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Antimicrobial Agents and Chemotherapy, September 1998, p. 2342-2346, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Synergy of Nitric Oxide and Azoles against
Candida Species In Vitro
Gail E.
McElhaney-Feser,1
Robert E.
Raulli,2 and
Ronald L.
Cihlar1,*
Department of Microbiology and Immunology,
Georgetown University,1 and
Amulet
Pharmaceuticals Inc.,2 Washington, D.C. 20007
Received 9 April 1998/Returned for modification 5 May 1998/Accepted 29 June 1998
 |
ABSTRACT |
The candidacidal activity of nitric oxide (NO) as delivered by a
class of compounds termed diazeniumdiolates has been investigated. Diazeniumdiolates are stable agents capable of releasing NO in a
biologically usable form at a predicted rate, and three such compounds
were examined for activity. One compound,
(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), proved to be most suitable for examining NO activity due to
its relatively long half-life (20 h) and because of limited candidacidal activity of the uncomplexed DETA nucleophile. DETA-NO was
active against six species of Candida for which the MICs
necessary to inhibit 50% growth (MIC50s) ranged from 0.25 to 1.0 mg/ml. C. parapsilosis and C. krusei
were the most susceptible to the compound. In addition to a
determination of NO effects alone, the complex was utilized to
investigate the synergistic potential of released NO in combination
with ketoconazole, fluconazole, and miconazole. Activity was
investigated in vitro against representative strains of Candida
albicans, C. krusei, C. parapsilosis,
C. tropicalis, C. glabrata, and C. dubliniensis. Determination of MIC50,
MIC80 and MICs indicated that DETA-NO inhibits all strains
tested, with strains of C. parapsilosis and C. krusei being consistently the most sensitive. The combination of
DETA-NO with each azole was synergistic against all strains tested as
measured by fractional inhibitory concentration indices that ranged
from 0.1222 to 0.4583. The data suggest that DETA-NO or compounds with
similar properties may be useful in the development of new therapeutic
strategies for treatment of Candida infections.
| |
INTRODUCTION |
The antimicrobial activity of nitric
oxide (NO) and other reactive nitrogen intermediates against a wide
array of microorganisms is well documented (3, 7, 13). On
the other hand, NO has been implicated in diverse physiological
processes in humans (17). Thus, the therapeutic value of NO
as an antimicrobial agent may be compromised by the likelihood of
unacceptable side effects accompanying usage. Likewise, its instability
and limited solubility in aqueous environments, as well as the lack of
a reliable delivery system, have made investigations concerning the
potential of NO as an antimicrobial agent problematic.
A class of compounds termed diazeniumdiolates might be useful in
resolving these problems. Diazeniumdiolates are NO-nucleophile complexes capable of releasing NO in an aqueous environment or in
response to a shift in the local pH (12, 14, 16, 18). Importantly, the complexes do not require activation through a redox
reaction or electron transfer as do glyceryl trinitrate and sodium
nitroprusside (5). Finally, the complexes are stable and
capable of delivering NO in a biologically usable form at a predictable
rate (14). As a first step in assessing the potential of
diazeniumdiolates for use in treatment of fungal infections, the
current study investigated the inhibitory effect of the compounds on in
vitro growth of Candida species, including C. albicans, C. dubliniensis, C. glabrata,
C. krusei, C. parapsilosis, and C. tropicalis. In addition, the potential for synergistic activity of
the compounds in combination with test azoles has been evaluated.
| |
MATERIALS AND METHODS |
Candida strains.
C. albicans strains
included 4918 (15) and ATCC strains 28366 and 62376. C. krusei 30672 and C. tropicalis 13803 were also obtained from the American Type Culture Collection. Clinical isolates of C. albicans, C. krusei, C. parapsilosis, and C. tropicalis, C. glabrata, and C. dubliniesis were isolated from
patients at Georgetown University Hospital, Washington, D.C. All
strains were evaluated on CHROMagar (22) for presumptive
identification, and identity was verified by the RapID Yeast Plus
System (Innovative Diagnostics Systems). Strains were maintained in
20% glycerol stocks at 
70°C and subcultured on modified Sabouraud
agar (Difco) at 27°C for inoculum preparation.
