Echinocandin Drugs Research Articles

In Vivo Pharmacodynamic Characterization of Anidulafungin in a Neutropenic Murine Candidiasis Model

Multiple in vivo studies have characterized the pharmacodynamics of drugs from the triazole and polyene antifungal drug classes. Fewer studies have investigated these pharmacodynamic relationships for the echinocandin drug class. We used a neutropenic murine model of disseminated Candida albicans, Candida tropicalis, and Candida glabrata infection to characterize the time course of activity of the new echinocandin anidulafungin. The pharmacokinetic-pharmacodynamic (PK-PD) indices (the percentage of time that the drug concentration was above the MIC, the ratio of the area under the concentration-time curve from 0 to 24 h [AUC(0-24)] to the MIC, and the ratio of the maximum serum drug concentration [C(max)] to the MIC) were correlated with in vivo efficacy, as measured by organism numbers in kidney cultures after 96 h of therapy. The kinetics following intraperitoneal anidulafungin dosing in neutropenic infected mice were monitored. Peak levels and AUCs were linear over the 16-fold dose range studied. The drug elimination half-life in serum ranged from 14 to 24 h. Single-dose postantifungal-effect studies demonstrated prolonged suppression of organism regrowth after serum anidulafungin levels had fallen below the MIC. Of the four dosing intervals studied, treatment with the more widely spaced dosing regimens was most efficacious, suggesting the C(max)/MIC ratio as the PK-PD index most predictive of efficacy. Nonlinear regression analysis suggested that both the C(max)/MIC and AUC/MIC ratios were strongly predictive of treatment success. Studies were then conducted with 13 additional C. albicans, C. tropicalis, and C. glabrata isolates with various anidulafungin susceptibilities (MICs of anidulafungin for these strains, 0.015 to 2.0 microg/ml) to determine if similar C(max)/MIC and AUC(0-24)/MIC ratios for these isolates were associated with efficacy. The anidulafungin exposures associated with efficacy were similar among Candida species.

The Echinocandins: Total and Semi-Synthetic Approaches in Antifungal Drug Discovery

The echinocandins are a new class of antifungal lipopeptides for the treatment of serious nosocomial mycoses. The three currently approved drugs, caspofungin, micafungin, and anidulafungin, were each discovered through the syn- thetic modification of echinocandin natural products obtained from fermentation. This review is intended for the medici- nal chemist who is actively pursuing or has a general interest in the synthetic modification of natural products as a means to identify drug candidates. It provides a survey of the synthetic strategies that produced the approved echinocandin thera- peutics and a discussion of more recent efforts to identify a new generation of echinocandin drug candidates. Both total synthetic approaches starting from the constituent amino acids and semi-synthetic approaches relying on fermentation- produced lipopeptide are addressed. These various efforts by chemists from industry and academia have not only illumi- nated the interesting chemistry of these natural products, but have provided the means by which improvements in antifun- gal potency and spectrum, pharmacokinetic profile, solubility, stability, and safety can be realized. The ultimate success of these efforts can be judged by considering the important role the echinocandins are already playing in the treatment of se- rious fungal infection.

Resistance to echinocandin-class antifungal drugs

Invasive fungal infections cause morbidity and mortality in severely ill patients, and limited drug classes restrict treatment choices. The echinocandin drugs are the first new class of antifungal compounds that target the fungal cell wall by blocking beta-1,3-d-glucan synthase. Elevated MIC values with occasional treatment failure have been reported for strains of Candida. Yet, an uncertain correlation exists between clinical failure and elevated MIC values for the echinocandin drugs. Fungi display several adaptive physiological mechanisms that result in elevated MIC values. However, resistance to echinocandin drugs among clinical isolates is associated with amino acid substitutions in two "hot-spot" regions of Fks1, the major subunit of glucan synthase. The mutations, yielding highly elevated MIC values, are genetically dominant and confer cross-resistance to all echinocandin drugs. Prominent Fks1 mutations decrease the sensitivity of glucan synthase for drug by 1000-fold or more, and strains harboring such mutations may require a concomitant increase in drug to reduce fungal organ burdens in animal infection models. The Fks1-mediated resistance mechanism is conserved in a wide variety of Candida spp. and can account for intrinsic reduced susceptibility of certain species. Fks1 mutations confer resistance in both yeasts and moulds suggesting that this mechanism is pervasive in the fungal kingdom.

Serum Differentially Alters the Antifungal Properties of Echinocandin Drugs

Antifungal efficacies of the echinocandin drugs caspofungin, micafungin, and anidulafungin were reduced significantly in the presence of 50% human serum, which yielded nearly equivalent MICs or minimum effective concentrations against diverse Candida spp. and Aspergillus spp. Consistent with a direct drug interaction, serum decreased the sensitivity of glucan synthase to echinocandin drugs.

