Current and emerging azole antifungal agents.

Major developments in research into the azole class of antifungal agents during the 1990s have provided expanded options for the treatment of many opportunistic and endemic fungal infections. Fluconazole and itraconazole have proved to be safer than both amphotericin B and ketoconazole. Despite these advances, serious fungal infections remain difficult to treat, and resistance to the available drugs is emerging. This review describes present and future uses of the currently available azole antifungal agents in the treatment of systemic and superficial fungal infections and provides a brief overview of the current status of in vitro susceptibility testing and the growing problem of clinical resistance to the azoles. Use of the currently available azoles in combination with other antifungal agents with different mechanisms of action is likely to provide enhanced efficacy. Detailed information on some of the second-generation triazoles being developed to provide extended coverage of opportunistic, endemic, and emerging fungal pathogens, as well as those in which resistance to older agents is becoming problematic, is provided.

Mechanism of fluconazole resistance in Candida krusei.

The mechanisms of fluconazole resistance in three clinical isolates of Candida krusei were investigated. Analysis of sterols of organisms grown in the absence and presence of fluconazole demonstrated that the predominant sterol of C. krusei is ergosterol and that fluconazole inhibits 14alpha-demethylase in this organism. The 14alpha-demethylase activity in cell extracts of C. krusei was 16- to 46-fold more resistant to inhibition by fluconazole than was 14alpha-demethylase activity in cell extracts of two fluconazole-susceptible strains of Candida albicans. Comparing the carbon monoxide difference spectra of microsomes from C. krusei with those of microsomes from C. albicans indicated that the total cytochrome P-450 content of C. krusei is similar to that of C. albicans. The Soret absorption maximum in these spectra was located at 448 nm for C. krusei and at 450 nm for C. albicans. Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not greater than that of C. albicans. Thus, there are significant qualitative differences between the 14alpha-demethylase of C. albicans and C. krusei. In addition, fluconazole resistance in these strains of C. krusei appears to be mediated predominantly by a reduced susceptibility of 14alpha-demethylase to inhibition by this drug.

The search for new triazole antifungal agents.

The first generation antifungal agent triazoles, fluconazole and itraconazole, have revolutionised the treatment of serious fungal infections such as mucosal and invasive candidiasis and cryptococcal meningitis. However, the treatment of some fungal infections, particularly aspergillosis, is still far from satisfactory and thus there is an important requirement for new broad-spectrum antifungal agents. The new second generation triazoles voriconazole and SCH-56592 show considerable promise in achieving this goal in the near future.

Comparison of voriconazole (UK-109,496) and itraconazole in prevention and treatment of Aspergillus fumigatus endocarditis in guinea pigs.

Left-sided Aspergillus fumigatus endocarditis was established in the guinea pig heart by catheterization and inoculation with conidia via a tributary of the femoral vein. This animal model was used to compare the efficacy of the triazole antifungal agents voriconazole (UK-109,496) and itraconazole. In the prophylaxis experiments, voriconazole at a dosage of 10 mg/kg of body weight given intraperitoneally twice daily prevented A. fumigatus endocarditis in all but 1 animal (11 of 12 animals were cured). Itraconazole did not prevent Aspergillus endocarditis when it was given at the same dosage and by the same route (0 to 12 animals were cured). In the treatment experiments with 10 animals per group, voriconazole at 10, 7.5 and 5 mg/kg given orally twice daily for 7 days produced cure rates of 100, 70 and 0%, respectively. In contrast, itraconazole at 10 mg/kg given orally twice daily did not cure A. fumigatus endocarditis in the guinea pig. It is concluded that voriconazole is highly efficacious in the prevention and treatment of Aspergillus endocarditis in the guinea pig and is superior to itraconazole in these respects.

Microdilution antifungal susceptibility testing of Candida albicans and Cryptococcus neoformans with and without agitation: an eight-center collaborative study.