Antifungal drugs.
Azoles utilized were fluconazole (a gift
of Pfizer Ltd.), miconazole, and ketoconazole (Sigma Chemical
Co.). NO-generating compounds DETA-NO,
(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; DEA-NO,
sodium(Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; and MAHMA-NO,
(Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate; and their base compounds DETA (diethylenetriamine), DEA
(diethylamine), and MAHMA (dimethylhexanediamine) were gifts from Larry
Keefer, National Cancer Institute, Frederick Cancer Research Center,
Frederick, Md.
Growth media and microdilution assays.
Drug susceptibility
assays and checkerboard microdilution assays were performed in 96-well
flat-bottom microtiter plates (Falcon) following the recommendations of
the National Committee for Clinical Laboratory Standards
(20). Assays were performed in RPMI 1640 media (GIBCO)
buffered to pH 7 with 0.165 M morpholinepropanesulfonic acid (MOPS)
(Sigma Chemical Co.) containing 18-h yeast cells at 104
cells/ml (determined by optical density at 600 nm
[OD600]). Appropriate amounts of azoles (ketoconazole,
10
4 to 20 µg/ml; fluconazole, 102 to 200 µg/ml; miconazole, 103 to 20 µg/ml) and/or
NO-generating compounds (DETA, 102 to 15 mg/ml; DETA-NO,
102 to 5.0 mg/ml) prediluted in RPMI were delivered to
achieve a final volume of 200 µl. Microtiter plates were incubated at
37°C on a nuctating mixer (Shelton Scientific). Absorbance was read at OD492 on a Titertek plate reader at 24 and 48 h.
The data are reported as the concentrations of each antifungal agent
necessary to inhibit 50% growth (MIC50) and
MIC80 as described previously (23). Growth was
determined by OD and verified by plating 2 µl from each microtiter
well after 48 h of incubation onto YEPD agar (2% [wt/vol] yeast
extract, 1% [wt/vol] Bacto Peptone containing 2% [wt/vol]
glucose). After incubation for 24 h at 27°C, CFUs were
determined. The final data reported are the average of six independent
experiments.
Experiments to determine the effect of hemoglobin on inhibitory
activity of DETA-NO were performed by microdilution assays exactly as
described above except that assay mixtures contained DETA-NO at 0.7 mg/ml (this concentration results in 40% inhibition of growth) and
variable amounts of hemoglobin (0.001 to 1.0 mg/ml). In synergy
studies, the fractional inhibitory concentration index (FIX) was
calculated and its significance assessed as described previously
(2, 27). Briefly, FIX is equal to (MIC of drug A in
combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of
drug B alone). Drug interactions were judged on the basis of the
following criteria: synergistic effect, 
0.5; additive effect, >0.5
but 1; indifferent effect, >1 but 4; and antagonistic effect, >4
(2, 33).
| |
RESULTS |
Diazeniumdiolate inhibition.
Several diazeniumdiolates of
various NO release times were tested to determine which would be most
effective in studies to measure the candidacidal activity of NO. The
compounds and their respective uncomplexed nucleophiles are listed in
Table 1. MAHMA-NO and DEA-NO proved
unsuitable, as their respective uncomplexed nucleophiles were toxic and
exhibited candidacidal activity only slightly lower than that observed
during NO release from the complexed nucleophile (data not shown). In
contrast, the uncomplexed nucleophile, DETA, had only limited
candidacidal activity at concentrations up to 5 mg/ml (Fig. 1). Thus,
DETA-NO was used in all subsequent experiments.
The data in Table
2 summarize the effect
of DETA-NO against all strains tested. Figure
1 shows susceptibility curves of DETA-NO
and DETA for representative strains of each
Candida species
under
study. The data suggest that
C. albicans was the least
susceptible
of the species tested at a MIC
50; however, at
MIC
80 and MICs,
C. albicans,
C. tropicalis,
C. glabrata, and
C. dubliniensis
had
approximately equal sensitivities.
C. parapsilosis
and
C. krusei were consistently the most susceptible to
DETA-NO.
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|
FIG. 1.
Inhibition of representative strains of
Candida spp. grown in the presence of DETA-NO and DETA.
