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.
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, 10−2 to 200 μg/ml; miconazole, 10−3 to 20 μg/ml) and/or NO-generating compounds (DETA, 10−2 to 15 mg/ml; DETA-NO, 10−2 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.
TABLE 1.
Diazeniumdiolates and uncomplexed nucleophile bases used in the present study
| Acronym | Systematic name | Formula | Formula weight | Half-lifea |
|---|---|---|---|---|
| MAHMA | N,N′-dimethlyhexanediamine | |||
| MAHMA-NO | (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]} diazen-1-ium-1,2-diolate | CH3NH2+(CH2)6N(CH3)[N(O)NO]− | 144 | 30 s |
| DEA | Diethylamine | |||
| DEA-NO | Sodium(Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate | Et2N[N(O)NO]Na | 155 | 2 min |
| DETA | Diethylenetriamine | |||
| DETA-NO | (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate | H2NCH2CH2N(CH2CH2NH3+)[N(O)NO]− | 163 | 20 h |
At pH 7.4 and 37°C (20).
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.
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 MIC50; however, at MIC80 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.
TABLE 2.
MICs of DETA-NO (mg/ml) for Candida strains
| Strain | MIC50 | MIC80 | MIC |
|---|---|---|---|
| C. albicans 4918 | 1.00 | 1.30 | 2.00 |
| C. albicans patient isolate | 0.70 | 1.20 | 1.80 |
| C. albicans ATCC 28366 | 1.00 | 1.30 | 2.00 |
| C. albicans ATCC 62376 | 0.83 | 1.10 | 1.60 |
| C. albicans patient isolate 5 | 0.83 | 1.10 | 1.60 |
| C. albicans patient isolate 27 | 0.94 | 1.30 | 2.00 |
| C. krusei ATCC 30676 | 0.38 | 0.80 | 1.20 |
| C. krusei patient isolate | 0.40 | 0.80 | 1.20 |
| C. parapsilosis patient isolate | 0.25 | 0.45 | 0.80 |
| C. parapsilosis patient isolate | 0.28 | 0.46 | 1.00 |
| C. parapsilosis patient isolate | 0.26 | 0.45 | 0.80 |
| C. parapsilosis patient isolate | 0.25 | 0.45 | 0.80 |
| C. tropicalis ATCC 13803 | 0.55 | 1.00 | 2.20 |
| C. tropicalis patient isolate | 0.60 | 1.20 | 2.20 |
| C. glabrata patient isolate | 0.70 | 1.00 | 1.60 |
| C. glabrata patient isolate | 0.80 | 1.20 | 1.80 |
| C. glabrata patient isolate | 0.65 | 1.00 | 1.60 |
| C. glabrata patient isolate | 0.75 | 1.20 | 1.60 |
| C. dubliniensis patient isolate | 0.90 | 1.40 | 2.00 |
| C. dubliniensis patient isolate | 0.85 | 1.30 | 2.00 |
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.
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).
TABLE 3.
MICs of azoles (μg/ml) for Candida strains
| Strain | Ketoconazole
|
Fluconazole
|
Miconazole
|
||||||
|---|---|---|---|---|---|---|---|---|---|
| MIC50 | MIC80 | MIC | MIC50 | MIC80 | MIC | MIC50 | MIC80 | MIC | |
| C. albicans 4918 | 0.001 | 0.004 | 2.00 | 0.20 | 0.60 | 16.0 | 0.02 | 0.08 | 2.00 |
| C. albicans patient isolate | 0.001 | 0.005 | 2.00 | 0.06 | 0.12 | 16.0 | 0.04 | 0.20 | 2.00 |
| C. albicans ATCC 28366 | 0.001 | 0.003 | 4.00 | 0.10 | 0.30 | 16.0 | 0.01 | 0.06 | 2.00 |
| C. albicans ATCC 62376 | 0.002 | 0.003 | 3.00 | 0.10 | 0.30 | 18.0 | 0.02 | 0.06 | 2.00 |
| C. albicans patient isolate 5 | 0.003 | 0.008 | 2.00 | 0.10 | 0.30 | 30.0 | 0.01 | 0.05 | 2.00 |
| C. albicans patient isolate 27 | 0.001 | 0.004 | 8.00 | 0.10 | 0.30 | 120.0 | 0.02 | 0.06 | 2.00 |
| C. krusei ATCC 30676 | 0.720 | 3.200 | 8.00 | 10.00 | 40.00 | 120.0 | 4.00 | 7.00 | 15.00 |
