In-vitro activity of dicationic aromatic compounds and fluconazole against Cryptococcus neoformans and Candida spp.

Maurizio Del Poetaa,,b, Amy S. Bixela, Francesco Barchiesib, Richard R. Tidwellc, David Boykind, Giorgio Scaliseb and John R. Perfecta,*

a Department of Medicine, Division of Infectious Diseases and International Health, Duke University Medical Center, PO Box 3353, Durham, NC 27710, USA; b Institute of Infectious Diseases and Public Health, University of Ancona, Ospedale Umberto I, 60121 Ancona, Italy; c Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; d Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We investigated the in-vitro activity of three selected dicationic aromatic compounds for nine clinical isolates of Cryptococcus neoformans and 93 clinical isolates of Candida spp., representing 12 different species, using a broth macrodilution method following NCCLS recommendations. All the clinical isolates were also tested for fluconazole susceptibility. The in-vitro data demonstrate that compounds 39 and 57 have excellent in-vitro activity for all tested strains (MIC 0.19–1.56 mg/L) except Candida pelliculosa. Moreover, compound 39 showed excellent in-vitro fungicidal activity against Candida krusei, Candida glabrata, Candida lusitaniae and Cryptococcus neoformans with MFCs in the range 0.39–6.25 mg/L. Both compounds 39 and 57 showed excellent in-vitro activity against fluconazole-resistant Candida albicans isolates, including a C. albicans strain that contains all known fluconazole-resistant mechanisms. Comparing MIC data from compounds 21, 39 and 57 with fluconazole, we found a statistically significant difference only with compound 39 (P= 0.043). However, comparing MFC data from compounds 21, 39 and 57 with fluconazole, we found statistically significant differences with all three compounds (P < 0.00001). These data indicate the potential antifungal breadth of two bis-benzimidazoles (compounds 39 and 57) as antifungal agents against yeasts. If it can be determined that compounds 39 and 57 are effective and non-toxic in vivo, the prospect of these compounds as clinically useful antifungal agents will be enhanced.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The incidence of fungal infections has increased significantly in the last 20 years.1 In immunocompromised patients, the emergence of candida infections with bothprimary drug resistance and the secondary development of azole-resistant Candida spp. isolates has been described.2,3,4,5 The development of new antifungal agents with potent and broad-spectrum fungicidal activity is needed for effective management of these infections.

Dicationic aromatic compounds (DACs) are agents that have been shown to possess excellent in-vitro and in-vivo activity against a number of pathogenic microorganisms, including Giardia lamblia,6,7 Toxoplasma gondii,8 Pneumocystis carinii,9,10,11,12 Plasmodium falciparum,9 Leishmania mexicana amazonensis,9 Trypanosoma brucei,13 Candida albicans and Cryptococcus neoformans.14,15

Our initial studies of in-vitro structure–activity relationships have shown that some of these compounds, especially one furan (compound 21) and two bis-benzimidazoles (compounds 39 and 57), possess potent in-vitro antifungal activity for both yeasts and moulds. However, in these preliminary studies we tested only one strain of several Candida spp. and only a few C. neoformans strains.14,15 In this report, we expand the evaluation of the in-vitro antifungal activity of compounds 21, 39 and 57 for nine clinical isolates of C. neoformans and 93 clinical isolates of Candida spp., representing 12 different species: 21 isolates of C. albicans, 10 isolates each of Candida glabrata, Candida parapsilosis and Candida tropicalis, seven isolates of Candida krusei, and five isolates each of Candida kefyr, Candida lusitaniae, Candida famata, Candida incospicua, Candida pelliculosa, Candida guilliermondii and Candida lipolytica. We also confirm that all known azole-resistant mechanisms including drug efflux pumps do not impact on the antifungal activity of this class of compounds.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Drugs

Three dicationic aromatic compounds, a furan 2,5-bis[4-(N-cyclopentylamidino)phenyl]furan (compound 21) and two bis-benzimidazoles, 2,5-bis[2-(5-amidino)benzimidazoyl] pyrrole hydrochloride (compound 39) and 2,7-bis[2-(5-N-isopropylamidino)benzimidazoyl] fluorene hydrochloride (compound 57) were provided as pure powders by the Laboratory of Chemistry at Georgia State University in Atlanta, GA, USA. Synthesis of compound 21 has been previously described by Boykin et al.;16 synthesis of compounds 39 and 57 was recently described in detail by Del Poeta et al.14 Fluconazole was provided by Pfizer, Inc. (New York, NY, USA) as a pure powder. Stock solutions of 10 mg/mL were made in sterile distilled water for compounds 39, 57 and fluconazole, and in dimethyl sulphoxide (DMSO) for compound 21. The stocks were then filter sterilized by passage through a 0.22 µm Millex-GV Durapore membrane filter (Bedford, MA, USA) and stored at -70°C until used.