Assays were performed as detailed in Materials and Methods. Percent
inhibition was calculated against medium controls and represents the
average of six independent experiments. Strains grown in the presence
of DETA-NO are depicted by solid symbols as follows: C. albicans 4918 ( ), C. krusei ATCC 30672 ( ),
C. parapsilosis patient isolate 9 ( ), C. tropicalis ATCC 13803 ( ), C. glabrata patient
isolate 15 ( ), and C. dubliniensis patient isolate 19 ( ). Growth of each strain in the presence of DETA is indicated by
the companion open symbols.
|
|
To obtain evidence that the effects observed were due to released NO
and not to a structural pharmacophore inherent in DETA-NO,
the ability
of the NO scavenger, hemoglobin, to eliminate the
inhibitory effect of
DETA-NO against
C. albicans was investigated.
The data
depicted in Fig.
2 show that hemoglobin
at concentrations
less than 0.1 mg/ml abolished the activity of
DETA-NO, indicating
that the inhibitory effects are related to released
NO.
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|
FIG. 2.
Effect of hemoglobin on the inhibitory effects of
DETA-NO against C. albicans. Assays were performed as
described in Materials and Methods. Assay mixtures contained either 0.7 mg of DETA-NO per ml (), hemoglobin ( ), or hemoglobin and DETA-NO
at 0.7 mg/ml ().
|
|
Azole resistance.
Prior to synergy investigations, the
susceptibilities of test strains to selected azoles alone were
established. The data in Table 3
summarize the effects of selected azoles against all the strains
examined. As with DETA-NO, kinetics were similar for all strains of a
particular species, except that fluconazole exhibited more variability
with C. albicans and C. glabrata.
Susceptibilities of different strains of the same species showed
significant variation in some instances, but overall trends could be
determined. The general order of Candida sp.
susceptibilities to ketoconazole demonstrated by MIC80
results was C. albicans > C. dubliniensis 
C. parapsilosis > C. tropicalis > C. glabrata > C. krusei (Table 3). The order of susceptibilities to fluconazole
(Table 3) as indicated by MIC80 results was C. albicans > C. parapsilosis > C. dubliniensis > C. glabrata > C. tropicalis C. krusei. MIC80s of
miconazole (Table 3) indicated the following order of susceptibility: C. albicans > C. glabrata > C. dubliniensis C. tropicalis C. parapsilosis > C. krusei. C. krusei strains were
consistently the least susceptible to all azoles tested, while C. albicans strains were the most sensitive to azoles in vitro. The
data obtained are in general agreement with the results reported in
similar investigations by others (4, 9, 19, 21, 26, 32).
Synergy.
Synergy studies were performed with DETA-NO as the NO
donor in combination with fluconazole, ketoconazole, or miconazole. One
representative of each Candida species was used in the
investigation, and the data are summarized in Table
4. DETA-NO and each of the azoles used
acted in synergy against all strains, as indicated by FIX values of
<0.5. Differences in sensitivities to the drug combinations were
evident, with C. parapsilosis and C. krusei having the higher FIX values (least sensitive) and C. tropicalis consistently having the lowest FIX value (most
sensitive). Thus, while FIX values indicate synergistic effects for all
azole-NO combinations, the data also suggest complex interactions
between the two drugs as well as interspecies variability in responses to drug combinations.
| |
DISCUSSION |
NO-nucleophile adducts have been shown previously to exhibit
vasorelaxant effects in aortic ring experiments (16), to
serve as an inhibitors of platelet aggregation (24), and to
block tumor necrosis factor alpha-induced apoptosis and toxicity in the
liver (25). The present study has demonstrated that the diazeniumdiolate DETA-NO also has clear candidacidal effects against strains of at least six Candida spp. In addition, synergy
was demonstrated with the combinations of DETA-NO and ketoconazole, fluconazole, and miconazole (indicated by a FIX of 0.5 in all cases).
