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
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Abstract |
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Introduction |
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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 structureactivity 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.
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Materials and methods |
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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 strainsC. neoformans ATCC 90113, C. neoformans H99, C. albicansATCC 76615, C. albicans A39, C. krusei ATCC 6258 and C. albicansisolates 1 and 17were 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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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 (1000.09 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.
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Results and discussion |
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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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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 structureactivity 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 structureactivity relationships and will require future synthesis and antifungal testing. It is clear that these compounds warrant further studies on structureactivity relationships, mechanism(s) of action and toxicity, and in-vivo efficacy to determine their clinical potential as a class of antifungal agents.
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Acknowledgments |
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Notes |
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References |
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Del Poeta, M., Toffaletti, D. L., Rude, T. H., Dykstra,
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Received 4 November 1998; returned 5 February 1999; revised 25 February 1999; accepted 23 March 1999