Roberta S. Hare

In Vitro Activities of Posaconazole, Fluconazole, Itraconazole, Voriconazole, and Amphotericin B against a Large Collection of Clinically Important Molds and Yeasts

ABSTRACT 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 MIC50 and MIC90 values for posaconazole were 0.063 μg/ml and 1 μg/ml, respectively. MIC90 values against all yeasts (18,351 MICs) and molds (4,499 MICs) were both 1 μg/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.

Antibiotic Susceptibility Profiles of Escherichia coli Strains Lacking Multidrug Efflux Pump Genes

ABSTRACT The contribution of seven known and nine predicted genes or operons associated with multidrug resistance to the susceptibility of Escherichia coli W3110 was assessed for 20 different classes of antimicrobial compounds that include antibiotics, antiseptics, detergents, and dyes. Strains were constructed with deletions for genes in the major facilitator superfamily, the resistance nodulation-cell division family, the small multidrug resistance family, the ATP-binding cassette family, and outer membrane factors. The agar dilution MICs of 35 compounds were determined for strains with deletions for multidrug resistance (MDR) pumps. Deletions in acrAB or tolC resulted in increased susceptibilities to the majority of compounds tested. The remaining MDR pump gene deletions resulted in increased susceptibilities to far fewer compounds. The results identify which MDR pumps contribute to intrinsic resistance under the conditions tested and supply practical information useful for designing sensitive assay strains for cell-based screening of antibacterial compounds.

Mutations in Aspergillus fumigatus Resulting in Reduced Susceptibility to Posaconazole Appear To Be Restricted to a Single Amino Acid in the Cytochrome P450 14α-Demethylase

ABSTRACT 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 108 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 [14C]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 14α-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.

Posaconazole for the Treatment of Azole-Refractory Oropharyngeal and Esophageal Candidiasis in Subjects with HIV Infection

BACKGROUND We evaluated the efficacy and safety of oral posaconazole for human immunodeficiency virus (HIV)-infected subjects with oropharyngeal candidiasis (OPC) and/or esophageal candidiasis (EC) who were clinically refractory to treatment with oral fluconazole or itraconazole. METHODS Subjects with confirmed OPC or EC who did not improve after receiving standard courses of fluconazole or itraconazole treatment were eligible for study enrollment. Subjects received either oral posaconazole (400 mg twice daily) for 3 days followed by oral posaconazole (400 mg once daily) for 25 days (regimen A; 103 patients) or oral posaconazole (400 mg twice daily) for 28 days (regimen B; 96 patients). The primary end point was cure or improvement after 28 days. Primary efficacy analyses were performed on the subset of treated subjects with refractory disease (e.g., baseline culture positive for fluconazole- or itraconazole-resistant Candida species or persistent or progressive clinical signs or symptoms consistent with treatment failure). RESULTS Of the modified intent-to-treat population, 132 (75%) of 176 subjects achieved a clinical response to posaconazole treatment. Clinical response rates were similar between regimen A recipients (75.3%) and regimen B recipients (74.7%). Clinical responses occurred in 67 (73%) of 92 subjects with baseline isolates resistant to fluconazole, 49 (74%) of 66 subjects with baseline isolates resistant to itraconazole, and 42 (74%) of 57 subjects with isolates resistant to both. Clinical response was achieved in 32 (74.4%) of 43 subjects with endoscopically documented EC. The most common treatment-related adverse events were diarrhea (11%), neutropenia (7%), flatulence (6%), and nausea (6%). Eight subjects (4%) discontinued therapy as a result of a treatment-related adverse event. CONCLUSIONS Posaconazole offers a safe and effective treatment option for HIV-infected subjects with azole-refractory OPC and/or EC.

