Posaconazole (Noxafil, SCH 56592), a new azole antifungal drug, was a discovery based on the isolation and mass spectral characterization of a circulating metabolite of an earlier lead (SCH 51048).

Posaconazole (SCH 56592) is a novel triazole antifungal drug that is marketed in Europe and the United States under the trade name 'Noxafil' for prophylaxis against invasive fungal infections. SCH 56592 was discovered as a possible active metabolite of SCH 51048, an earlier lead. Initial studies have shown that serum concentrations determined by a microbiological assay were higher than those determined by HPLC from animals dosed with SCH 51048. Subsequently, several animals species were dosed with (3)H-SCH 51048 and the serum was analyzed for total radioactivity, SCH 51048 concentration and antifungal activity. The antifungal activity was higher than that expected based on SCH 51048 serum concentrations, confirming the presence of active metabolite(s). Metabolite profiling of serum samples at selected time intervals pinpointed the peak that was suspected to be the active metabolite. Consequently, (3)H-SCH 51048 was administered to a large group of mice, the serum was harvested and the metabolite was isolated by extraction and semipreparative HPLC. LC-MS/MS analysis suggested that the active metabolite is a secondary alcohol with the hydroxyl group in the aliphatic side chain of SCH 51048. All corresponding monohydroxylated diastereomeric mixtures were synthesized and characterized. The HPLC retention time and LC-MS/MS spectra of the diastereomeric secondary alcohols of SCH 51048 were similar to those of the isolated active metabolite. Finally, all corresponding individual monohydroxylated diasteriomers were synthesized and evaluated for in vitro and in vivo antifungal potencies, as well as pharmacokinetics. SCH 56592 emerged as the candidate with the best overall profile.

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.

Posaconazole as salvage treatment for invasive fusariosis in patients with underlying hematologic malignancy and other conditions.

BACKGROUND: Conventional amphotericin B-based antifungal therapy for invasive fusariosis in patients with a hematologic malignancy results in a > or = 70% failure rate. Posaconazole is a broad-spectrum antifungal agent with in vitro and in vivo activity against Fusarium species. METHODS: In this retrospective analysis of patients from 3 open-label clinical trials, we evaluated posaconazole for the treatment of invasive fusariosis. Twenty-one patients with proven or probable invasive fusariosis who had disease refractory to or who were intolerant of standard antifungal therapy received oral posaconazole suspension (800 mg per day in divided doses) as salvage therapy. RESULTS: Successful outcome occurred in 10 (48%) of all 21 patients. Among patients with leukemia who received posaconazole therapy for >3 days, the overall success rate was 50%; for patients who recovered from myelosuppression, the success rate was 67%, compared with 20% for those with persistent neutropenia. CONCLUSION: These results suggest that posaconazole is useful for the treatment of invasive fusariosis.

Posaconazole is effective as salvage therapy in zygomycosis: a retrospective summary of 91 cases.

To evaluate the activity of posaconazole for treatment of zygomycosis, a disease for which therapeutic options are limited, we conducted a retrospective study including 91 patients with zygomycosis (proven zygomycosis, 69 patients; probable zygomycosis, 22 patients). Patients had infection that was refractory to prior antifungal treatment (n=81) or were intolerant of such treatment (n=10) and participated in the compassionate-use posaconazole (800 mg/day) program. The rate of success (i.e., either complete or partial response) at 12 weeks after treatment initiation was 60%, and 21% of patients had stable disease. The overall high success and survival rates reported here provide encouraging data regarding posaconazole as an alternative therapy for zygomycosis.

Hydroxylated analogues of the orally active broad spectrum antifungal, Sch 51048 (1), and the discovery of posaconazole [Sch 56592; 2 or (S,S)-5].

As part of a detailed study, the syntheses, biological activities, and pharmacokinetic properties of hydroxylated analogues of the previously described broad spectrum antifungal agents, Sch 51048 (1), Sch 50001 (3), and Sch 50002 (4), are described. Based on an overall superior profile, one of the alcohols, Sch 56592 (2), was selected for clinical studies.

Activity of posaconazole in the treatment of central nervous system fungal infections.

