Fumiaki Ikeda

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.

In Vitro Antifungal Activity of Micafungin (FK463) against Dimorphic Fungi: Comparison of Yeast-Like and Mycelial Forms

ABSTRACT The characteristics of in vitro micafungin (FK463) antifungal activity against six species of dimorphic fungi were investigated in accordance with the NCCLS M27-A microdilution methods. MICs of micafungin, amphotericin B, itraconazole, and fluconazole for Histoplasma capsulatum var. capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Penicillium marneffei, and Sporothrix schenckii were determined both for the yeast-like form and mycelial form. Coccidioides immitis was tested only in its mycelial form. We have clearly demonstrated that the in vitro activity of micafungin depends considerably on the growth form of dimorphic fungi. Micafungin exhibited potent activity against the mycelial forms of H. capsulatum, B. dermatitidis, and C. immitis (MIC range, 0.0078 to 0.0625 μg/ml), while it was very weakly active against their yeast-like forms (MIC range, 32 to >64 μg/ml). Micafungin was also more active against the mycelial forms than the yeast-like forms of Paracoccidioides brasiliensis, Penicillium marneffei, and S. schenckii. The MICs of amphotericin B were 2 to 5 dilutions lower for the mycelial forms than for the yeast-like forms of B. dermatitidis and Paracoccidioides brasiliensis. There was no apparent difference in the activity of itraconazole between the two forms. The MICs of fluconazole for the yeast-like forms were generally lower than those for the mycelial forms, and considerably so for B. dermatitidis. These results suggest that the growth form employed in antifungal susceptibility testing of dimorphic fungi can considerably influence the interpretation of results. At present, it cannot be judged whether micafungin has clinical usefulness for dimorphic fungus infections, since for most fungi it remains uncertain which growth form correlates better with therapeutic outcome. However, the results of this study warrant further investigations of micafungin as a therapeutic agent for infections caused by dimorphic fungi.

Evidence for the involvement of GABAA receptor blockade in convulsions induced by cephalosporins

There is accumulating evidence that most beta-lactam antibiotics (i.e., cephalosporins and penicillins) have some degree of convulsive activity, both in laboratory animals as well as in clinical settings. The proposed mechanism is suppression of inhibitory postsynaptic responses, mainly mediated by gamma-amino butyric acid (GABA)(A)-receptors (GABA(A)-R). However, comprehensive studies on the convulsive activities of various beta-lactam antibiotics in vivo and in vitro have not been performed. We have therefore examined the convulsive activities of seven different cephalosporins using both in vivo and in vitro models: intracerebroventricular (ICV) administration in mouse; [(3)H]muscimol binding assay (BA) in mouse brain synaptosome; and inhibition of recombinant mouse alpha1beta2gamma2s GABA(A)-Rs in Xenopus oocyte (GR). The rank orders of convulsive activities in mouse (cefazolin>cefoselis>cefotiam>cefpirome>cefepime>ceftazidime>cefozopran) correlated with those of inhibitory potencies on [(3)H]muscimol binding and GABA-induced currents of GABA(A)-R in vitro, with correlation coefficients of ICV:GR, ICV:BA and BA:GR of 0.882, 0.821 and 0.832, respectively. In contrast, none of the antibiotics had affinities for N-methyl-D-aspartate (NMDA) receptors nor facilitatory actions on NMDA receptor-mediated current in oocytes. These results clearly demonstrate that the mechanism of cephalosporin-induced convulsions is mediated predominantly through the inhibition of GABA(A)-R function and not through NMDA receptor modulation.

Efficacy of FK463, a New Lipopeptide Antifungal Agent, in Mouse Models of Pulmonary Aspergillosis

ABSTRACT The efficacy of FK463, a novel water-soluble lipopeptide, was evaluated in mouse models of pulmonary aspergillosis and was compared with that of amphotericin B (AMPH-B). In the pulmonary aspergillosis models induced by intranasal inoculation, FK463 exhibited good efficacy, with 50% effective doses in the range of 0.26 to 0.51 mg/kg of body weight; these values were comparable to those of AMPH-B. In anAspergillus target organ assay with immunosuppressed mice, under conditions of constant plasma levels of FK463, using a subcutaneously implanted osmotic pressure pump, a significant reduction in viable fungal cells was observed at plasma FK463 levels of 0.55 to 0.80 μg/ml or higher. We conclude that FK463 is highly effective in the treatment of pulmonary aspergillosis in this animal model. These results indicate that FK463 may be a potent parenterally administered antifungal agent for pulmonary aspergillosis.

