Candida albicans and Saccharomyces cerevisiae switch from a yeast to a filamentous form. In Saccharomyces, this switch is controlled by two regulatory proteins, Ste12p and Phd1p. Single-mutant strains, ste12/ste12 or phd1/phd1, are partially defective, whereas the ste12/ste12 phd1/phd1 double mutant is completely defective in filamentous growth and is noninvasive. The equivalent cph1/cph1 efg1/efg1 double mutant in Candida (Cph1p is the Ste12p homolog and Efg1p is the Phd1p homolog) is also defective in filamentous growth, unable to form hyphae or pseudohyphae in response to many stimuli, including serum or macrophages. This Candida cph1/cph1 efg1/efg1 double mutant, locked in the yeast form, is avirulent in a mouse model.

The in vitro activity of the novel triazole antifungal agent posaconazole (Noxafil; SCH 56592) was assessed in 45 laboratories against approximately 19,000 clinically important strains of yeasts and molds. The activity of posaconazole was compared with those of itraconazole, fluconazole, voriconazole, and amphotericin B against subsets of the isolates. Strains were tested utilizing Clinical and Laboratory Standards Institute broth microdilution methods using RPMI 1640 medium (except for amphotericin B, which was frequently tested in antibiotic medium 3). MICs were determined at the recommended endpoints and time intervals. Against all fungi in the database (22,850 MICs), the MIC 50 and MIC 90 values for posaconazole were 0.063 g/ml and 1 g/ml, respectively. MIC 90 values against all yeasts (18,351 MICs) and molds (4,499 MICs) were both 1 g/ml. In comparative studies against subsets of the isolates, posaconazole was more active than, or within 1 dilution of, the comparator drugs itraconazole, fluconazole, voriconazole, and amphotericin B against approximately 7,000 isolates of Candida and Cryptococcus spp. Against all molds (1,702 MICs, including 1,423 MICs for Aspergillus isolates), posaconazole was more active than or equal to the comparator drugs in almost every category. Posaconazole was active against isolates of Candida and Aspergillus spp. that exhibit resistance to fluconazole, voriconazole, and amphotericin B and was much more active than the other triazoles against zygomycetes. Posaconazole exhibited potent antifungal activity against a wide variety of clinically important fungal pathogens and was frequently more active than other azoles and amphotericin B.

The cytochrome P450 sterol 14␣-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole. Mutations in and around the CYP51 active site have resulted in azole resistance. In this work, homology models of the CYP51 enzymes from Aspergillus fumigatus and Candida albicans were constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis. Using these models, binding modes for voriconazole (VOR), fluconazole (FLZ), itraconazole (ITZ), and posaconazole (POS) were predicted from docking calculations. Previous work had demonstrated that mutations in the vicinity of the heme cofactor had a greater impact on the binding of FLZ and VOR than on the binding of POS and ITZ. Our modeling data suggest that the long side chains of POS and ITZ occupy a specific channel within CYP51 and that this additional interaction, which is not available to VOR and FLZ, serves to stabilize the binding of these azoles to the mutated CYP51 proteins. The model also predicts that mutations that were previously shown to specifically impact POS susceptibility in A. fumigatus and C. albicans act by interfering with the binding of the long side chain.

Real-time quantitative PCR was used to measure expression levels of genes encoding efflux pumps, ERG11 and two control genes, ACT1 and PMA1, in a collection of 14 fluconazole-susceptible Candida albicans isolates. For each gene, average expression levels and variations within the population were determined. These values were then used as reference points to make predictions about the molecular basis of resistance in 38 clinical isolates (the majority of which were resistant to fluconazole) obtained from 18 patients treated with posaconazole for refractory oropharyngeal candidiasis. For each of the 38 isolates, the expression levels of genes encoding efflux pumps, ERG11 and the control genes, were measured as above. Comparison of the two data sets revealed that expression of ACT1 and PMA1 did not vary significantly between the two sets of isolates. In contrast, MDR1, ERG11, CDR1, and CDR2 were overexpressed in 3, 4, 14, and 35, respectively, of the isolates from patients treated with azoles. In addition to these changes, the patient isolates all had at least one and often multiple missense mutations in ERG11. Select ERG11 alleles were expressed in Saccharomyces cerevisiae; all of the alleles tested conferred reduced susceptibility to fluconazole. Despite both the increases in pump expression and the ERG11 mutations, only one of the patient isolates exhibited a large decrease in posaconazole susceptibility.