The growth patterns observed in the trailing wells when fluconazole is being tested may give rise to readings that suggest resistance or increased MICs for known susceptible strains. We conducted a multicenter study to evaluate the intralaboratory and interlaboratory reproducibilities of a method that uses agitation to disperse these types of growth. Ten strains of Candida albicans and five strains of Cryptococcus neoformans were tested against fluconazole, flucytosine, and amphotericin B by using a microdilution adaptation of the proposed reference method of the National Committee for Clinical Laboratory Standards for yeasts (M27-T). The endpoint criterion used before agitation was consistent with the M27-T recommendation, while a criterion of 50% or more reduction of growth compared with the control was used after agitation. The results of this study showed that use of agitation and the modified endpoint criterion both improved intralaboratory and inter-laboratory agreement and increased the frequency of interpretable MICs. The MICs obtained by this method were comparable to those obtained by the broth macrodilution M27-T method. Like M27-T, this method was not able to definitely distinguish amphotericin B-susceptible from -resistant strains, although the MICs for the resistant strains were consistently higher than those for the susceptible ones. The findings imply that agitation should be seriously considered when antifungal agents, particularly fluconazole, are tested in a microdilution format.

Increased cytochrome P-450 activity in Aspergillus fumigatus after xenobiotic exposure.

Microsomal fractions were isolated from Aspergillus fumigatus cultures that had been treated with dimethylsulphoxide (DMSO), ethanol, benzopyrene, phenobarbital or naphthalene. All these xenobiotics increased microsomal cytochrome P-450 content. Cytochrome P-450 reductase activity remained unaffected and no alteration in the microsomal protein profile was detectable by SDS PAGE. Benzopyrene, dimethylaniline, hexobarbitol and p-nitroanisole were metabolised by A. fumigatus microsomes, while aniline, ethoxyresorufin and pentoxyresorufin were not. No xenobiotic elicited a more specific oxidation of any of the substrates. A fumigatus microsomes also catalyzed the cell-free biosynthesis of ergosterol and quantitative analysis of assay metabolites showed that exposure to ethanol, benzopyrene or phenobarbitone in vivo resulted in enhanced ergosterol biosynthesis in vitro.

Fluconazole therapy of oropharyngeal candidiasis in a patient with multiple endocrine failure does not correlate with Candida albicans susceptibility to fluconazole in vitro.

We report a case of oropharyngeal and oesophageal candidiasis in a 23-year-old man with endocrinopathy syndrome. Multiple episodes of infection were treated with topical miconazole, oral ketoconazole (200 mg daily) or oral fluconazole (50 mg daily) over a period of 7 years. The final episode failed to respond to ketoconazole (200 mg daily) or fluconazole (200 mg daily), but was treated successfully by increasing the fluconazole dose to 400 mg daily for 6 months. The patient was maintained on fluconazole 200 mg daily without relapse. Serial Candida albicans isolates from the oral cavity were clonally related by RFLP analyses of genomic DNA, and were resistant to fluconazole, ketoconazole and itraconazole in vitro. We conclude that fluconazole 400 mg daily is effective against oropharyngeal and oesophageal candidiasis in a patient with endocrinopathy syndrome, despite the infecting Candida albicans strains being resistant to azole antifungals in vitro.

Correlation between rhodamine 123 accumulation and azole sensitivity in Candida species: possible role for drug efflux in drug resistance.

A wide variety of prokaryotic and eukaryotic cells exhibit a multidrug resistance (MDR) phenotype, indicating that resistance to potentially toxic compounds is mediated by their active efflux from the cell. We have sought to determine whether resistance to azoles in some strains of Candida species may be due in part to active drug efflux. Rhodamine 123 (Rh123) is a fluorescent compound that is transported by a wide variety of MDR cell types. We have shown that certain azole-resistant strains of Candida albicans, C. glabrata, and C. krusei accumulate less Rh123 than azole-susceptible ones. In C. albicans, Rh123 accumulation was growth phase and temperature dependent and was increased by proton uncouplers and by reserpine, an MDR modulator. This is consistent with an energy-dependent efflux mechanism for Rh123, mediated by an MDR transporter. In C. glabrata, but not in C. albicans, there was competition between Rh123 and fluconazole for efflux. Thus, in C. glabrata, Rh123 and fluconazole appear to be transported via a common MDR-like transporter, whereas in C. albicans, the Rh123 transporter does not appear to transport azoles.

Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility.

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae.

Fluconazole resistance due to energy-dependent drug efflux in Candida glabrata.

We report on the mechanism of fluconazole resistance in Candida glabrata from a case of infection in which pre- and posttreatment isolates were available for comparison. The resistant, posttreatment isolate was cross-resistant to ketoconazole and itraconazole, in common with other azole-resistant yeasts. Resistance was due to reduced levels of accumulation of [3H]fluconazole rather than to changes at the level of ergosterol biosynthesis. Studies with metabolic or respiratory inhibitors showed that this phenomenon was a consequence of energy-dependent drug efflux, as opposed to a barrier to influx. Since energy-dependent efflux is a characteristic of multidrug resistance in bacteria, yeasts, and mammalian cells, we investigated the possibility that fluconazole resistance is mediated by a multidrug resistance-type mechanism. Benomyl, a substrate for the Candida albicans multidrug resistance protein, showed competition with fluconazole for efflux from resistance C. glabrata isolates, consistent with a common efflux mechanism for these compounds. By contrast, other standard substrates or inhibitors of multidrug resistance proteins had no effect on fluconazole efflux. In conclusion, we have identified energy-dependent efflux of fluconazole, possibly via a multidrug resistance-type transporter, as the mechanism of resistance to fluconazole in C. glabrata.

Combination therapy of murine invasive candidiasis with fluconazole and amphotericin B.

A study was performed to assess the in vivo relevance of the in vitro antagonism between fluconazole and amphotericin B against Candida albicans. Combinations of fluconazole and amphotericin B were explored for their efficacies against acute (100% mortality in 2 to 5 days) or less acute (100% mortality in 30 days) invasive candidiasis infections in mice with healthy immune systems and immunocompromised mice. Treatment efficacy was assessed by protection from mortality and/or a reduction in the fungal burden in tissue. In models of acute infection in mice with healthy immune systems or less acute infection in immunocompromised mice, combinations of fluconazole and amphotericin B were superior to fluconazole alone, and the effects were at least additive. Combination therapy was at least as efficacious as amphotericin B alone. In a different model of less acute infection in mice with healthy immune systems, combinations of fluconazole and amphotericin B showed no interactions and were no better than either drug alone. We conclude that combination therapy with fluconazole and amphotericin B is not antagonistic in vivo, in contrast to published in vitro studies, and, consequently, suggest that combination therapy should be considered in the management of clinical candidiasis.

The oncogenic potential of Candida in the female genital tract.

The possible role of Candida species in carcinogenesis at the uterine cervix was investigated in 226 females attending a colposcopy clinic. Approximately 34% of the 226 subjects harbored Candida species in cervical/vaginal secretions, but there was no association with any particular histologic abnormality. Two independent analytical procedures were used for strain discrimination of the isolates of C. albicans, but again no relationship was found between individual strains and histologic diagnoses. Only three C. glabrata strains were isolated, but they were all in association with cervical intraepithelial neoplasia (CIN) II or III. A total of 18 strains of C. albicans, one C. glabrata and one C. parapsilosis all inhibited the formation of the nitrosamine nitrosodimethylamine (NDMA) from precursors. Furthermore, C. albicans strains did not convert NDMA to carcinogenic metabolites. The results of this study do not suggest that C. albicans has a role in cervical carcinogenesis.

Fluconazole resistance in Candida glabrata.