OBJECTIVES: A multinational, multicentre, open-label clinical trial was conducted to evaluate the safety and efficacy of posaconazole, an extended-spectrum triazole antifungal agent, in subjects with invasive fungal infections who had refractory disease or who were intolerant of standard antifungal therapy. In this subanalysis, we report on those subjects in this trial who had a fungal infection that involved the CNS. METHODS: Subjects received posaconazole oral suspension 800 mg/day in divided doses for up to 1 year; however, subjects could receive additional therapy as part of a treatment-use extension protocol. A blinded, third-party data review committee determined subject eligibility and outcome. RESULTS: Of the 330 subjects who enrolled in the study, 53 had infections of the CNS, of which 39 were considered evaluable for efficacy. Most had refractory disease (37 of 39) and underlying HIV infection (29 of 39). Twenty-nine subjects had cryptococcal infections, and 10 had infections caused by other fungal pathogens [Aspergillus spp. (four), Pseudallescheria boydii (two), Coccidioides immitis (one), Histoplasma capsulatum (one), Ramichloridium mackenziei (one), and Apophysomyces elegans plus a Basidiomycetes sp. (one)]. Successful outcomes were observed in 14 of 29 (48%) subjects with cryptococcal meningitis and five of 10 (50%) subjects with CNS infections due to other fungal pathogens. Posaconazole was well tolerated. CONCLUSIONS: These data suggest that posaconazole, as an oral medication, has clinical activity against fungal infections of the CNS and may provide a valuable alternative to parenteral therapy in patients failing existing antifungal agents.

Posaconazole is a potent inhibitor of sterol 14alpha-demethylation in yeasts and molds.

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 14alpha-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14alpha-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 14alpha-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.

Effect of granulocyte colony-stimulating factor combination therapy on efficacy of posaconazole (SCH56592) in an inhalation model of murine pulmonary aspergillosis.

Using an inhalation model of pulmonary aspergillosis, we observed modest differences in the survival rates of mice treated with granulocyte colony-stimulating factor (G-CSF) and posaconazole (POS) and those treated with POS alone. This finding is in contrast to a previous report that suggested that G-CSF had a significant antagonistic effect on the antifungal activity of POS.

Changes in susceptibility to posaconazole in clinical isolates of Candida albicans.

OBJECTIVES: To characterize the molecular mechanisms responsible for reduced susceptibility to azoles in Candida albicans clinical isolates. MATERIALS AND METHODS: Seven sequential C. albicans isolates were cultured from an AIDS patient treated with posaconazole for refractory oropharyngeal candidiasis. Expression levels of the CDR1, CDR2 and MDR1 genes, encoding efflux pumps previously implicated in azole resistance, and ERG11, encoding the azole target site, were monitored using northern blot and real-time PCR. The ERG11 genes from all seven isolates were sequenced. RESULTS: The seven closely related isolates exhibited significant decreases in susceptibility to fluconazole (MIC >or= 32 mg/L) and voriconazole (MIC >or= 2 mg/L) and progressive decreases in susceptibility to both posaconazole (isolates 1-4 MIC 0.25 mg/L, isolates 5-7 MIC 2 mg/L) and itraconazole (isolates 1-4 MIC 1 mg/L, isolates 5-7 MIC > 8 mg/L). None of the isolates exhibited any significant changes in the expression levels of ERG11 or the efflux pump genes. All seven isolates had multiple mutations in ERG11; isolates one through four each had five missense mutations; four of the resultant amino acid changes were previously associated with azole resistance. The fifth isolate had an additional novel mutation in one copy of ERG11, resulting in a Pro-230 to Leu substitution. This mutation was present in both ERG11 genes in the last two isolates. Select ERG11 genes were expressed in Saccharomyces cerevisiae, the ERG11 allele with all six mutations conferred the highest level of posaconazole resistance. CONCLUSIONS: Multiple mutations in ERG11 are required to confer decreased susceptibility to posaconazole.

Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome P450 14alpha-demethylase.

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 14alpha-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.

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus.

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.

Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis.

A liquid chromatography/mass spectrometry (LC/MS) method for separation and characterization of ergosterol biosynthetic precursors was developed to study the effect of Posaconazole on sterol biosynthesis in fungi. Ergosterol biosynthetic precursors were characterized from their electron ionization mass spectra acquired by a normal-phase chromatography, particle beam LC/MS method. Fragment ions resulting from cleavage across the D-ring and an abundant M - 15 fragment ion were diagnostic for methyl substitution at C-4 and C-14. Comparison of the sterol profile in control and treated Candida albicans incubations showed depletion of ergosterol and accumulation of C-4 and C-14 methyl-substituted sterols following treatment with Posaconazole. These C-4 and C-14 methyl sterols are known to be incapable of sustaining cell growth. The results demonstrate that Posaconazole exerts its antifungal activity by inhibition of ergosterol biosynthesis. Furthermore, Posaconazole appears to disrupt ergosterol biosynthesis by inhibition of lanosterol 14alpha-demethylase.