To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 10 8 and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [ 14 C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14␣-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.

We have studied the in vivo activity of the new experimental triazole derivative SCH 56592 (posaconazole) against a variety of strains of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, in both immunocompetent and immunosuppressed murine hosts. The T. cruzi strains used in the study were previously characterized as susceptible (CL), partially resistant (Y), or highly resistant (Colombiana, SC-28, and VL-10) to the drugs currently in clinical use, nifurtimox and benznidazole. Furthermore, all strains are completely resistant to conventional antifungal azoles, such as ketoconazole. In the first study, acute infections with the CL, Y, and Colombiana strains in both normal and cyclophosphamide-immunosuppressed mice were treated orally, starting 4 days postinfection (p.i.), for 20 consecutive daily doses. The results indicated that in immunocompetent animals SCH 56592 at 20 mg/kg of body weight/day provided protection (80 to 90%) against death caused by all strains, a level comparable or superior to that provided by the optimal dose of benznidazole (100 mg/kg/day). Evaluation of parasitological cure revealed that SCH 56592 was able to cure 90 to 100% of the surviving animals infected with the CL and Y strains and 50% of those which received the benznidazole-and nifurtimox-resistant Colombiana strain. Immunosuppression markedly reduced the mean survival time of untreated mice infected with any of the strains, but this was not observed for the groups which received SCH 56592 at 20 mg/kg/day or benznidazole at 100 mg/kg/day. However, the overall cure rates were higher for animals treated with SCH 56592 than among those treated with benznidazole. The results were confirmed in a second study, using the same model but a longer (43-dose) treatment period. Finally, a model for the chronic disease in which oral treatment was started 120 days p.i. and consisted of 20 daily consecutive doses was investigated. The results showed that SCH 56592 at 20 mg/kg/day was able to induce a statistically significant increase in survival of animals infected with all strains, while benznidazole at 100 mg/kg/day was able to increase survival only in animals infected with the Colombiana strain. Moreover, the triazole was able to induce parasitological cures in 50 to 60% of surviving animals, irrespective of the infecting strain, while no cures were obtained with benznidazole. Taken together, the results demonstrate that SCH 56592 has in vivo trypanocidal activity, even against T. cruzi strains naturally resistant to nitrofurans, nitroimidazoles, and conventional antifungal azoles, and that this activity is retained to a large extent in immunosuppressed hosts.Chemotherapy of Chagas' disease (American trypanosomiasis), a parasitic disease caused by the kinetoplastid protozoan Trypanosoma (Schizotrypanum) cruzi which afflicts 16 to 18 million people in Latin America, remains an enormous scientific and social challenge, as the drugs currently available, nitrofurans (nifurtimox; Bayer) and nitroimi...

Previous in vivo studies have characterized the pharmacodynamic characteristics of two triazole compounds, fluconazole and ravuconazole. These investigations demonstrated that the 24-h area under the concentrationtime curve (AUC)/MIC ratio is the critical pharmacokinetic-pharmacodynamic (PK-PD) parameter associated with treatment efficacy. Further analysis demonstrated that a free-drug triazole 24-h AUC/MIC ratio of 20 to 25 was predictive of treatment success in both experimental models and clinical trials. We used a neutropenic murine model of disseminated Candida albicans infection to similarly characterize the time course activity of the new triazole, posaconazole. The PK-PD parameters (percent time above MIC, AUC/MIC ratio, and peak serum drug level/MIC ratio) were correlated with in vivo efficacy, as measured by organism number in kidney cultures after 48 h of therapy. Kinetics and protein binding following oral posaconazole dosing were performed in neutropenic infected mice. Peak levels and AUC from 0 h to ؕ values were nonlinear over the 16-fold dose range studied. Serum drug elimination half-life ranged from 12.0 to 17.7 h. Protein binding was 99%. Single dose postantifungal effect studies demonstrated prolonged suppression of organism regrowth after serum posaconazole levels had fallen below the MIC. Treatment efficacy with the four dosing intervals studied was similar, supporting the AUC/MIC ratio as the PK-PD parameter predictive of efficacy. Nonlinear regression analysis also suggested that the AUC/MIC ratio was strongly predictive of treatment outcomes (AUC/MIC ratio R 2 ‫؍‬ 83%; peak serum drug/MIC ratio R 2 ‫؍‬ 85%; time that serum levels of posaconazole remained above the MIC R 2 ‫؍‬ 65%). Similar studies were conducted with 11 additional C. albicans isolates with various posaconazole susceptibilities (MIC, 0.015 to 0.12 g/ml) to determine if a similar 24-h AUC/MIC ratio was associated with efficacy. The posaconazole free-drug AUC/MIC ratios were similar for all of the organisms studied (6.12 to 26.7, mean ؎ SD ‫؍‬ 16.9 ؎ 7.8, P value, 0.42). These free-drug AUC/MIC ratios are similar to those observed for other triazoles in this model. Antimicrobial pharmacodynamic characterizations have provided an understanding of the relationship between drug exposure and treatment efficacy. Therapeutic outcome predictions based upon these pharmacodynamic studies have correlated well in treatment against both susceptible and resistant pathogens (3). In addition, the strength of these in vivo predictions has been shown to be independent of animal species, infection site, and duration of treatment studied. These pharmacodynamic analyses in animal infection models have proven useful for the design of optimal dosing regimens and the validation of susceptibility breakpoint guidelines (8, 9, 18).Prior in vivo studies have demonstrated that the pharmacokinetic-pharmacodynamic (PK-PD) parameter predictive of triazole efficacy against Candida albicans is the 24-h area under the concentration-time curve (AUC)/MIC rati...

