In-vitro interactions of itraconazole with flucytosine against clinical isolates of Cryptococcus neoformans

Francesco Barchiesi*, Domenico Gallo, Francesca Caselli, Luigi Falconi Di Francesco, Daniela Arzeni, Andrea Giacometti and Giorgio Scalise

Istituto di Malattie Infettive e Medicina Pubblica Università degli Studi di Ancona, Ospedale Umberto I°, Largo Cappelli 1, 60121, Ancona, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Treatment failures can occur in AIDS patients infected with Cryptococcus neoformans,despite aggressive antifungal therapy. Combination regimens with additive or synergic drugs could provide additional options for treating cryptococcosis. We studied the effects of itraconazole combined with flucytosine against 16 strains of C. neoformansvar. neoformans. Combination therapy revealed different results for the various strains, including synergy (fractional inhibitory concentration (FIC) index 0.5, 63% of the interactions), addition (FIC >0.5 to 1.0, 31% of the interactions) and indifference (FIC >1.0 to <2.0, 6% of the interactions). Antagonism (FIC >2.0) was not observed. The efficacy of combination therapy was confirmed by quantitative cfu and killing curve assays. In particular, killing curves conducted in replicating cells showed that the addition of itraconazole prevented the development of flucytosine-resistant mutants of C. neoformans. These data show that the combination of itraconazole and flucytosine is significantly more active than either drug alone against C. neoformans in vitro.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cryptococcus neoformans is an important cause of morbidity and mortality in immunocompromised patients. 1 Cryptococcal meningoencephalitis in AIDS patients is sometimes incurable despite aggressive antifungal therapy. 2 Although amphotericin B, alone or associated with flucytosine, remains the standard therapy for cryptococcosis, this approach has important clinical limitations including toxic side-effects and incovenience of intravenous dosing. 3 Recently, several antifungal combination therapies against C. neoformans have been tested in both in-vitro and in-vivo models. 4,5,6,7,8,9,10,11,12,13,14 One of the most promising combination strategy seems to be flucytosine in association with an azole derivative, mainly with fluconazole. 4,5,8,9,10,11

Little is known about the interaction of flucytosine with the other commercially available triazole, itraconazole, against isolates of C. neoformans. Thus far, only a few isolates of C. neoformans have been tested and, generally, by means of unstandardized procedures. 7,13,14

The main aim of this study was to evaluate the in-vitro interactions of itraconazole with flucytosine against clinical isolates of C. neoformans by a broth microdilution method performed according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations.


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

Sixteen isolates of Cryptococcus neoformans were included in this study. They comprised 15 clinical strains isolated from blood, CSF, or skin biopsy of AIDS patients, and one strain belonging to the American Type Culture Collection: C. neoformans ATCC 90112. All the strains were maintained on Sabouraud dextrose agar (Difco Laboratories, Detroit, MI, USA) slants at 4°C.

Antifungal agents

Stock solution of itraconazole (Janssen, Beerse, Belgium) was prepared in polyethylene glycol 400 (Janssen Chimica, Geel, Belgium). Stock solution of flucytosine (Sigma, Milan, Italy) was prepared in sterile distilled water. Further dilutions of both drugs were performed in the test medium.

Antifungal susceptibility testing

MICs. Drug interactions were assessed using a chequerboard titration broth microdilution-based method, adhering to the NCCLS recommendations. 15,16,17 Testing was performed in RPMI 1640 medium (Sigma) buffered to pH 7.0 with 0.165 M morpholinepropanesulphonic acid buffer (MOPS, Gibco Laboratories, Milan, Italy). Final concentrations of the antifungal agents ranged from 0.0078 to 0.5 mg/L for itraconazole and from 0.03 to 16 mg/L for flucytosine. Yeast inocula (1 to 5 x 10 3 cfu/mL) were added to each well of the microdilution trays. The trays were incubated in air at 35°C and read at 48 and 72 h. Readings were performed spectrophotometrically with an automatic plate reader (Dynatech, model MR 700) set at 490 nm. MIC endpoints were determined as the first concentration of the antifungal agent tested alone and in combination at which turbidity in the well was >50% less than that in the control well. 16,17 Drug interaction was classified as synergic, additive, indifferent or antagonistic on the basis of the fractional inhibitory concentration (FIC) index. The FIC index is the sum of the FICs for each drug; the FIC is defined as the MIC of each drug when used in combination divided by the MIC of the drug when used alone. The interaction was defined as synergic if the FIC index was 0.50, additive if the FIC index was >0.50 to 1.0, indifferent if the FIC index was >1.0 to 2.0 and antagonistic if the FIC index was >2.0. 18

Determination of cfu/mL. After the MICs were recorded, aliquots of 100 µL of each well were spread on Sabouraud dextrose agar plates to determine the number of colonies per millilitre. Plates were incubated from 48 to 72 h at 35°C and then the number of cfus counted.