We report a case of infection with Candida glabrata in which the organism became resistant to fluconazole and in which pre- and posttreatment isolates were available for comparison. The organism was cross-resistant to ketoconazole and itraconazole, in common with other azole-resistant yeasts. Fluconazole was a potent inhibitor of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) in lysates of cells from both susceptible and resistant cultures (50% inhibitory concentration, 0.2 microM), indicating that resistance was unrelated to changes in P-450DM. Instead, it appeared to arise from a permeability barrier to fluconazole, since resistant cells were unable to take up radiolabelled drug.

Microsomal chitinase activity from Candida albicans.

Chitinase (E.C. 3.2.1.14) was characterized in microsomal fractions from yeast cells of Candida albicans. Following six washes with buffer (50 mM Bis-Tris.Cl, pH 6.5), enzyme activity of microsomes fell markedly to 0.3% of total and 6% of the specific activity detected in the low-speed supernatant (9000 X g) of a cell lysate. An apparently zymogenic, microsomal chitinase activity became more readily detectable with washing and after six washes enzyme activity was activated 1.7-fold following pre-incubation with trypsin. The following properties of microsomal chitinase were closely comparable with those for cytosolic chitinase (indicated in parentheses): Km = 2.1 mg chitin per ml (2.9 mg chitin per ml); temperature optimum = 45 degrees C (45 degrees C); inhibition by allosamidin competitive, Ki = 0.29 microM (competitive, Ki = 0.23 microM). A range of detergents solubilized and activated microsomal chitinase in a highly specific manner. Following density gradient centrifugation of microsomes, chitinase was distributed approximately evenly throughout the gradient suggesting that microsomal chitinase is not associated exclusively with any one membrane component. The possible morphogenetic role of microsomal chitinase is discussed in relation to the potential of this enzyme as a target for highly specific antifungal agents.

Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14 alpha-sterol demethylase purified from Candida albicans.

The interaction of azole antifungal antibiotics with purified Candida albicans cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) was measured spectrophotometrically and by inhibition of enzyme activity. Ketoconazole and ICI 153066 (a triazole derivative) formed low-spin complexes with the ferric cytochrome and induced type II difference spectra. These spectra are indicative of an interaction between the azole moiety and the sixth co-ordination position of P-450DM haem. Both azoles inhibited the binding of CO to the sodium dithionite-reduced ferrous cytochrome, and inhibited reconstituted P-450DM activity by binding to the cytochrome with a one-to-one stoichiometry. Similarly, total inhibition of enzyme activity occurred when equimolar amounts of clotrimazole, miconazole or fluconazole were added to reconstituted P-450DM. These results correlated with the inhibition of P-450DM in broken cell preparations, confirming that all five azoles are potent inhibitors of ergosterol biosynthesis in C. albicans.

Purification and properties of cytochrome P-450-dependent 14 alpha-sterol demethylase from Candida albicans.

The purification of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) from the important opportunistic fungal pathogen, Candida albicans, is described. Optimal purification (875-fold) was achieved by extracting the cytochrome from microsomes with sodium cholate followed by hydroxyapatite, octyl-Sepharose and CM-Sepharose chromatographies, giving a cytochrome preparation of 17.5 nmol/mg of protein. By the use of SDS/polyacrylamide-gel electrophoresis the cytochrome was judged to be highly purified on the basis of Coomassie Brilliant Blue staining of protein. The Mr of P-450DM was estimated to be 51,000. The absorption spectrum of oxidized P-450DM was characteristic of a low-spin cytochrome, and its reduced CO complex had a Soret absorption peak at 447 nm. When reconstituted in a model membrane system of dilauroylphosphatidylcholine with NADPH and O2, P-450DM catalysed the complete 14 alpha-demethylation of lanosterol, which was inhibited by CO. The cytochrome appeared to have a high degree of substrate specificity; it was unable to oxidize a number of xenobiotic compounds in the reconstituted assay.