The in vivo activities of posaconazole, itraconazole, and amphotericin B in neutropenic mice with zygomycosis were compared. The in vitro MICs of posaconazole and itraconazole for the strains of Mucor spp. used in this study ranged from 0.125 to 8 g/ml and 0.25 to 8 g/ml, respectively. The in vitro MIC range for amphotericin B is 0.125 to 0.25 g/ml. At twice-daily doses of >15 mg/kg of body weight, posaconazole prolonged the survival of the mice and reduced tissue burden.Zygomycosis is a relatively uncommon but highly aggressive fungal infection that affects diabetics, neutropenic patients, and subjects with burns or iron overload but that only rarely affects healthy people (15,19). Illness may rapidly progress with angioinvasion and tissue infarction. The existing methods for treatment are often ineffective (4, 9, 17). Presently, therapy utilizes aggressive surgical measures and high doses of amphotericin B. There are scattered reports of lipid-associated amphotericin B in salvage, but there is no evidence that these forms are more efficacious than amphotericin B (2, 3, 5). Even with aggressive therapy, mortality is often above 50% (6). Of the alternative antifungals, itraconazole has been effective in vitro, particularly against Absidia spp. One mouse study has also shown some activity against Absidia, but the clinical evidence of efficacy is less clear (2, 6, 10, 16; E. Dannaoui, J. Meletiadis, J. Meis, J. W. Moulton, and P. E. Verweij, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 939, 2000). Posaconazole is a new broad-spectrum triazole with activity against many filamentous fungal pathogens (1,8,12,18). This study describes the in vivo activity of posaconazole against three Mucor spp. isolates in a neutropenic-mouse model.Three clinical Mucor isolates were tested in vitro by the NCCLS microdilution method for triazoles adapted to filamentous fungi (11). The endpoint was a visual MIC at which azoles were seen to reduce growth by 80% compared to that in the drug-free control tube. The MIC of amphotericin B was taken as the least drug producing a visually clear tube. MICs of posaconazole and itraconazole at 48 h were 0.125 and 0.25 g/ml for Mucor ramosissimus strain 98-1763, 0.25 and 0.25 g/ml for Mucor ramosissimus 95-2650, and 8 and 8 g/ml for Mucor circinelloides 00-1194, respectively. The 48-h MIC of amphotericin B was 0.25 g/ml for all three isolates. The isolates were grown on potato flake agar plates at room temperature for 1 week. They were harvested by scraping the plates with sterile isotonic saline and filtering the suspension through glass wool. The inoculum was calculated by determining hemacytometer counts.For the mouse model, we used 18-week-old BALB/c males. One day before infection, mice were rendered neutropenic with single doses of 5-fluorouracil administered intravenously at 150 mg/kg of body weight and with cyclophosphamide administered intraperitoneally at 200 mg/kg. In groups of five uninfected mice, this treatment reduced the neutrophil count from a median pretreatm...