficacy have involved infections due to Candida spp.11 Only a few reports have tried to correlate the in vitro activity of a given antifungal agent (mainly fluconazole) with the clinical outcome of cryptococcosis.12–14 In an early study, Casadevall et al.12 used a broth macrodilution method to analyse fluconazole and amphotericin B MICs for 13 strains of C. neoformans isolated from five AIDS patients, and showed a lack of correlation between in vitro data and clinical outcome. In contrast, Witt et al.14 used a modified broth microdilution method to test fluconazole MICs for clinical isolates of C. neoformans and found a statistically significant correlation between in vitro data and clinical success or failure.

The reason both posaconazole and amphotericin B were ineffective in prolonging the survival of mice infected with C. neoformans 491 is difficult to explain. It can only be hypothesized that failure to prolong survival of mice infected with this isolate might be due to the longer observation time (60 versus 40 days) than that applied in mice infected with the other two isolates of C. neoformans. This situation, owing to a progressive decrease of drug tissue levels over time, would facilitate the replication of the remaining fungi to a critical burden.

Unlike survival data, tissue burden experiments showed that posaconazole given at 10 mg/kg/day was effective at reducing the number of cfu per gram of brain and lung tissues in all isolates of C. neoformans. In particular, posaconazole was more effective than amphotericin B in the brain of mice infected with C. neoformans 491 and 2337 as in the lung of mice infected with C. neoformans 486 and 491. Determination of antigen serum levels of treated mice confirmed the potent in vivo efficacy of this new antifugal molecule.

Overall, our study underlines the excellent activity of posaconazole against this pathogenic yeast and indicates that this new antifungal molecule merits further investigation as a potentially useful agent for the treatment of human cryptococcosis.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported in part by a grant from Istituto Superiore di Sanità, Rome, Italy (III AIDS, project; grant no. 50C.29).


    Notes
 
* Correspondence address. Istituto di Malattie Infettive e Medicina Pubblica, Università degli Studi di Ancona, Azienda Ospedaliera Umberto I°, Via Conca –Torrette, 60020, Ancona, Italy. Tel: +39-71-5963467; Fax: +39-71-5963468; E-mail: cmalinf{at}popcsi.unian.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Mitchell, T. G. & Perfect, J. R. (1995). Cryptococcosis in the era of AIDS—100 years after the discovery of Cryptococcus neoformans. Clinical Microbiology Reviews 8, 515–48.[Abstract]

2 . Ghannoum, M. A. (2000). Potential role of phospholipases in virulence and fungal pathogenesis. Clinical Microbiology Reviews 13, 122–43.[Abstract/Free Full Text]

3 . Como, J. A. & Dismukes, W. E. (1994). Oral azole drugs as systemic antifungal therapy. New England Journal of Medicine 330, 263–72.[Free Full Text]

4 . Barchiesi, F., Arzeni, D., Fothergill, A. W., Falconi Di Francesco, L., Caselli, F., Rinaldi, M. G. et al. (2000). In vitro activities of the new antifungal triazole SCH 56592 against common and emerging yeast pathogens. Antimicrobial Agents and Chemotherapy 44, 226–9.[Abstract/Free Full Text]

5 . Barchiesi, F., Schimizzi, A. M., Caselli, F., Novelli, A., Fallani, S., Giannini, D. et al. (2000). Interactions between triazole and amphotericin B against Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy 44, 2435–41.[Abstract/Free Full Text]

6 . Galgiani, J. N. & Lewis, M. L. (1997). In vitro study of activities of antifungal triazole SCH 56592 and itraconazole against Candida albicans, Cryptococcus neoformans, and other pathogenic yeasts. Antimicrobial Agents and Chemotherapy 42, 2467–73.[Abstract/Free Full Text]

7 . Perfect, J. R., Cox, G. M., Dodge, R. K. & Schell, W. A. (1996). In vitro and in vivo efficacy of the azole SCH 56592 against Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy 40, 1910–3.[Abstract]

8 . Hossain, M. A., Maesaki, S., Mitsutake, K., Kekeya, H., Sasaki, E., Tomono, K. et al. (1999). In-vitro and in-vivo activities of SCH56592 against Cryptococcus neoformans. Journal of Antimicrobial Chemotherapy 44, 827–9.[Abstract/Free Full Text]

9 . Kwon-Chung, K. J., Polacheck, I. & Bennett, J. E. (1992). Improved diagnostic medium for separation of Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). Journal of Clinical Microbiology 15, 535–7.

10 . National Committee for Clinical Laboratory Standards. (1997). Reference for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA.

11 . Rex, J. H., Pfaller, M. A., Galgiani, J. N., Bartlett, M. S., Espinel-Ingroff, A., Ghannoum, M. A. et al. (1997). Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro–in vivo correlation data for fluconazole, itraconazole, and candida infection. Subcommittee on Antifungal Susceptibility Testing of the National Committee for Clinical Laboratory Standards. Clinical Infectious Diseases 24, 235–47.[ISI][Medline]

12 . Casadevall, A., Spitzer, E. D., Webb, D. & Rinaldi, M. G. (1993). Susceptibilities of serial Cryptococcus neoformans isolates from patients with recurrent cryptococcal meningitis to amphotericin B and fluconazole. Antimicrobial Agents and Chemotherapy 37, 1383–6.[Abstract]

13 . Velez, J. D., Allendoerfer, R., Luther, M., Rinaldi, M. G. & Graybill, J. R. (1993). Correlation azole susceptibility with in vivo response in a murine model of cryptococcal meningitis. Journal of Infectious Diseases 168, 508–10.[ISI][Medline]

14 . Witt, M. D., Lewis, R. J., Larsen, R. A., Milefchik, E. N., Leal, M. A. E., Haubrich, R. A. et al. (1996). Identification of patients with acute AIDS-associated cryptococcal meningitis suitable for fluconazole therapy: the role of antifungal susceptibility testing. Clinical Infectious Diseases 22, 322–8.[ISI][Medline]

Received 28 February 2001; returned 12 June 2001; revised 17 August 2001; accepted 30 August 2001