| C. krusei patient isolate | 0.900 | 2.200 | 8.00 | 8.00 | 25.00 | 180.0 | 3.50 | 8.00 | 20.00 |
| C. parapsilosis patient isolate | 0.005 | 0.020 | 0.08 | 0.20 | 0.40 | 2.0 | 0.30 | 1.50 | 6.00 |
| C. parapsilosis patient isolate | 0.005 | 0.020 | 0.08 | 0.20 | 0.40 | 2.0 | 0.50 | 0.60 | 6.00 |
| C. parapsilosis patient isolate | 0.004 | 0.010 | 0.08 | 0.20 | 0.40 | 2.0 | 0.10 | 0.50 | 2.00 |
| C. parapsilosis patient isolate | 0.004 | 0.010 | 0.08 | 0.20 | 0.40 | 2.0 | 0.10 | 0.50 | 2.00 |
| C. tropicalis ATCC 13803 | 0.009 | 0.170 | 4.00 | 15.00 | 30.00 | 160.0 | 0.20 | 0.60 | 4.00 |
| C. tropicalis patient isolate | 0.008 | 0.100 | 4.00 | 15.00 | 50.00 | 180.0 | 0.08 | 0.80 | 2.00 |
| C. glabrata patient isolate | 0.070 | 0.300 | 15.00 | 2.50 | 10.00 | 180.0 | 0.10 | 0.40 | 6.00 |
| C. glabrata patient isolate | 0.060 | 0.300 | 20.00 | 2.50 | 10.00 | 120.0 | 0.10 | 0.40 | 2.00 |
| C. glabrata patient isolate | 0.060 | 0.300 | 12.00 | 2.00 | 7.00 | 80.0 | 0.06 | 0.03 | 2.00 |
| C. glabrata patient isolate | 0.060 | 0.500 | 20.00 | 7.00 | 22.00 | 200.0 | 0.20 | 0.80 | 8.00 |
| C. dubliniensis patient isolate | 0.001 | 0.007 | 4.00 | 0.40 | 1.00 | 60.0 | 0.03 | 0.80 | 4.00 |
| C. dubliniensis patient isolate | 0.002 | 0.013 | 2.00 | 0.40 | 1.00 | 60.0 | 0.02 | 0.50 | 2.00 |
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.
TABLE 4.
Synergyc of azoles and DETA-NO against Candida strains
| Azole | Strain | MIC (μg/ml) of:
|
FICa | MIC (μg/ml) of:
|
FIC | FIXb | ||
|---|---|---|---|---|---|---|---|---|
| Azole alone | Azole with DETA-NO | DETA-NO alone | DETA-NO with azole | |||||
| Ketoconazole | C. albicans | 2.00 | 0.004 | 0.002 | 2,000 | 400 | 0.200 | 0.202 |
| C. krusei | 8.00 | 0.100 | 0.013 | 1,200 | 400 | 0.333 | 0.346 | |
| C. parapsilosis | 0.08 | 0.003 | 0.038 | 800 | 300 | 0.375 | 0.413 | |
| C. tropicalis | 4.00 | 0.200 | 0.050 | 2,200 | 300 | 0.136 | 0.186 | |
| C. glabrata | 15.00 | 0.500 | 0.033 | 1,600 | 400 | 0.250 | 0.283 | |
| C. dubliniensis | 4.00 | 0.250 | 0.063 | 2,000 | 300 | 0.150 | 0.213 | |
| Fluconazole | C. albicans | 16 | 0.24 | 0.015 | 2,000 | 500 | 0.250 | 0.265 |
| C. krusei | 120 | 15.00 | 0.125 | 1,200 | 400 | 0.333 | 0.458 | |
| C. parapsilosis | 2 | 0.25 | 0.125 | 800 | 200 | 0.250 | 0.375 | |
| C. tropicalis | 160 | 5.00 | 0.031 | 2,200 | 200 | 0.091 | 0.122 | |
| C. glabrata | 180 | 5.00 | 0.028 | 1,600 | 300 | 0.188 | 0.216 | |
| C. dubliniensis | 60 | 2.50 | 0.042 | 2,000 | 300 | 0.150 | 0.192 | |
| Miconazole | C. albicans | 2 | 0.020 | 0.010 | 2,000 | 400 | 0.200 | 0.210 |
| C. krusei | 15 | 1.400 | 0.093 | 1,200 | 400 | 0.333 | 0.426 | |
| C. parapsilosis | 6 | 0.100 | 0.017 | 800 | 300 | 0.375 | 0.392 | |
| C. tropicalis | 4 | 0.400 | 0.100 | 2,200 | 400 | 0.182 | 0.282 | |
| C. glabrata | 6 | 0.090 | 0.015 | 1,600 | 500 | 0.313 | 0.328 | |
| C. dubliniensis | 4 | 0.045 | 0.011 | 2,000 | 400 | 0.200 | 0.211 | |
FIC, fractional inhibitory concentration (MIC of drug in combination/MIC of drug alone).
FIX, fractional inhibitory concentration index (MIC of DETA-NO in combination with azole/MIC of DETA-NO alone) + (MIC of azole in combination with DETA-NO/MIC of azole alone).
All outcomes were synergistic (FIX, ≤0.5).
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.
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