Acquired Echinocandin Resistance in a Candida krusei Isolate Due to Modification of Glucan Synthase

A Candida krusei strain from a patient with acute myelogenous leukemia that displayed reduced susceptibility to echinocandin drugs contained a heterozygous mutation, T2080K, in FKS1. The resulting Phe655-->Cys substitution altered the sensitivity of glucan synthase to echinocandin drugs, consistent with a common mechanism for echinocandin resistance in Candida spp.

A case of invasive central nervous system aspergillosis treated with micafungin with monitoring of micafungin concentrations in the cerebrospinal fluid

Invasive aspergillosis has an extremely high mortality rate. In Japan, micafungin, an echinocandin drug that has a new mechanism of action as an antifungal agent and has a clinical effect against Aspergillus species, became available in 2002. However, little is known about its penetration into the central nervous system (CNS), or its efficacy for the treatment of invasive CNS aspergillosis. We report a 65-y-old female with diabetes mellitus and CNS aspergillosis who was treated with micafungin. During treatment, micafungin concentrations were measured in the cerebrospinal fluid and plasma. On a dose of 300 mg/d, the ratio of the micafungin concentration in the cerebrospinal fluid to that in plasma was extremely low (0.2%-0.05%); nevertheless, the patient did not have a relapse of invasive CNS aspergillosis after micafungin treatment.

Assessing Resistance to the Echinocandin Antifungal Drug Caspofungin in Candida albicans by Profiling Mutations in FKS1

Resistance of clinical isolates of Candida albicans to the echinocandin drug caspofungin is slowly emerging and is linked to mutations in short conserved regions in the FKS1 gene. The most prominent changes occurred at the serine 645 position in Fks1p with substitutions of proline, tyrosine, and phenylalanine. An allele-specific real-time PCR molecular-beacon assay was developed for rapid identification of drug resistance by targeting FKS1 mutations. Mutations altering serine 645 were reliably identified in both heterozygous and homozygous states. The molecular-beacon assay was used to evaluate two large collections of spontaneous mutants from separate strains of C. albicans with resistance (MICs, >16 microg/ml) to caspofungin with the goal of understanding the relationship between FKS1 mutations and echinocandin resistance. Of 85 resistant isolates recovered, all were identified with mutations in FKS1; 93% showed changes at Ser645, with 62% displaying a characteristic S645P substitution expressed as either a homozygous or a heterozygous mutation in FKS1. Two other prominent amino acid substitutions, S645Y and S645F, were found at frequencies of 22% and 8%, respectively. Three new mutations were also identified: T1922C, G1932T, and C1934G, encoding F641S, L644F, and S645C substitutions, respectively. One strain had the double amino acid substitution L644F and S645C. Allele-specific probes were combined in a multiplex assay for reliable screening of known FKS1 mutations. These data support the importance of FKS1p substitutions in echinocandin resistance and demonstrate the feasibility of applying molecular screening for routine resistance assessment.

Progressive loss of echinocandin activity following prolonged use for treatment of Candida albicans oesophagitis

To illustrate the progressive loss of cross-echinocandin activity on Candida albicans isolates with strong clonal homology from a patient with advanced HIV infection and chronic oesophagitis progressively resistant to uninterrupted micafungin treatment. Antifungal susceptibility profiles for different antifungal agents were determined against serial C. albicans isolates retrieved before and during therapy. Multilocus sequencing typing (MLST) was performed on each of the isolates. FKS1 mutations conferring reduced susceptibility to echinocandin drugs were determined by DNA sequence analysis. Four C. albicans isolates showing identical allelic homology were retrieved from the patient at the initiation and during therapy with micafungin. The progressive lack of clinical response to micafungin therapy was associated with increased MICs of all three echinocandin drugs (caspofungin, micafungin and anidulafungin) in association with the acquisition of mutations in the FKS1 gene. This report documents for the first time a progressive loss of activity of all three echinocandin drugs against clonally related C. albicans isolates following long-term clinical exposure to this new class of antifungal agents.

Specific Substitutions in the Echinocandin Target Fks1p Account for Reduced Susceptibility of Rare Laboratory and Clinical Candida sp. Isolates

An association between reduced susceptibility to echinocandins and changes in the 1,3-beta-d-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.

Micafungin: the US perspective

Invasive fungal infections cause considerable morbidity and mortality in nosocomial settings and amongst immunocompromised hosts. Invasive candidiasis and aspergillosis remain the most common invasive fungal infections, with Candida spp. constituting the fourth most common bloodstream infection in the USA. Currently available antifungal therapies include the polyene, antimetabolite, allylamine, azole and echinocandin drug classes. Micafungin, approved in March 2005 by the Food and Drug Administration for use in the USA, has shown safety and efficacy for the treatment of candidiasis and aspergillosis in clinical trials in Japan, Europe and South Africa. Micafungin holds promise as a first-line treatment option for candidiasis, as well as prophylaxis against invasive fungal infections during periods of neutropenia in high-risk patients.