Isolates

One hundred and two yeast clinical isolates were used in this study. Each strain represented a unique isolate coming from a different patient, with the exception of the C. albicans strains, displayed in Table I, several of which were isolated from two different episodes of oropharyngeal and/or oesophageal candidiasis in the same patient.17 Seven reference strains—C. neoformans ATCC 90113, C. neoformans H99, C. albicansATCC 76615, C. albicans A39, C. krusei ATCC 6258 and C. albicansisolates 1 and 17—were included in each run of the experiments. C. neoformans H99 and C. albicans A39 represented two reference strains of the Duke University Mycology Research Unit.14,15 The fluconazole-sensitive C. albicans isolate 1 and the fluconazole-resistant C. albicans isolate 17 have been used for studies of the molecular mechanisms of drug resistance and were kindly provided by Dr Theodore C. White.18,19


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Table I. MIC geometric means and ranges (mg/L) of three dicationic aromatic compounds and fluconazole against C. neoformans and Candida spp.
 
In-vitro susceptibility testing

Antifungal susceptibility testing was performed using RPMI-1640 medium (Sigma Chemical Co., St Louis, MO, USA) with glutamine, without sodium bicarbonate, and buffered at pH 7.0 with 0.165 M morpholine propane-sulphonic acid (MOPS). Drug dilutions were prepared at 10 times the strength of the final drug concentration (100–0.09 mg/L) by a serial drug dilution scheme for minimizing systematic pipetting errors.20 The x10 drug dilutions were dispensed as 0.1 mL volumes into sterile polystyrene tubes (12 mm x 75 mm; Falcon 2054; Becton Dickinson, Lincoln Park, NJ, USA) and stored at -20°C until used. Yeast isolates were grown on yeast-extract peptone dextrose agar (YEPD) at 30°C and subcultured twice to ensure viability. The yeast inocula were prepared as described in the NCCLS document.20 For each test, colony counts were performed and the strain was retested if the inoculum was not 0.5 x103 to 2.5 x103 cfu/mL. All tubes were incubated at 35°C and were read after 48 h for Candida spp., and after 72 h for C. neoformans. The MIC was defined as the lowest drug concentration that resulted in a visual turbidity of <=80% inhibition compared with that produced by the growth control tube (0.2 mL of growth control plus 0.8 mL of uninoculated RPMI 1640).20 MFC experiments were adapted from a method of McGinnis.21 Briefly, 100 µL aliquots from tubes with growth inhibition were plated on to Sabouraud agar plates. The lowest drug concentration that yielded three or fewer yeast colonies was recorded as the MFC.

Statistical analysis

The significance of the differences between compounds 21, 39 and 57 versus fluconazole for geometric means of MICs and MFCs was determined by Student's t-test.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
All of the Candidaspp. and C. neoformans isolates produced adequate growth after 48 h and 72 h, respectively, and all the inocula of the pathogenic yeasts and reference strains yielded between 0.5 x103 and 2.5 x103 cfu/mL. Clear MIC 80% endpoints were observed with all three new compounds and for all tested strains.

The MIC and MFC geometric means and ranges of compounds 21, 39, 57 and fluconazole against all tested clinical isolates are summarized in Tables II and III, respectively. The MICs and MFCs for seven reference strains and two other sequential C. albicans clinical isolates from five patients are displayed in Table I.


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Table II. MFC geometric means and ranges (mg/L) of three dicationic aromatic compounds and fluconazole against C. neoformans and Candida spp.
 

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Table III. MICs and MFCs of three dicationic aromatic compounds and fluconazole against sequential isolates of C. albicansa and seven reference strains
 
The in-vitro data show that, except for C. pelliculosa, the two bis-benzimidazoles, compounds 39 and 57, have excellent in-vitro antifungal activity, with MICs in the range 0.19–1.56 mg/L. There was little variation among unique strains within the species. Furthermore, compound 39 also produced in-vitro fungicidal activity, especially against the yeasts with known antifungal drug resistance, such as C. krusei, C. glabrata and C. lusitaniae. With these yeasts, MFCs ranged from 0.39 to 6.25 mg/L.

In a general comparison of MIC data for compounds 21, 39 and 57 versus fluconazole for all yeast strains, a statistically significant difference was found only with compound 39 versus fluconazole (P= 0.043, Table II). However, in MFC comparisons, compounds 21, 39 and 57 all were found to be significantly more potent than fluconazole (P < 0.00001, Table III). According to Table I, compounds 39 and 57 demonstrated good in-vitro activity for C. albicans fluconazole-resistant isolates, but compound 21 did not. In addition, compounds 21, 39 and 57 showed identical in-vitro activity against the fluconazole-sensitive C. albicans isolate 1 and the fluconazole-resistant isolate 17 (Table I). C. albicansisolate 17 is a fluconazole-resistant strain that contains several different drug-resistant mechanisms including both overexpression and alteration of the target enzyme (Erg11), and the amplification of both CDR efflux pumps and MDR efflux pumps.18,19 Since strain 17 represents all known fluconazole resistance mechanisms presently identified, the DACs should possess excellent in-vitro antifungal activity against most fluconazole-resistant yeast strains.

