Efficacy of laninamivir octanoate in mice with advanced inflammation stage caused by infection of highly lethal influenza virus.

Four neuraminidase (NA) inhibitors and an RNA synthesis inhibitor were recently approved and are currently in clinical use for influenza. Among NA inhibitors, oseltamivir phosphate (OSE, Tamiflu®) and zanamivir are approved worldwide, whereas peramivir and laninamivir octanoate (LAN, Inavir®) are regionally approved for human use. Therefore, OSE has been used to treat infections of highly pathogenic influenza viruses, such as H5N1 and H7N9, which caused epidemic in southeast Asia and Egypt, and China, respectively. Generally, OSE is administered twice daily for 5 days by oral administration, and LAN once by inhalation for completing influenza therapy. In this study, we compared the efficacy of OSE and LAN administered according to the regimens in mice infected with highly lethal influenza viruses. The drugs were administered at the early and late stages of infection, which correspond to mild and severe inflammation in the lungs, respectively. Based on the drugs' regimens for human, a single administration of LAN at both stages of inflammation showed superior efficacy to repeated administration of OSE. LAN, as in OSE, could also be efficacious in treating severe influenza in humans.

In vivo antianaerobe activity of DS-8587, a new fluoroquinolone, against Fusobacterium necrophorum in a mouse model.

DS-8587 is a novel parenteral fluoroquinolone, which has an activity equivalent to sitafloxacin against various pathogens including anaerobes. We examined the in vivo anti-anaerobic activity of DS-8587, and compared it with that of levofloxacin (LVFX), using a murine model of Fusobacterium necrophorum-induced liver abscess developed via blood borne infection. Mice with liver abscess infection caused by F. necrophorum were treated with saline (control), DS-8587 (0.8, 4, and 20 mg/kg twice daily), or LVFX (20 and 100 mg/kg) for a day. After treatment, the number of viable bacteria in liver was analyzed. We also analyzed the pharmacokinetics of these agents in plasma and the liver after initial treatment. The MICs of DS-8587 and LVFX were 0.015 and 1 mg/mL, respectively. DS-8587 eradicated the viable bacteria in the liver even at doses as low as 4 mg/kg. In contrast, the liver bacteria were not eradicated in any of the LVFX-treated mice even at a dose of 100 mg/kg (P < 0.05 compared with DS-8587, 4 or 20 mg/kg). The pharmacokinetic parameter AUC/MIC ratios for DS-8587 (4 mg/kg) and LVFX (100 mg/kg) were 96.7 and 60.8 in plasma and 600 and 145.6 in the liver, respectively. The AUC/MIC ratio showed the best correlation with efficacy of DS-8587. DS-8587 significantly reduced the number of viable bacteria in a murine model of F. necrophorum-induced liver abscess compared to LVFX. Our study demonstrated that the anti-anaerobic activity of quinolones in vivo was different from the MICs in vitro.

Characteristics of antibiotic resistance and sequence type of Acinetobacter baumannii clinical isolates in Japan and the antibacterial activity of DS-8587.

DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the antibacterial activity and mechanism of DS-8587 in 31 quinolone-resistant Acinetobacter baumannii clinical isolates. Efflux pump and qnr genes, mutations in quinolone resistance-determining regions of target enzymes, and sequence types determined by multilocus sequence typing were analyzed. Forty-two quinolone-susceptible clinical isolates were analyzed for comparison. For susceptibility testing, DS-8587 exhibited more effective antibacterial activity when compared with ciprofloxacin and levofloxacin. When combined with the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, the MIC of DS-8587 was less affected when compared with the MIC exhibited by combined ciprofloxacin and 1-(1-napthylmethyl)-piperazine. The efflux pump genes adeA/adeB/adeC and regulatory elements adeR/adeS were detected in 23 of 31 quinolone-resistant isolates. The qnrA/qnrB/qnrS genes were not detected in any A. baumannii isolates analyzed. Mutations in quinolone resistance-determining regions were observed in all 31 quinolone-resistant isolates. Multilocus sequence typing analyses revealed that 22 of 31 quinolone-resistant isolates belonged to ST-2, corresponding to international clonal lineage II. In conclusion, DS-8587 exhibits potent antibacterial activity against quinolone-resistant A. baumannii isolates that harbor mutations in quinolone resistance-determining regions. In the presence of the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, no significant changes were observed in the MIC for DS-8587. DS-8587 should be considered as a treatment option for A. baumannii including ST-2 strains that are predominant among the quinolone-resistant A. baumannii isolates found in Japan.