Delineation of drug susceptibilities of Candida species or
strains to antifungal drugs has become of increased importance due to
the identification of multi-azole-resistant strains and the emergence
of species other than C. albicans as pathogens (9, 23,
32). However, as noted elsewhere, the precise definition of
relative resistance levels remains unresolved, due in part to technical
difficulties in the reproducibility of susceptibility testing (21,
31). Nonetheless, it has been observed that strains of C. albicans are usually more susceptible to azoles than are C. glabrata and C. krusei (4, 21). The results
of the current study confirm these observations. In addition, while the
sensitivity of the organisms to azoles varies greatly, the sensitivity
of the organisms to released NO is within 1/2 order of magnitude for
the entire range of tested Candida species, suggesting a
mechanism of action that is highly conserved. Moreover, the steepness
of the DETA-NO inhibition curve is indicative of a threshold effect of
NO and is different from the typical dose-response relationship exhibited by drugs acting on receptor or enzyme active sites.
The mechanism(s) whereby NO shows candidacidal activity was not
investigated. It has been observed that NO produced by the inducible NO
synthase of macrophages is associated with candidacidal activity
(3, 8, 11, 13), but the basis of killing was not determined.
Candidacidal activity, however, could be related to any of a number of
the documented effects of NO (7, 28). For example,
reactivity of NO and its multiple redox states may cause inactivation
of a variety of cellular enzymes, including ribonucleotide reductase,
aconitase, and ubiquinone reductase (3, 6, 8). Similarly, NO
has been reported to disrupt respiration, alter protein function, or
cause lipid peroxidation and oxidation of sulfhydryl groups (3, 8,
29). NO may also interact with DNA, resulting in deamination or
cross-linking (3, 30).
The results suggest that the use of diazeniumdiolates
[nucleophile-NO adducts with the structure
XN(O)N = O, where X is a nucleophile residue]
might overcome certain obstacles that preclude the in vivo use of NO as
an antimicrobial agent. For example, NO in its pure form is a highly
reactive gas and has limited solubility in aqueous environments. In
contrast, diazeniumdiolates stabilize NO in a solid form that is
highly soluble under such conditions (14). The rate
and amount of NO generated can be adjusted over a wide range,
depending on the characteristics of the nucleophile. In particular, as
much as 2 mol of NO can be liberated rapidly (1 to 2 min) or slowly
(several days) per mole of complex (16, 18) as a function of
the NO-nucleophile adduct (12, 18).
On the other hand, since NO is an important biological mediator,
playing a role in the cardiovascular, immune, and central and
peripheral nervous systems (1-3, 10, 17), it is possible that diazeniumdiolates will have unacceptable toxic effects
that could limit their antimicrobial value to topical applications. It
should be noted that in vitro studies demonstrated that treatment of
rat vascular smooth-muscle cell with DETA-NO had an antiproliferative effect but that cell viability was greater than 95%, suggesting that the compound was not cytotoxic (18). NO donors with
shorter half-lives did not inhibit DNA synthesis (18). The
MIC of DETA-NO in the present assays was approximately 2 mg/ml, also raising questions as to whether such concentrations can be
reached in vivo. No data are currently available to address this issue;
however, topical use, at least, should be feasible, since the favorable solubility of DETA-NO suggests that these concentrations can easily be
obtained in such preparations. Problems that are associated with
internal use might be overcome with delivery systems that activate the
compounds upon encountering the target organism or site. In this
regard, prodrug derivatives of diazeniumdiolates constructed such that
activation and release of NO occur primarily in the liver have been
developed to treat hepatic disorders (25). No toxic effects
were reported (25). Similar strategies, including linkage of
diazeniumdiolates to an appropriate monoclonal antibody, might allow
specific targeting and delivery to treat fungal or bacterial
infections. Studies are in progress to investigate this possibility.
| |
ACKNOWLEDGMENTS |
We are indebted to Larry Keefer for providing us with the
diazeniumdiolates used in this study and for his advice concerning their use. We thank Pfizer Ltd. for the gift of fluconazole.
This work was supported in part by Public Health Service grant PO1
AI37251 from the National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Georgetown University, Washington, D.C. 20007. Phone: (202) 687-1802. Fax: (202) 687-1800. E-mail:
cihlarr{at}gunet.georgetown.edu.
| |
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Antimicrobial Agents and Chemotherapy, September 1998, p. 2342-2346, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