Time-kill studies. Three to five colonies of an isolate from a 72 h growth plate were suspended in 10 mL of sterile distilled water and the turbidity was adjusted using spectrophotometric methods to a 0.5 McFarland standard (approximately 1 x 10 6 to 5 x 10 6 cfu/mL). One millilitre of the adjusted fungal suspension was added to 9 mL of either RPMI-1640 medium buffered with MOPS buffer, or a solution of growth medium plus an appropriate amount of each drug alone or in combination. Test solutions were placed on a shaker and incubated at 35°C. At time points 0, 24 and 48 h following the introduction of the test isolate into the system, 100 µL aliquots were removed from each test solution. After 10-fold serial dilutions, a 50 µL aliquot from each dilution was streaked in triplicate onto Sabouraud dextrose agar plates for colony count determination. Following incubation at 35°C for 48 to 72 h, the number of cfus on each plate was determined. Time-kill studies were also performed with non-growing cells. Experiments were performed exactly as reported above with the exception of RPMI-1640 which was replaced with PBS pH 7.0. For time-kill study, synergy was defined as a >100-fold increase in killing as compared with the most active single agent, while antagonism was defined as at least a 100-fold decrease in killing as compared with the most active single agent. If less than a 10-fold change from the effect of the most active single drug was observed, the interaction was considered additive. 18

Statistical analysis

The MIC data were transformed logarithmically to approximate a normal distribution before statistical analysis. Continous variables were compared with Student's t-test or the Mann- Whitney test. The results of colony count were obtained as means and standard deviations of at least three repetitions carried out for each compound, alone and in combination. Statistical significance was evaluated by analysis of variance followed by Bonferroni t test. A value of P < 0.05 was considered to be significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antifungal susceptibility data at 48 h

Flucytosine MICs ranged from 0.125 to 8.0 mg/L, with an MIC 50 and an MIC 90 of 2.0 mg/L (see Table). Itraconazole MICs ranged from 0.125 mg/L to >0.5 mg/L, with an MIC 50 and an MIC 90 of 0.125 mg/L and 0.5 mg/L, respectively. When flucytosine and itraconazole were given in combination, there were significant reductions in the geometric mean of the flucytosine MIC (from 1.18 mg/L to 0.18 mg/L; P = 0.005) and of the itraconazole MIC (from 0.21 to 0.05 mg/L; P = 0.001). Thirty-seven per cent (6/16) of the interactions were synergic, 44% (7/16) were additive, 19% (3/16) were indifferent, while antagonism was not observed. When synergy was documented, the median reductions in MICs were two-fold (range, two-to five-fold) for flucytosine and two-fold (range, two-to five-fold) for itraconazole. When additivity was documented, the median reductions in MICs were two-fold (range, one-to three-fold) for flucytosine and one-fold (range, one-to three-fold) for itraconazole. Isolates 2341 and 2853, showing an indifferent interaction, had their initial flucytosine MICs reduced six-and four-fold, respectively, upon combination with itraconazole. Isolate 2337 had its initial itraconazole MIC reduced one-fold upon combination with flucytosine (seeTable).


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Table. Mode of interaction of itraconazole with flucytosine against 16 isolates of C. neoformansa
 
Antifungal susceptibility data at 72 h

Flucytosine MICs ranged from 0.5 to 8.0 mg/L, with an MIC 50 and an MIC 90 of 2.0 and 4.0 mg/L, respectively (seeTable). Itraconazole MICs ranged from 0.125 to >0.5 mg/L, with an MIC 50 and an MIC 90 of 0.25 and 0.5 mg/L, respectively. When flucytosine and itraconazole were given in combination, there were significant reductions in the geometric mean of the flucytosine MIC (from 2.0 to 0.19 mg/L; P = 0.0001) and of the itraconazole MIC (from 0.26 to 0.04 mg/L; P = 0.0001). Sixty-three per cent (10/16) of the interactions were synergic, 31% (5/16) were additive, 6% (1/16) were indifferent, while antagonism was not observed. When synergy was documented, the median reductions in MICs were four-fold (range, two-to six-fold) for flucytosine and three-fold (range, two-to four-fold) for itraconazole. When additivity was documented, the median reductions in MICs were two-fold (range, one-to four-fold) for flucytosine and one-fold (range, one- to three-fold) for itraconazole. Combination therapy for isolate 2337 still showed as indifferent at 72 h; however, initial itraconazole MIC was reduced four-fold upon combination with flucytosine (seeTable).