Anti-multidrug-resistant Acinetobacter baumannii activity of DS-8587: In vitro activity and in vivo efficacy in a murine calf muscle infection model.

DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the in vitro and in vivo antibacterial activity of DS-8587 against multidrug-resistant (MDR) Acinetobacter baumannii. The MIC range of DS-8587 against MDR A. baumannii was 0.25-2 mg/L. These DS-8587 MICs were a minimum of 16-fold or 8-fold more potent than ciprofloxacin or levofloxacin, respectively. Bactericidal activity, a 3 log10 reduction from the initial bacterial counts, was observed within 2 h for 1593644 and 4 h for 1593684 after exposure to DS-8587. Therapeutic efficacy of DS-8587 in the murine calf muscle model was observed at 256 mg/kg. The analysis of the pharmacokinetic and pharmacodynamic index revealed that the AUC/MIC ratio showed the best correlation with efficacy. The total and free drug AUC/MIC value required for a static effect was 29.4 and 14.1, respectively. These data indicate DS-8587 would be an effective agent against MDR A. baumannii infection.

Structural basis for the inhibition of bacterial multidrug exporters.

The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π-π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.

Rapid bactericidal activity of sitafloxacin against Streptococcus pneumoniae.

The initial bactericidal activity of quinolones against Streptococcus pneumoniae at the concentration equivalent to their respective peak serum concentration (C(max)) and free drug fraction of C(max) (fC(max)) were investigated. The bactericidal activity of sitafloxacin (STFX), levofloxacin (LVFX), moxifloxacin (MFLX), and garenoxacin (GRNX) were compared by determining the actual killing of bacteria at C(max) and fC(max) for 1 and 2 hours based on the Japanese maximum dose per administration (100, 500, 400, and 400 mg, respectively). Against 4 quinolone-susceptible clinical isolates (wild-type), STFX with C(max) and fC(max) exhibited the most rapid bactericidal activity resulting in an average reduction of > or = 3.0 log10 colony forming units (CFU)/ mL in 1 hour. STFX with C(max) and fC(max) also showed the most rapid and potent bactericidal activity against 9 clinical isolates with single par (C/E) mutation, resulting in > or = 3.0 log10 CFU/mL average reduction in viable cells in 1 hour. STFX showed a statistically significant advantage in initial bactericidal activity over other quinolones for single mutants (P < 0.001). The propensity that the difference in the initial bactericidal activity between STFX and other quinolones was higher in single mutants than wild-type strains, was confirmed using S. pneumoniae ATCC49619 (wild-type) and its laboratory single parC mutant. As a result, STFX showed a similar rapid and potent initial bactericidal activity against both strains, while initial bactericidal activity for other quinolones was significantly reduced in the single mutant (P < 0.05). In conclusion, STFX has the most rapid and potent initial bactericidal activity against wild-type and single mutants of S. pneumoniae and its bactericidal activity is not affected by the presence of a single par mutation compared to LVFX, MFLX, and GRNX.

Potent in vitro antibacterial activity of DS-8587, a novel broad-spectrum quinolone, against Acinetobacter baumannii.

We investigated the in vitro activity of DS-8587, a novel fluoroquinolone, against Acinetobacter baumannii. The MICs of DS-8587 against clinical isolates and its inhibitory activity against target enzymes were superior to those of ciprofloxacin and levofloxacin. Furthermore, the antibacterial activity of DS-8587 was less affected by adeA/adeB/adeC or abeM efflux pumps than was that of ciprofloxacin and the frequency of single-step mutations with DS-8587 was lower than that with ciprofloxacin. DS-8587 might be an effective agent against A. baumannii infection.

[Antimicrobial and rapid bactericidal activities of sitafloxacin and other agents against Streptococcus pyogenes].

