Sepantronium bromide (YM155) induces disruption of the ILF3/p54(nrb) 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.

Unique profiles of changes in cell membrane fluidity during ethanol-induced yeast-to-pseudohyphal transition in Candida tropicalis.

A dimorphic transition from the yeast form to filamentous one in Candida tropicalis pK233 is triggered by the addition of ethanol into the glucose semi-defined liquid medium and the process of filamentation accompanies temporal depolarization of yeast cells. The transition is completely prevented by further supplementation of myo-inositol at the start of cultivation. The addition of ethanol caused an increase in membrane fluidity during the process of depolarization, and then fluidity was gradually lowered to the level equivalent with that of the stationary-phase yeast cells in accordance with filamentation. The increase in membrane fluidity of ethanol-induced cells appeared parallel with reduction in the content of membrane phosphatidylinositol, which was rich in saturated palmitic acid. Introduction of exogenous myo-inositol or 1 M sorbitol into the ethanol-supplemented culture at the start of cultivation restored yeast growth and the reduction of membrane fluidity occurred, coupled with the recovery of the phosphatidylinositol content.

Antibacterial activity of cefixime against Salmonella typhi and applicability of Etest.

Recent clinical studies have shown the excellent efficacy of cefixime for the treatment of typhoid fever. In this study, the in vitro antibacterial activity of various antibiotics including cefixime against 73 clinical isolates of Salmonella typhi from a variety of sources was evaluated by the conventional agar-dilution method and Etest. Eighteen strains of these 73 isolates were chloramphenicol- and cotrimoxazole- (sulfamethoxazole-trimethoprim) resistant, and 12 of these 18 strains were also resistant to amoxicillin because of beta-lactamase production. Cefixime showed excellent activity against all 73 strains with an MIC90 value of 0.25 &mgr;g/ml. Reflecting its high beta-lactamase stability, cefixime also had excellent activity against beta-lactamase-producing amoxicillin-resistant strains. Antibacterial activity of cefixime was comparable to ceftriaxone, ofloxacin, and ciprofloxacin, which are often used for the treatment of typhoid fever. The MIC values obtained from the Etest correlated well with the results of the conventional agar-dilution method, suggesting the usefulness of the Etest as a new easy MIC determination method. The microbiological results supplement the previous clinical data, which showed that oral cefixime provides a safe and effective alternative for the treatment of typhoid fever, even in cases of multidrug-resistant S. typhi.

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.

Direct comparison of the pharmacodynamics of four antifungal drugs in a mouse model of disseminated candidiasis using microbiological assays of serum drug concentrations.

The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum-based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ-based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs.

Determination of antifungal activities in serum samples from mice treated with different antifungal drugs allows detection of an active metabolite of itraconazole.

To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole-treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.

Studies on anti-Helicobacter pylori agents. Part 3: A novel, efficacious cephem derivative, FR193879.

The synthesis, therapeutic efficacy against H. pylori, and preliminary safety of the novel cephem derivative, FR193879 (8a) are described. Compound 8a having a (4-carbamoylmethylthiazol-2-yl)thio moiety at the 3-position and a phenylacetamido at the 7-position was found to have good safety showing a nontoxic dose of > 100 mg/kg in dogs in a 4-week repeat dose toxicity study and extremely potent therapeutic efficacy against H. pylori, showing 30 times superior activity compared to AMPC, and did not display cross-resistance with CAM or MNZ.

In vitro antifungal activity of Micafungin (FK463) against dimorphic fungi: comparison of yeast-like and mycelial forms.

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 micro g/ml), while it was very weakly active against their yeast-like forms (MIC range, 32 to >64 micro g/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.

Orally active cephalosporins. Part 4: synthesis, structure--activity relationships and oral absorption of novel 3-(4-pyrazolylmethylthio)cephalosporins with various C-7 side chains.

A series of 3-(4-pyrazolylmethylthio)cephalosporins with various C-7 side chains was designed, synthesized and evaluated for antibacterial activity and oral absorption in rats. Antibacterial activity against Haemophilus influenzae was markedly increased by the C-7 oxime moiety. Deamination at the 2 position of, or introduction of a substituent such as halogen or methyl to, the 5 position of the (Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino) moiety improved oral absorption. Among these compounds, FR192752 having a (Z)-2-(2-amino-5-chlorothiazol-4-yl)-2-hydroxyiminoacetamido moiety, showed potent antibacterial activity against both Gram-positive and Gram-negative bacteria including H.influenzae and penicillin G-resistant Streptococcus pneumoniae (PRSP). Further, it showed higher oral absorption than CFDN and FK041.