Determination of cfu/mL

Determination of cfu/mL was performed only with C. neoformans 486. The effects of the combination of subinhibitory concentrations of flucytosine and itraconazole on the cfu of C. neoformans 486 are shown in Figure 1. Marked reductions in the number of cfu were observed when flucytosine (at 0.25, 0.5 and 1.0 mg/L) was combined with itraconazole (at 0.015, 0.03 and 0.06 mg/L). For all combinations, the reduction in cfu was significantly greater than that observed with single drugs alone (P values ranging from 0.0001 to 0.031).



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Figure 1. Effects of flucytosine alone (white bars) and in combination with itraconazole on number of C. neoformans 486. Itraconazole concentrations were 0.015 mg/L (black bars), 0.03 mg/L (light grey bars), and 0.06 mg/L (dark grey bars); cfus were determined after 72 h of incubation under NCCLS 15 conditions. Data are the averages of three experiments, and error bars denote standard deviations.

 
Time-kill experiments

Time-kill experiments with both growing or non-growing cells of C. neoformans 486 are shown in Figure 2. In both experiments flucytosine was used at 5 x MIC (10 mg/L) while itraconazole was used either at 4 x MIC (0.5 mg/L) or 16 x MIC (2.0 mg/L). No antifungal carryover was observed with either flucytosine or itraconazole. Figure 2a shows the killing rates of flucytosine and itraconazole, alone or in combination, against growing cells. Flucytosine alone showed to be the most effective antifungal regimen at 24 h. On the other hand, quantitative cultures at 48 h showed an approximately 10-fold increase in the number of cfu/mL of cells treated with flucytosine alone, possibly due to the development of flucytosine-resistant mutants. At this time interval, combination therapy showed to be the most effective antifungal regimen regardless of the dose of itraconazole combined with flucytosine. In order to avoid the development of flucytosine-resistant mutants we carried out time-kill studies in nongrowing cells (Figure 2b). In this system, flucytosine combined with itraconazole 2.0 mg/L was the most effective antifungal regimen at 24 h. At 48 h both combination therapies yielded the lowest number of cfu/mL. Overall, neither flucytosine nor itraconazole, alone or in combination, were demonstrated to be fungicidal over 48 h. Although both systems suggested that combination therapies were the most effective in reducing the number of cfu/mL, interactions were only additive.



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Figure 2. Time-kill studies on C. neoformans 486: (a) cells were incubated in RPMI-1640 at 35°C with gently shaking (growing cells); (b) cells were incubated in PBS at 35°C without shaking (nongrowing cells). Flucytosine was used at 53 MIC (10 mg/L); itraconazole was used either at 43 MIC (0.5 mg/L) or 16× MIC (2.0 mg/L). {diamond} No drugs; {triangleup} itraconazole 0.5; {triangledown} itraconazole 2.0; {square} flucytosine; • flucytosine + itraconazole 0.5; {circ} flucytosine + itraconazole 2.0.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although most infections respond satisfactorily to a single antimicrobial agent, there are clinical circumstances in which a combination of antimicrobial agents is advantageous. Achievement of synergy is one of the major theoretical justifications for combination therapy. In the last few years, several antifungal combination therapies, other than the classic association of amphotericin B/flucytosine, have been tried against C. neoformans. Combination of a polyene and an azole has always been questioned because of the potential for antagonism. 19 However, in a rabbit model of experimental cryptococcal meningitis, amphotericin B and ketoconazole had an additive effect. 12 Both animal data and human trials have supported the combination of flucytosine and fluconazole. 4,5,8,9,10,11 Both agents achieve similar penetration into the CSF. However, in the last few years, isolates of C. neoformans resistant to fluconazole in vitro as well in vivo have been reported more frequently. 1,20 Recently, Franzot and Casadevall found that the echinocandin derivative, L-743,872, enhances the activities of both amphotericin B and fluconazole against C. neoformans in vitro. 6 Literature data regarding the interactions of flucytosine and itraconazole against C. neoformans are controversial. Polak examined several antifungal combination therapies in mouse models of candidiasis, cryptococcosis, aspergillosis and wangiellosis, and found that flucytosine-itraconazole combination was definitely synergic or additive in candidosis and aspergillosis but indifferent in cryptococcosis. 15 Iovanitti et al. studied this combination in a hamster model of cryptococcosis and found that it was less effective than administering the drugs separately. 7 Conversely, Van-Custem et al. found that this combination was more efficacious than monotherapy in an experimental model of murine cryptococcosis. 14 However, in these studies only few isolates have been tested and, in general, no data about in-vitro interactions were available.

