Katsuyuki Maki

In vitro activities of a new lipopeptide antifungal agent, FK463, against a variety of clinically important fungi

ABSTRACT The in vitro antifungal activity and spectrum of FK463 were compared with those of amphotericin B, fluconazole, and itraconazole by using a broth microdilution method specified by National Committee for Clinical Laboratory Standards document M27-A (National Committee for Clinical Laboratory Standards, Wayne, Pa., 1997). FK463 exhibited broad-spectrum activity against clinically important pathogens including Candida species (MIC range, ≦0.0039 to 2 μg/ml) and Aspergillus species (MIC range, ≦0.0039 to 0.0313 μg/ml), and its MICs for such fungi were lower than those of the other antifungal agents tested. FK463 was also potently active against azole-resistant Candida albicans as well as azole-susceptible strains, and there was no cross-resistance with azoles. FK463 showed fungicidal activity against C. albicans, i.e., a 99% reduction in viability after a 24-h exposure at concentrations above 0.0156 μg/ml. The minimum fungicidal concentration (MFC) assays indicated that FK463 was fungicidal against most isolates of Candida species. In contrast, the MFCs of FK463 for A. fumigatus isolates were much higher than the MICs, indicating that its action is fungistatic against this species. FK463 had no activity against Cryptococcus neoformans,Trichosporon species, or Fusarium solani. Neither the test medium (kind and pH) nor the inoculum size greatly affected the MICs of FK463, while the addition of 4% human serum albumin increased the MICs for Candida species and A. fumigatus more than 32 times. Results from preclinical in vitro evaluations performed thus far indicate that FK463 should be a potent parenteral antifungal agent.

Efficacy of FK463, a New Lipopeptide Antifungal Agent, in Mouse Models of Disseminated Candidiasis and Aspergillosis

ABSTRACT The efficacy of intravenous injection of FK463, a novel water-soluble lipopeptide, was evaluated in mouse models of disseminated candidiasis and aspergillosis and was compared with those of fluconazole (FLCZ) and amphotericin B (AMPH-B). In the candidiasis model, FK463 significantly prolonged the survival of intravenously infected mice at doses of 0.125 mg/kg of body weight or higher. In disseminated candidiasis caused by Candida species, including FLCZ-resistant Candida albicans, FK463 exhibited an efficacy 1.4 to 18 times inferior to that of AMPH-B, with 50% effective doses (ED50s) ranging from 0.21 to 1.00 mg/kg and 0.06 to 0.26 mg/kg, respectively, and was much more active than FLCZ. The protective effect of FK463 was not obviously influenced by the fungal inoculum size, the starting time of the treatment, or the immunosuppressed status of the host. The reduction in efficacy was less than that observed with FLCZ or AMPH-B. The efficacy of FK463 was also evaluated in the disseminated candidiasis target organ assay and was compared with those of FLCZ and AMPH-B. Efficacies were evaluated on the basis of a comparison between the mean log10 CFU in kidneys in the groups treated with antifungal agents and that in control group. A single dose of FK463 at 0.5 mg/kg or higher significantly reduced the viable counts in kidneys compared with the numbers of yeast cells before treatment, and its efficacy was comparable to that of AMPH-B, while FLCZ at 4 mg/kg showed only a suppressive effect on the growth of C. albicans in the kidneys. In the disseminated aspergillosis model, FK463 given at doses of 0.5 mg/kg or higher significantly prolonged the survival of mice infected intravenously with Aspergillus fumigatus conidia. The efficacy of FK463 was about 2 times inferior to that of AMPH-B, with ED50s ranging from 0.25 to 0.50 mg/kg and 0.11 to 0.29 mg/kg, respectively. These results indicate that FK463 may be a potent parenterally administered therapeutic agent for disseminated candidiasis and aspergillosis.

