Shuichi Tawara

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.

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.

Sepantronium Bromide (YM155) induces disruption of the ILF3/p54nrb complex, which is required for survivin expression

YM155, a small-molecule survivin suppressant, specifically binds to the transcription factor ILF3, which regulates the expression of survivin[1]. In this experiment we have demonstrated that p54(nrb) binds to the survivin promoter and regulates survivin expression. p54(nrb) forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54(nrb) complex, which results in a different subcellular localization between ILF3 and p54(nrb). Thus, identification of molecular targets of YM155 in suppression of the survivin pathway, might lead to development of its use as a novel potential target in cancers.

Effect of protein binding in serum on therapeutic efficacy of cephem antibiotics.

The effect of protein binding in serum of eight cephem antibiotics (ceftazidime, ceftizoxime, cefotiam, cefmetazole, cefpiramide, cefazolin, cefuzonam, ceftriaxone) on their therapeutic efficacies was examined in mice with experimentally induced intraperitoneal infections or pneumonia. The relationship among therapeutic activity, in vitro antibacterial activity, total or free (unbound) levels in serum, and homogenized whole lung levels was investigated. In the intraperitoneal infection caused by Staphylococcus aureus or Klebsiella pneumoniae, the 50% effective doses (ED50s) of the cephem antibiotics correlated with the area under the concentration-time curve (AUC) values of free levels in serum and the MICs but not with those of total levels in serum. A linear relationship was seen between 1/ED50 values and AUC of free levels in serum/MIC values. On the other hand, in mice with pneumonia caused by K. pneumoniae, the number of bacteria in the lung closely correlated with the AUC of the antibiotic concentration in lung tissue. There was a direct correlation between the levels in lung tissue and total levels in serum but not free levels in serum. The cephem antibiotics tested in this study were bound only slightly to homogenates of mouse lung. These results indicate that the effect of protein binding in serum on therapeutic efficacy against intraperitoneal infection differs from that against pulmonary infection. Images

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.

Orally active cephalosporins. Part 3: synthesis, structure-activity relationships and oral absorption of novel C-3 heteroarylmethylthio cephalosporins.

A series of 7beta-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3-(heteroarytmethylthio)cephalosporins was designed, synthesized and evaluated for antibacterial activity and oral absorption in rats. Antibacterial activity was markedly influenced by the structure of the heteroaromatic ring moiety. Oral absorption was influenced by the heteroaromatic ring moiety as well as by the arrangement of heteroatoms. Among these compounds, FK041 (2o), having a 4-pyrazolylmethylthio moiety, showed potent antibacterial activity against both gram-positive and gram-negative bacteria including Haemophilus influenzae. Further, it showed higher oral absorption than CFDN.

Synthesis and antibacterial activity of novel 4-pyrrolidinylthio carbapenems Part IV. 2-Alkyl substituents containing cationic heteroaromatics linked via a C–C bond

The synthesis and biological activity of a novel series of 2-alkyl-4-pyrrolidinylthio-beta-methylcarbapenems containing a variety of cationic heteroaromatic substituents linked via a C-C bond is described. As a result of these studies, we selected FR21818 (In) as a candidate compound for development. FR21818 exhibited a well balanced spectrum of antibacterial activity, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), excellent urinary recovery, good stability against renal dehydropeptidase-I (DHP-I). no antigenicity and mutagenicity, weak toxicities, and good efficacy and therapeutic effect on mice systemic infections. Affinities to PBPs, permeability of outer membrane, and plasma levels in mice, dog, and cynomolgous monkey of FR21818 are also reported.

Mechanism of Therapeutic Effectiveness of Cefixime against Typhoid Fever

ABSTRACT β-Lactams have been considered ineffective against organisms growing inside mammalian cells because of their poor penetration into cells. However, cefixime has been shown to be clinically effective against typhoid fever. The probable mechanism of therapeutic effectiveness of cefixime against typhoid fever was investigated usingSalmonella enterica serovar Typhimurium instead of S. enterica serovar Typhi both in a cellular and in a mouse infection model. Cefixime was able to inhibit the growth of serovar Typhimurium inhabiting monocyte-derived THP-1 cells. Elongation of serovar Typhimurium in THP-1 cells was observed microscopically. Apparent morphological changes of serovar Typhimurium in THP-1 cells were also observed by electron microscopy. The concentration of cefixime inside THP-1 cells was almost half (46 to 48%) of the concentration outside the cells when serovar Typhimurium coexisted in the solution. The length of time after oral dosing (8 mg/kg) that cefixime was present—calculated from levels in serum—at a concentration above the MIC at which 90% of the serovar Typhi organisms inside human cells were inhibited was presumed to be more than 12 h. Cefixime also showed excellent activity in the mouse systemic and oral infection models based on infections caused by serovar Typhimurium. It is concluded that a fair amount of cefixime can enter mammalian cells and inhibit the growth of bacteria inside cells when the bacteria are sensitive enough to cefixime, as are serovars Typhimurium and Typhi.