Induction of ulceration and severe gastritis in Mongolian gerbil by Helicobacter pylori infection

Specific pathogen-free Mongolian gerbils were infected orally with Helicobacter pylori to establish a new small animal model of severe gastritis H. pylori was recovered by culture from both antrum and body over a 16-week period after a single inoculation. The number of H. pylori colonising the antrum was about 100-fold higher than in the body, and this was consistent throughout the experiment. Histological examination showed that all animals developed severe inflammation with infiltration of polymorphonuclear leucocytes and mononuclear cells into the lamina propria and submucosa of the antrum from 4 weeks after infection. From 8 weeks after infection, multifocal lymphoid follicles appeared in the lamina propria and submucosa, and micro-erosions were also observed in the epithelial layer. At 16 weeks after infection, ulceration with disruption of the lamina muscularis mucosae was observed in the antral mucosa. To determine whether H. pylori caused gastritis or not, infected gerbils were treated with amoxycillin. After the treatment, gastritis could not be seen in the gastric mucosa. Therefore, the Mongolian gerbil is a useful small animal model to study the pathogenesis of H. pylori in gastric ulceration and severe gastritis and to assess anti-H. pylori treatment.

Prophylactic Effect of FK463, a Novel Antifungal Lipopeptide, against Pneumocystis carinii Infection in Mice

ABSTRACT The prophylactic effect of FK463, a new water-soluble echinocandin-like lipopeptide with inhibitory activity against 1,3-β-d-glucan synthase, against Pneumocystis carinii infection was investigated with the severe combined immunodeficient (SCID) mouse model. Treatment with FK463, pentamidine, and saline only was performed for 6 weeks from the day after the SCID mice were inoculated intranasally with infected lung homogenates. FK463 at 0.2 or 1.0 mg/kg of body weight, pentamidine at 4 mg/kg, or saline was subcutaneously administered daily into the backs of the SCID mice. The effects of the drugs were evaluated by detection of P. carinii cysts in mouse lung homogenates by toluidine blue O staining, lung histology, and PCR amplification of a P. carinii-specific DNA fragment from the lungs. P. carinii cysts were detected in the lungs of all mice administered saline. In contrast, no cysts were detected in mice administered both doses of FK463 and pentamidine. A specific DNA fragment was amplified from all mice administered saline and at least half or more of the mice administered FK463 and pentamidine. These results indicate that FK463 acts on cyst wall formation but not on trophozoite proliferation and is extremely effective in preventing P. carinii-associated pneumonia. These results suggest that FK463 is potentially useful as a prophylactic agent against P. carinii infection.

Killing Activity of Micafungin against Aspergillus fumigatus Hyphae Assessed by Specific Fluorescent Staining for Cell Viability

ABSTRACT We studied the anti-Aspergillus activity of micafungin by using two fluorescent dyes to detect cell viability. Micafungin induced flattened hyphae, caused by the bursting of cells, which had lost their viability. Micafungin has killing activity against actively growing hyphae, even though it is not fungicidal against the whole burden of Aspergillus fumigatus.

Low Prevalence of Helicobacter pylori Infection in Patients with Hamartomatous Fundic Polyps

Helicobacter pylori infection of the gastricmucosal surface was investigated in patients withhamartomatous fundic polyps or hyperplastic polyps andin patients without endoscopic evidence of disease(healthy subjects). Presence of H. pylori infection wasdetermined by culture, histologic examination, and theendoscopic phenol red test. Adherence of H. pylori wasevaluated with scanning electron microscopic examination of antral biopsy specimens. Bothprevalence of H. pylori infection (P < 0.001) and H.pylori adherence (P < 0.05) were less in patientswith hamartomatous fundic polyps than in healthy subjects and patients with hyperplastic polyps.However, the percentages of plasma cells in gastricmucosa that contained IgA and of gastric epithelialcells that expressed Lewis b did not differsignificantly among the three groups. These findings suggestthat defense mechanisms against the attachment of H.pylori other than IgA or Lewis b antigen are present inpatients with hamartomarous fundic polyps.

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.