Our findings show that: (i) compound 21 demonstrated better in-vitro activity for C. neoformans than for Candida spp.; (ii) compound 39 showed excellent in-vitro antifungal activity for both C. neoformans and Candida spp.; (iii) compound 57 and fluconazole showed similar in-vitro antifungal activity for all tested strains, except that compound 57 produced a lower MFC endpoint; (iv) compound 39 had better in-vitro antifungal activity than fluconazole for all tested strains; (v) compound 21 had less in-vitro antifungal activity than fluconazole; (vi) compounds 39 and 57 showed excellent in-vitro activity for C. krusei and C. incospicua, which are considered to be fluconazole-resistant species (Table II).

Compound 21 was chosen for this study because of its excellent in-vivo activity in the treatment of Pneumocystis carinii infections in rats at 0.5 mg/kg/day and because it was shown to be non-toxic at 5.0 mg/kg/day in animals.22 Compound 39 was chosen because it too is non-toxic in animals in vivo (data unpublished). Compound 57 was studied as a derivative of compound 39,14 and for use in structure–activity relationships. However, in-vivo data for this compound are not yet available.

In general, many dicationic-substituted bis-benzimidazole derivatives have shown good in-vivo activity for P. carinii pneumonia in the rat model,22 and also excellent in-vitro fungicidal activity for C. albicans and C. neoformans.14 Moreover, the compounds used in this study show both broad-spectrum antifungal activity and in-vitro fungicidal properties for all Candida spp.

A potential key to the further antifungal development of these compounds will be to establish their mechanism(s) of action. It has been proposed that the activity of the bis-benzimidazoles for G. lamblia is through inhibition of topoisomerase II.6 Although this mechanism appears likely for G. lamblia, no direct correlation was found for topoisomerase inhibition for Cryptosporidium parvum23 or P. carinii.16,24,25 A major problem in determining the mechanism of action for P. carinii and C. parvum is the lack of effective in-vitro culture systems. This problem does not exist with yeasts, such as Saccharomyces cerevisiae and/or C. neoformans. Our studies to determine the mechanism(s) of action of these compounds for selected fungi continue. DNA binding is a requirement for antifungal activity from these compounds. However, our initial studies on C. neoformans have confirmed that the compounds' antifungal activity for this yeast is not mediated through an effect on topoisomerase I.26

In conclusion, these data indicate the potential of two bis-benzimidazoles (compound 39 and 57) as antifungal agents. They are consistently fungicidal for the majority of clinically relevant yeast species. Several new agents have been identified from this initial study of structure–activity relationships and will require future synthesis and antifungal testing. It is clear that these compounds warrant further studies on structure–activity relationships, mechanism(s) of action and toxicity, and in-vivo efficacy to determine their clinical potential as a class of antifungal agents.


    Acknowledgments
 
We are grateful to Mary Ann Howard for help in preparing the manuscript. This work is part of the Duke University Mycology Research Unit. This work was supported by an NIH Program Project, NAIAD AI-33363, and awards by Pharm-Eco Pharmaceutical, Inc., Lexington, MA, USA, and Immtech International, Inc., Evanston, IL, USA. Giorgio Scalise and Francesco Barchiesi are recipients of a grant from the Istituto Superiore di Sanita', Rome, Italy (AIDS project, 50A.0.32).


    Notes
 
* Corresponding author. Tel: +1-919-684-2660; Fax: +1-919-684-8902; E-mail: perfe001{at}mc.duke.edu Back


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 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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2 . Cameron, M. L., Schell, W. A., Bruch, S., Bartlett, J. A., Waskin, H. A. & Perfect, J. R. (1993). Correlation of in-vitro fluconazole resistance of Candida isolates in relation to therapy and symptoms of individuals seropositive for human immunodeficiency virus type 1. Antimicrobial Agents and Chemotherapy 3, 2449–53.

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14 . Del Poeta, M., Schell, W. A., Dykstra, C. C., Jones, S. K., Tidwell, R. R., Czarny, A. et al. (1998). Structure–in vitro activity relationships of pentamidine analogous and dication substituted bis-benzimidazoles as new antifungal agents. Antimicrobial Agents and Chemotherapy 42, 2495–502.[Abstract/Free Full Text]

15 . Del Poeta, M., Schell, W. A., Dykstra, C. C., Jones, S. K., Tidwell, R. R., Kumar, A., et al. (1998). In vitro antifungal activity of a series of dication substitute carbazoles, furans and benzimidazoles. Antimicrobial Agents and Chemotherapy 42, 2503–10.[Abstract/Free Full Text]

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Received 4 November 1998; returned 5 February 1999; revised 25 February 1999; accepted 23 March 1999





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