Cytokine Networking in Lungs of Immunocompetent Mice in Response to Inhaled Aspergillus fumigatus

ABSTRACT Cytokine networking in the lung in response to inhaledAspergillus fumigatus was assessed using a murine model of primary pulmonary aspergillosis in immunocompetent Crl:CF-1 mice. Inhalation of virulent A. fumigatus (6 × 106 CFU) resulted in the induction of interleukin 18 (IL-18), tumor necrosis factor alpha (TNF-α), IL-12, and gamma interferon (IFN-γ) protein in bronchoalveolar lavage fluid and/or lung tissue. Induction of immunoreactive IL-18 preceded induction of TNF-α protein, which preceded induction of immunoreactive IL-12 and IFN-γ. Real-time reverse transcriptase (RT) PCR analysis of infected lung tissue demonstrated that induction of IL-18 protein also preceded induction of pulmonary TNF-α, IL-12, and IFN-γ mRNAs. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to the IL-18 receptor (anti-IL-18R MAb), TNF-α (anti-TNF-α MAb), IL-12 (anti-IL-12 MAb), and/or IFN-γ (anti-IFN-γ MAb), and effects on intrapulmonary cytokine activity and growth of A. fumigatuswere assessed in infected lung homogenates. Simultaneous neutralization of IL-12 and IL-18 resulted in decreased levels of immunoreactive TNF-α, while neutralization of IL-18, TNF-α, or IL-12 alone or of IL-18 and IL-12 together resulted in decreased levels of immunoreactive IFN-γ. Simultaneous neutralization of IL-12 and IL-18 or neutralization of TNF-α alone or in combination with IL-12, IL-18, or IFN-γ also resulted in a significant increase inA. fumigatus CFU in lung tissue. Taken together, these results demonstrate that endogenous IL-18, IL-12, and TNF-α, through their modulatory effects on both intrapulmonary cytokine activity and growth of A. fumigatus, play key roles in host defense against primary pulmonary aspergillosis.

Comparison of Pathogenesis and Host Immune Responses to Candida glabrata and Candida albicans in Systemically Infected Immunocompetent Mice

ABSTRACT Cytokine-mediated host defense against Candida glabratainfection was compared to that against C. albicans, using immunocompetent murine models of systemic candidiasis. The pathogenesis of infection was evaluated morphologically and by culture of target organs, while the kinetics of induction of cytokine mRNAs and corresponding proteins were determined in kidneys by real-time reverse transcription-PCR and cytokine-specific murine enzyme-linked immunosorbent assays, respectively. Systemic infection with C. glabrata resulted in a chronic, nonfatal infection with recovery of organisms from kidneys, while intravenous inoculation with C. albicans resulted in rapid mortality with logarithmic growth of organisms in kidneys and recovery of C. albicans from the spleen, liver, and lungs. Survival of C. glabrata-infected mice was associated with rapid induction of mRNAs and corresponding immunoreactive proteins for the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and gamma interferon (IFN-γ) and the lack of induction of protein for the anti-inflammatory cytokine IL-10. In contrast, mortality in C. albicans-infected mice was associated with induction of mRNA and corresponding protein for IL-10 but delayed (i.e., TNF-α) or absent (i.e., IL-12 and IFN-γ) induction of immunoreactive proinflammatory cytokines. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to TNF-α, IL-12, or IFN-γ, and the effect on growth of C. glabrata in kidneys was assessed. Neutralization of endogenous TNF-α resulted in a significant increase in C. glabrata organisms compared to similarly infected mice administered an isotype-matched control MAb, while neutralization of endogenous IL-12 or IFN-γ had no significant effect on C. glabrata replication. These results demonstrate that in response to intravenous inoculation of C. glabrata, immunocompetent mice develop chronic nonfatal renal infections which are associated with rapid induction of the proinflammatory cytokines TNF-α, IL-12, and IFN-γ. Furthermore, TNF-α plays a key role in host defense against systemic candidiasis caused by either C. glabrata or C. albicans, as the absence of endogenous TNF-α activity was associated with enhanced tissue burden in both infection models.

In vitro and in vivo activities of SCH 56592 (Posaconazole), a new triazole antifungal agent, against Aspergillus and Candida