We evaluated the in vitro activity of sitafloxacin against Japanese clinical isolates of Streptococcus pyogenes by broth microdilution susceptibility testing and time-kill studies to elucidate its eradication potential against S. pyogenes. One hundred and nineteen clinical isolates of S. pyogenes isolated from pharynx were tested to sitafloxacin and seven other agents in the susceptibility testing. The time-kill studies were conducted with five strains, one of which was resistant to clarithromycin, one resistant to levofloxacin and one type strain of S. pyogenes. In the time-kill studies, sitafloxacin, garenoxacin, amoxicillin and clarithromycin were assessed at static concentrations of their respective peak concentrations in plasma (C(max)) when administered as oral single doses for adult patients with S. pyogenes infections. We found the rank order of antimicrobial activity against S. pyogenes isolates was: cefcapene (MIC90, 0.015 microg/mL) > amoxicillin (0.03 microg/mL) > sitafloxacin (0.12 microg/mL) > garenoxacin (0.25 microg/mL) > levofloxacin (4 microg/mL) > minocycline (16 microg/mL). Macrolide-resistant isolates accounted for 72 (60.5%), resulting in clarithromycin and azithromycin MIC90s of > 32 and > 128 microg/mL, respectively. Sitafloxacin exhibited the most rapid bactericidal activity (> or = log reduction from the initial inoculum) within 2h against all tested strains, including even one levofloxacin-resistant strain. For garenoxacin, bactericidal activity was achieved between 2 and 6 h. Amoxicillin revealed no significant bactericidal activity up to 6 h. Clarithromycin showed no bactericidal activity and did not inhibit growth of a clarithromycin-resistant strain. These data indicate the potential usefulness of sitafloxacin for the treatment of S. pyogenes eradication.

[Bactericidal effect of levofloxacin injection in combination with meropenem against Pseudomonas aeruginosa using an in vitro simulation model with a hollow fiber system].

An in vitro human plasma concentration simulation model with a hollow fiber system was established and used to evaluate the bactericidal effect of levofloxacin (LVFX) 500mg q.d. in combination with meropenem (MEPM) 1000mg t.i.d. against Pseudomonas aeruginosa. Six clinical isolates of P. aeruginosa which had MEPM MICs of 2 - 16 microg/mL, LVFX MICs of 2 microg/mL, and fractional inhibitory concentration (FIC) indices by the in vitro checkerboard method of 0.625-1 were used. In the treatment with MEPM alone, initial viable counts (10(6)-10(7) CFU/ mL) decreased, but did not reach below the detection limit (100 CFU/mL) and the regrowth of bacteria was observed. In the treatment with LVFX alone, viable counts decreased once below the detection limit, although increased after treatment for 24 hours. On the other hand, in the treatment with LVFX-MEPM combination, more potent bactericidal effects were observed compared to LVFX or MEPM alone in all strains. Especially, in the strains with MEPM MICs of 2 and 4 microg/mL, viable counts rapidly decreased below the detection limit and no regrowth was observed until 24 hours. These results suggested that LVFX-MEPM has a potential to be an effective combination against P. aeruginosa by synergistic rapid bactericidal action in clinical settings, even in the strain against which no significant synergy is confirmed by the traditional in vitro checkerboard method.

Efflux pump inhibitors reduce the invasiveness of Pseudomonas aeruginosa.

Efflux systems are thought to contribute to antimicrobial resistance in Pseudomonas aeruginosa. The mexAB-oprM deletion strain of P. aeruginosa PAO1 is compromised in its capacity to invade Madin-Darby canine kidney (MDCK) cells, suggesting that P. aeruginosa exports invasion determinants using a MexAB-OprM system. The influences of efflux pump inhibitors (EPIs), including the broad-spectrum EPI Phe-Arg-beta-naphthylamide (PAbetaN) and MexAB-OprM-specific EPI D13-9001, on the invasion of wild-type (WT) P. aeruginosa PAO1 and its MexAB-OprM-overproducing nalB strain were examined. The invasiveness of PAO1 WT and nalB strains was inhibited in the presence of EPIs in a concentration-dependent manner. Reduction of the invasiveness of both strains was greater for D13-9001 compared with PAbetaN. EPIs are thought to be useful in reducing the invasiveness and antimicrobial resistance of P. aeruginosa and thus may be promising as new anti-infectious agents.