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, Ϲ0.0039 to 2 g/ml) and Aspergillus species (MIC range, Ϲ0.0039 to 0.0313 g/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 g/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.

No 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 yeastlike 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 g/ml), while it was very weakly active against their yeast-like forms (MIC range, 32 to >64 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.

FR264205 is a novel parenteral 3-aminopyrazolium cephalosporin. This study evaluated the in vitro and in vivo activities of FR264205 against Pseudomonas aeruginosa. The MIC of FR264205 at which 90% of 193 clinical isolates of P. aeruginosa were inhibited was 1 g/ml, 8-to 16-fold lower than those of ceftazidime (CAZ), imipenem (IPM), and ciprofloxacin (CIP). FR264205 also exhibited this level of activity against CAZ-, IPM-, and CIP-resistant P. aeruginosa. The reduction in the susceptibility of FR264205 by AmpC ␤-lactamase was lower than that of CAZ, indicating a relatively high stability of FR264205 against AmpC ␤-lactamase, the main resistance mechanism for cephalosporins. Neither expression of efflux pumps nor deficiency of OprD decreased the activity of FR264205. No spontaneous resistance mutants were selected in the presence of FR264205, and the reduction in susceptibility to FR264205 was lower than that to CAZ, IPM, and CIP after serial passage, suggesting that FR264205 has a low propensity for selecting resistance. In murine pulmonary, urinary tract, and burn wound models of infection caused by P. aeruginosa, the efficacy of FR264205 was superior or comparable to those of CAZ and IPM. These results indicate that FR264205 should have good potential as an antibacterial agent for P. aeruginosa.Nosocomial infections with gram-negative bacteria are a major problem for immunocompromised patients. Pseudomonas aeruginosa exhibits considerable inherent resistance, caused by low outer membrane permeability, multiple efflux pumps, and chromosomal AmpC ␤-lactamase (12). P. aeruginosa can also acquire additional resistance mechanisms, such as constitutive production of AmpC ␤-lactamase, OprD loss, and overproduction of efflux pumps. Although ceftazidime (CAZ) has been used as a first-line drug for P. aeruginosa infection, resistant mutants showing constitutive AmpC ␤-lactamase production can be selected in clinical settings, leading to therapeutic failure (2). P. aeruginosa has developed resistance not only to cephalosporins but also to carbapenems and quinolones. In 2003, the National Nosocomial Infections Surveillance System reported that resistance rates of P. aeruginosa to imipenem, quinolone, and broad-spectrum cephalosporins were 21.1, 29.5, and 31.9%, respectively. Compared to rates in the period between 1998 and 2002, these rates were increased by 15, 9, and 20%, respectively (15). Therefore, there is a critical need for new anti-P. aeruginosa agents that have no cross-resistance to currently marketed antibacterial agents and low propensities for inducing resistance.In order to generate promising anti-P. aeruginosa agents, research by our group has been directed toward the development of novel cephalosporins. As a result of exploration of structure-activity relationships of 3-(2,4-disubstituted 3-aminopyrazolio)methyl cephalosporins, FR264205 was discovered (Fig. 1). The antibacterial spectrum of FR264205 was similar to that of CAZ, and the MICs of FR264205 for Staphylococcus aureus ATCC 29213, Streptococcus pn...

A highly cephem-resistant Escherichia coli strain, FP1546, isolated from the fecal flora of laboratory dogs previously administered 1-lactam antibiotics was found to produce a P-lactamase, FEC-1, of 48-kilodalton size and pl 8.2. FEC-1 hydrolyzed cefuroxime, cefotaxime, cefmenoxime, and ceftriaxone, as well as the enzymatically less-stable antibiotics cephaloridine, cefotiam, and cefpiramide. Of the oxyimino-cephalosporins, ceftizoxime was fairly stable to FEC-1. FEC-1 differed notably from chromosomal E. coli cephalosporinase, especially in its broad-spectrum substrate profile and its high inhibition by clavulanic acid, sulbactam, and imipenem. A conjugation study revealed that FEC-1 was encoded by a 74-megadalton plasmid, pFCX1. This may be the first instance of a plasmid-mediated oxyimino-cephalosporinase from E. coli.Over 30 plasmid-mediated f-lactamases (mostly penicillinases) have been found in gram-negative bacteria and classified according to characteristics such as substrate specificity, isoelectric point, and molecular weight. Common are the TEM, OXA, SHV, and PSE types (10,(17)(18)(19)24), with the TEM type of penicillinase being the most frequently isolated plasmid P-lactamase from such gram-negative bacteria as members of the family Enterobacteriaceae, Pseudomonas aeruginosa, Haemophilus influenzae, and Neisseria gonorrhoeae. On the other hand, there have been only two reports of plasmid-mediated cephalosporinases, from Proteus mirabilis (2) and Achromobacter spp. (14).We evaluated the possible resistance mechanisms of oxyimino-cephalosporin-resistant Escherichia coli isolated from the fecal flora of laboratory dogs, and we identified a 3-lactamase which hydrolyzes oxyimino-cephalosporins such as cefuroxime, cefotaxime, cefmenoxime, and ceftriaxone in addition to cephaloridine. The physiological properties of this enzyme were quite distinct from those of chromosomal cephalosporinases from E. coli (20,23). We confirmed that this enzyme was plasmid mediated and had properties similar to those of oxyimino-cephalosporinase type I (7). MATERIALS AND METHODSBacterial strains. E. coli FP1546 was isolated from the fecal flora of a laboratory dog being used for pharmacokinetic studies of P-lactam antibiotics. The nalidixic acidresistant derivative of E. coli CSH2 (metB F-) was kindly provided by T. Yokota of Juntendo University.Antibiotics. Commercially available cephaloridine, cephalothin, cefamandole, cefotiam, cefmetazole, cefsulodin, cefuroxime, cefotaxime, cefmenoxime, ceftriaxone, ceftazidime, cefoperazone, cefpiramide, cefoxitin, moxalactam, and imipenem were used. Cefazolin and ceftizoxime were from Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan. * Corresponding author.Clavulanic acid, sulbactam, and nitrocefin were synthesized in our laboratories.Susceptibility testing. Antibacterial activity of test antibiotics was determined by the agar dilution method. Hundredfold dilutions of overnight cultures in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) were inoculated with a multipoint replic...

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 (ED 50 s) 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 log 10 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 ED 50 s 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.People who have impaired immune systems are susceptible to fungal infections which can be life-threatening. Immune deficiencies resulting from AIDS, aggressive cancer treatment, the growing use of organ transplants, and other nosocomial situations have greatly increased the incidence of serious fungal infections (2,3,4,6) and have created a critical need for new, safe fungicidal agents that can be used to treat disseminated infections. Systemic mycoses are not easily diagnosed, and the patient usually has been infected for quite some time before symptoms appear. Thus, empiric therapy needs to begin immediately, but currently available treatments have problems with toxicity or resistance. Amphotericin B (AMPH-B) is the first-line therapy for systemic infections because of its broadspectrum and fungicidal activity. However, significant side effects limit its clinical utility to controlled intravenous administration (16). Lipid AMPH-B formulations have recently attracted much attention due to significantly lower toxic...

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.

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.