Overexpression of Candida albicans CDR1, CDR2, or MDR1 Does Not Produce Significant Changes in Echinocandin Susceptibility

ABSTRACT The micafungin and caspofungin susceptibilities of Candida albicans laboratory and clinical isolates and of Saccharomyces cerevisiae strains stably hyperexpressing fungal ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters involved in azole resistance were determined using three separate methods. Yeast strains hyperexpressing individual alleles of ABC transporters or an MFS transporter from C. albicans gave the expected resistance profiles for the azoles fluconazole, itraconazole, and voriconazole. The strains hyperexpressing CDR2 showed slightly decreased susceptibility to caspofungin in agar plate drug resistance assays, as previously reported, but increased susceptibility to micafungin compared with either the strains hyperexpressing CDR1 or the null parent deleted of seven ABC transporters. The strains hyperexpressing CDR1 showed slightly decreased susceptibility to micafungin in these assays. A C. albicans clinical isolate overexpressing both Cdr1p and Cdr2p relative to its azole-sensitive isogenic progenitor acquired resistance to azole drugs and showed reduced susceptibility to caspofungin and slightly increased susceptibility to micafungin in agar plate drug resistance assays. None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays. The antifungal activities of micafungin and caspofungin were similar in agarose diffusion assays, although the shape and size of the caspofungin inhibitory zones were affected by medium composition. The assessment of micafungin and caspofungin potency is therefore assay dependent; the differences seen with agar plate drug resistance assays occur over narrow ranges of echinocandin concentrations and are not of clinical significance.

Clinically significant micafungin resistance in Candida albicans involves modification of a glucan synthase catalytic subunit GSC1 (FKS1) allele followed by loss of heterozygosity

OBJECTIVES To determine the mechanism of intermediate- and high-level echinocandin resistance, resulting from heterozygous and homozygous mutations in GSC1 (FKS1), in both laboratory-generated and clinical isolates of Candida albicans. METHODS The DNA sequences of the entire open reading frames of GSC1, GSL1 (FKS3) and RHO1, which may contribute to the beta-1,3-glucan synthase of a micafungin-susceptible strain and a resistant clinical isolate, were compared. A spontaneous heterozygous mutant isolated by selection for micafungin resistance, and a panel of laboratory-generated homozygous and heterozygous mutants that possessed combinations of the echinocandin-susceptible and -resistant alleles, or mutants with individual GSC1 alleles deleted, were used to compare levels of echinocandin resistance and inhibition of glucan synthase activity. RESULTS DNA sequence analysis identified a mutation, S645P, in both alleles of GSC1 from the clinical isolate. GSL1 had two homozygous amino acid changes and five non-synonymous nucleotide polymorphisms due to allelic variation. The predicted amino acid sequence of Rho1p was conserved between strains. Reconstruction of the heterozygous (S645/S645F) and homozygous (S645F/S645F) mutation showed that the homozygous mutation conferred a higher level of micafungin resistance (4 mg/L) than the heterozygous mutation (1 mg/L). Exposure of the heterozygous mutant to micafungin resulted in a loss of heterozygosity. Kinetic analysis of beta-1,3-glucan synthase activity showed that the homozygous and heterozygous mutations gave echinocandin susceptibility profiles that correlated with their MIC values. CONCLUSIONS A homozygous hot-spot mutation in GSC1, caused by mutation in one allele and then loss of heterozygosity, is required for high-level echinocandin resistance in C. albicans. Both alleles of GSC1 contribute equally and independently to beta-1,3-glucan synthase activity.