ABSTRACT SCH 56592 (posaconazole), a new triazole antifungal agent, was tested in vitro, and its activity was compared to that of itraconazole against 39 Aspergillus strains and to that of fluconazole against 275 Candida and 9 Cryptococcus strains. The SCH 56592 MICs for Aspergillus ranged from ≤0.002 to 0.5 μg/ml, and those of itraconazole ranged from ≤0.008 to 1 μg/ml. The SCH 56592 MICs for Candida andCryptococcus strains ranged from ≤0.004 to 16 μg/ml, and those of fluconazole ranged from ≤0.062 to >64 μg/ml. SCH 56592 showed excellent activity against Aspergillus fumigatus andAspergillus flavus in a pulmonary mouse infection model. When administered therapeutically, the 50% protective doses (PD50s) of SCH 56592 ranged from 3.6 to 29.9 mg/kg of body weight, while the PD50s of SCH 56592 administered prophylactically ranged from 0.9 to 9.0 mg/kg; itraconazole administered prophylactically was ineffective (PD50s, >75 mg/kg). SCH 56592 was also very efficacious against fluconazole-susceptible, -susceptible dose-dependent, or -resistantCandida albicans strains in immunocompetent or immunocompromised mouse models of systemic infection. The PD50s of SCH 56592 administered therapeutically ranged from 0.04 to 15.6 mg/kg, while the PD50s of SCH 56592 administered prophylactically ranged from 1.5 to 19.4 mg/kg. SCH 56592 has excellent potential for therapy against seriousAspergillus or Candida infections.

Pharmacokinetics of SCH 56592, a new azole broad-spectrum antifungal agent, in mice, rats, rabbits, dogs, and cynomolgus monkeys.

ABSTRACT SCH 56592 is a new broad-spectrum azole antifungal agent that is in phase 3 clinical trials for the treatment of serious systemic fungal infections. The pharmacokinetics of this drug candidate were evaluated following its intravenous (i.v.) or oral (p.o.) administration as a solution in hydroxypropyl-β-cyclodextrin (HPβCD) or oral administration as a suspension in 0.4% methylcellulose (MC) in studies involving mice, rats, rabbits, dogs, and cynomolgus monkeys. SCH 56592 was orally bioavailable in all species. The oral bioavailability was higher with the HPβCD solution (range, 52 to ∼100%) than from the MC suspension (range, 14 to 48%) and was higher in mice (∼100% [HPβCD] and 47% [MC]), rats (∼66% [HPβCD] and 48% [MC]), and dogs (72% [HPβCD] and 37% [MC]) than in monkeys (52% [HPβCD] and 14% [MC]). In rabbits, high concentrations in serum suggested good oral bioavailability with the MC suspension. The i.v. terminal-phase half-lives were 7 h in mice and rats, 15 h in dogs, and 23 h in monkeys. In rabbits, the oral half-life was 9 h. In species given increasing oral doses (mice, rats, and dogs), serum drug concentrations were dose related. Food produced a fourfold increase in serum drug concentrations in dogs. Multiple daily doses of 40 mg of SCH 56592/kg of body weight for eight consecutive days to fed dogs resulted in higher concentrations in serum, indicating accumulation upon multiple dosing, with an accumulation index of approximately 2.6. Concentrations above the MICs and minimum fungicidal concentrations for most organisms were observed at 24 h following a single oral dose in MC suspension in all five species studied (20 mg/kg for mice, rats, and rabbits and 10 mg/kg for dogs and monkeys), suggesting that once-daily administration of SCH 56592 in human subjects would be a therapeutically effective dosage regimen.

Posaconazole Is a Potent Inhibitor of Sterol 14α-Demethylation in Yeasts and Molds

ABSTRACT Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata. In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C. albicans (both azole-sensitive and azole-resistant strains), C. glabrata, Aspergillus fumigatus, and Aspergillus flavus. Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy. Ergosterol was the major sterol in all but one of the strains; C. glabrata strain C110 synthesized an unusual sterol in place of ergosterol. Exposure to POS led to a decrease in the total sterol content of all the strains tested. The decrease was accompanied by the accumulation of 14α-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14α-demethylase enzyme. The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested. POS retained activity against C. albicans isolates with mutated forms of the 14α-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR. In addition, POS was a more potent inhibitor of sterol synthesis in A. fumigatus and A. flavus than either ITZ or VOR.

Correlation between aminoglycoside resistance profiles and DNA hybridization of clinical isolates.

DNA hybridization data and aminoglycoside resistance profiles (AGRPs) were determined for 4,088 clinical isolates from three studies (United States, Belgium, and Argentina). The correlation between susceptibility profiles and hybridization results was determined with nine DNA probes. For each of the seven aminoglycoside resistance profiles which we were able to test, the data suggested at least two distinct genes could encode enzymes which lead to identical resistance profiles. Furthermore, the DNA hybridization data showed that individual strains carried up to six unique aminoglycoside resistance genes. DNA hybridization revealed interesting differences in the frequencies of these genes by organism and by country.