To our knowledge, this is the first study in which a standardized in-vitro susceptibility testing method has been employed for studying the interactions between flucytosine and itraconazole against a large number of clinical isolates of C. neoformans. Our findings that 63% of itraconazole- flucytosine interactions were synergic and that antagonism was not observed are encouraging. Furthermore, even when a synergic interaction was not achieved, this combination still showed a beneficial effect, with at least a two-fold reduction in the MIC of both drugs in 83% (5/6) of interactions. In general, the geometric means (GMs) of the MICs of both drugs dropped dramatically when they were used in combination: GM of flucytosine dropped from 2.0 to 0.19 mg/L, GM of itraconazole dropped from 0.26 to 0.04 mg/L.

It has been reported that itraconazole serum levels range from 0.1 to 0.4 mg/L following the administration of 200 mg/day. 21 Recently, Prentice et al. showed that itraconazole oral solution given at 5 mg/kg/day produces serum levels ranging from 0.7 to 0.8 mg/L at steady state. 22 Interestingly, our itraconazole MICs, upon combination with flucytosine, were from 10-to 20-fold lower than the attainable serum levels described in vivo. In addition, because of the high lipophilicity of itraconazole, the tissue levels of this drug are substantially higher than the corresponding serum levels. This could explain the efficacy of itraconazole in cryptococcal meningoencephalitis despite the low measurable concentrations of this drug in cerebral spinal fluid. 21

Although the mechanism by which itraconazole enhances the activity of flucytosine was not investigated, one can speculate as to potential mechanisms based on the known effects of these drugs on fungal cells: itraconazole acts by damaging the fungal cell membrane, which could facilitate the in-vitro uptake of flucytosine. In addition, itraconazole might inhibit the development of flucytosine resistance, thereby conserving the potent activity of flucytosine against C. neoformans. Our killing curves conducted in replicating cells clearly showed that the addition of itraconazole prevented the development of flucytosine-resistant mutants of C. neoformans.

Since the in-vitro model, or models, that most closely mimic clinical infections are not known, we performed killing experiments by using both replicating and non-replicating cells. Both systems showed that, although a clear synergic interaction was not reached, combination treatments were more effective than either drug alone in reducing the number of cfu/mL at 48 h. Furthermore, we found that the degree of reduction of cfu was not dependent on the concentration of itraconazole employed in the system. It must be noted, however, that itraconazole-flucytosine combination did not show a fungicidal activity, even in experiments where both drugs were combined at several-fold their respective MICs. This fact, probably due to the fungistatic activity of both drugs, might imply that the sterilization of infected organs is difficult to reach by this therapeutic approach, even when high doses of both drugs are employed. Thus far, the only antifungal drug which has been reported to exert a fungicidal activity is amphotericin B. 23

In conclusion, our study demonstrates that the combination of itraconazole and flucytosine is significantly more active than either drug alone against C. neoformans in vitro. These findings suggest that this therapeutic approach could be useful in the treatment of cryptococcal infections. Clearly, clinical studies are warranted to elucidate further the potential utility of this combination therapy.


    Acknowledgments
 
This work was supported in part by a grant from Istituto Superiore di Sanità, Rome, Italy (I AIDS project, no. 50A.0.32).


    Notes
 
* Corresponding author. 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
 References
 
1 . Mitchell, T. G. & Perfect, J. R. (1995). Cryptococcosis in the era of AIDS— 100 years after discovery of Cryptococcus neoformans. Clinical Microbiology Reviews 8, 515–48.[Abstract]

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Received 25 August 1998; returned 10 November 1998; revised 28 January 1999; accepted 9 February 1999