Characterization ofAspergillus fumigatusmutants with reduced susceptibility to caspofungin

Caspofungin acetate (CAS) is a member of a new class of clinically-approved echinocandin drugs to treat invasive aspergillosis. CAS inhibits the activity of beta-1,3-D-glucan synthase (GS), thus damaging the fungal cell wall. Although no clinical resistance of Aspergillus to CAS has been reported as yet, the development of in vitro reduced susceptibility is presumed to be inevitable. By contrast, echinocandin resistance in laboratory strains of Candida albicans and Saccharomyces cerevisiae has been well documented. To study the potential for clinical resistance in Aspergillus, two classes of Aspergillus fumigatus mutant strains were isolated that exhibited reduced susceptibility to CAS. In the first class, a site-directed mutation within the target gene (AfFKS1, encoding the putative catalytic subunit of GS) was introduced and shown to confer low-level (16-fold) reduced susceptibility. A second class of spontaneous mutants were sensitive to low levels of drug but displayed nearly normal growth above 0.5 microg/ml, suggesting induction of an unknown resistance mechanism. At higher levels of drug (> or = 16 microg/ml), the mutants displayed partially restored sensitivity. Preliminary studies indicate that neither target site mutations, nor changes in target gene expression are present in these strains, as has been documented for several yeasts. Instead, preliminary results indicate that the molecular mechanism(s) underlying reduced susceptibility of CAS in the A. fumigatus strains is novel, possibly due to remodeling of the cell wall components.

Caspofungin uptake is mediated by a high-affinity transporter in Candida albicans.

The uptake of the echinocandin drug caspofungin acetate in Candida albicans was evaluated at drug levels at or near the MIC for the organism. Maximal uptake was achieved in 10 min and was energy independent. A saturable transport system, consistent with a facilitated-diffusion carrier, was observed with the unlabeled drug competing with the labeled drug for uptake and efflux. More than 90% of the transported drug was observed in a single kinetic compartment that was available for efflux, indicating that the drug was free in the cytoplasm following uptake. Efflux was also energy independent but was sensitive to the presence of a fully loaded carrier on both faces of the bilayer. Overall, the data presented are consistent with the presence of a high-affinity facilitated-diffusion transporter that mediates caspofungin uptake and could be a potential source of transport-related reduced susceptibility.

Caspofungin: the first licensed antifungal drug of the novel echinocandin class

Caspofungin is the first echinocandin drug licensed for treatment of fungal infections. The drug is active in vitro and in animal models against both albicans and non-albicans Candida species and Aspergillus species. Results from clinical trials support its efficacy as a first-line therapy for patients with esophageal or invasive candidiasis and salvage therapy for patients with invasive aspergillosis. In all studies, caspofungin has been generally well tolerated; serious drug-related adverse events and adverse events leading to discontinuation of therapy have been infrequent.

Evaluation of the echinocandin antifungal MK-0991 (L-743,872): efficacies in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis

The in vivo activity of the Merck antifungal echinocandin drug candidate MK-0991 (L-743,872) was evaluated in mouse models of disseminated candidiasis, aspergillosis, and cryptococcosis. The echinocandins are potent inhibitors of 1,3-beta-D-glucan synthase. Two models of disseminated candidiasis were used. In a Candida albicans mouse survival model with both DBA/2N and CD-1 mice, estimates of the 50% effective doses (ED50s) of MK-0991 were 0.04 and 0.10 mg/kg of body weight/dose at 21 days after challenge, respectively. In a C. albicans target organ assay (TOA) with DBA/2N mice, MK-0991 at levels of > or =0.09 mg/kg/dose significantly reduced the numbers of C. albicans CFU/g of kidneys compared to the numbers in the kidneys of control mice from 1 to 28 days after challenge. Even when given as a single intraperitoneal dose either 30 min or 24 h after challenge, MK-0991 was effective and significantly reduced the numbers of C. albicans CFU/g of kidney compared to those in the controls. MK-0991 was >300-fold less active when it was administered orally than when it was administered parenterally. MK-0991 was efficacious in mouse TOAs against other C. albicans strains and Candida species including Candida tropicalis, Candida (Torulopsis) glabrata, Candida lusitaniae, Candida parapsilosis, and Candida krusei. MK-0991 was ineffective against disseminated Cryptococcus neoformans infections. In the model of disseminated aspergillosis in mice, MK-0991 at doses of > or =0.02 mg/kg/dose significantly prolonged the survival of DBA/2N mice, with estimates of the ED50 and ED90 of MK-0991 being 0.03 and 0.12 mg/kg/dose, respectively, at 28 days after challenge. MK-0991 is a potent, parenterally administered therapeutic agent against disseminated candidiasis and aspergillosis that warrants further investigation in human clinical trials.