Role of Micafungin in the Antifungal Armamentarium

Serious infections caused by opportunistic molds remain a major problem for public health. Immune deficiency following organ transplantation and aggressive cancer treatment has greatly increased the incidence of systemic mycoses, and invasive aspergillosis in patients with AIDS is associated with significant morbidity and mortality. Amphotericin B is the first-line therapy for systemic infection because of its broad-spectrum and fungicidal activity. However, considerable side effects limit its clinical utility. The echinocandins are large lipopeptide molecules that inhibit the synthesis of 1,3-beta-D-glucan, a key component of the fungal cell wall. Three echinocandins have reached the market, and some others are in early clinical development. Caspofungin was the first echinocandin to be licensed for clinical use in most countries. Micafungin is licensed for clinical use in Japan, China, Taiwan, Jordan, Korea, Hong-Kong and the US, and anidulafungin is currently licensed in the US. The novel class of echinocandins represents a milestone in antifungal drug research that has further expanded our therapeutic options. Studies to date have shown that micafungin exhibits extremely potent antifungal activity against clinically important fungi, including Aspergillus and azole-resistant strains of Candida. In animal studies, micafungin is as efficacious as amphotericin B with respect to improvement of survival rate. Micafungin is also characterized by a linear pharmacokinetic profile and substantially fewer toxic effects. Micafungin is a poor substrate for the cytochrome P450 enzymes, and compared to azoles, fewer drug interactions are described. No dose adjustments of the drug are required in the presence of mycophenolate mofetil, cyclosporin, tacrolimus, prednisolone, or sirolimus. Strategies using this new echinocandin agent will benefit a large number of patients with severe immune dysfunction.

Novel echinocandin antifungals. Part 2: Optimization of the side chain of the natural product FR901379. Discovery of micafungin.

Further optimization of the potent antifungal activity of side chain analogs of the natural product FR901379 led to the discovery of compound 8 with an excellent, well-balanced profile. Potent compounds with reduced hemolytic potential were designed based upon a disruption of the linearity of the terphenyl lipophilic side chain. The optimized compound (8, FK463, micafungin) displayed the best balance and was selected as the clinical candidate.

FR290581, a novel sordarin derivative: synthesis and antifungal activity.

Sordarin is a unique natural product antifungal agent that is an inhibitor of elongation factor 2. To improve biological activity, we synthesized various compounds by novel modification of the aglycone, sordaricin. As a result, we have discovered the novel sordarin derivative FR290581. This compound exhibited superior activity and a good pharmacokinetic profile, and also displayed good in vivo activity against Candida albicans.

Use of a serum‐based antifungal susceptibility assay to predict the in vivo efficacy of novel echinocandin compounds

In vitro susceptibility assays of antifungal activity do not always accurately predict in vivo efficacy. As well as having a clear clinical importance, the ability to predict efficacy is also essential for effective screening of novel drug compounds. Initial screening of novel compounds must often be based on in vitro data. The present report describes the use of serum‐MIC, an in vitro test of antifungal susceptibility, to accurately predict in vivo efficacy of echinocandin drugs in a mouse model of disseminated candidiasis. The basis of the serum‐MIC method was to measure the inhibitory activity of a test compound against Candida albicans hyphal growth in the presence of pooled mouse serum. For 13 previously uncharacterized echinocandin compounds, as well as for the known echinocandin drugs, micafungin and caspofungin, serum‐MIC determinations were shown to give better correlation to efficacy in the animal model than conventional, CLSI standard, in vitro antifungal susceptibility tests. The most accurate prediction of efficacy was obtained when the serum‐MIC was adjusted in relation to the serum concentration at 30 min post‐treatment. Furthermore, when the efficacy of micafungin was determined by measuring C. albicans kidney burden in the mouse model of infection, the adjusted serum‐MIC consistently reflected the effective serum concentrations. Our data indicate that determination of serum‐MIC values will facilitate prediction of the in vivo potency of new antifungal compounds such as novel echinocandins.

Synthesis and antifungal activity of ASP9726, a novel echinocandin with potent Aspergillus hyphal growth inhibition

The synthesis and antifungal activity of ASP9726, a novel echinocandin with potent Aspergillus hyphal growth inhibition and significantly improved MIC against Candida parapsilosis and echinocandin resistant-Candida is described.

Novel echinocandin antifungals. Part 1: Novel side-chain analogs of the natural product FR901379

A series of novel acylated analogs of the novel water-soluble echinocandin FR901379 have been prepared and evaluated for antifungal and hemolytic activity. A relationship between antifungal activity and lipophilicity of the acyl side chain, expressed as ClogP was demonstrated, and an analog (3c) with 5.5- to 8-fold superior in vivo activity relative to the previously disclosed 4-(n-octyloxy)benzoyl side chain analog, FR131535 obtained.