Yasuaki Osada

In vitro and in vivo activity of DL-8280, a new oxazine derivative.

DL-8280, 9-fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H- pyrido-(1,2,3-de)1,4-benzoxazine-6-carboxylic acid, is a new nalidixic acid analog with a broad spectrum of antibacterial activity against gram-negative and gram-positive bacteria, including obligate anaerobes. The activity of DL-8280 against Enterobacteriaceae, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria gonorrhoeae, and Clostridium perfringens was roughly comparable to that of norfloxacin and far exceeded that of pipemidic acid and nalidixic acid. DL-8280 had greater activity against Staphylococcus spp., Streptococcus spp., Pseudomonas maltophilia, Acinetobacter spp., and Bacteroides fragilis than did norfloxacin, pipemidic acid, and nalidixic acid. Nalidixic acid-resistant Enterobacteriaceae, ampicillin-resistant gonococci, and clindamycin-resistant obligate anaerobes were also susceptible to DL-8280. The activity of DL-8280 was affected very little by inoculum size, and its action was bactericidal at two times the minimal inhibitory concentrations at most. Administered orally to mice experimentally infected with Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Proteus mirabilis, Serratia marcescens, or P. aeruginosa, DL-8280 was 2 to 7 times more effective than norfloxacin and 7 to more than 50 times more active than pipemidic acid.

In vitro activity of DR-3355, an optically active ofloxacin.

DR-3355, the S-(-)-isomer of ofloxacin, was generally twice as potent as ofloxacin against a variety of gram-positive and gram-negative pathogens, and its action was bactericidal. The compound was characterized by having the highest level of activity against staphylococci, Bacteroides fragilis, and Peptococcus spp. of the fluorinated quinolones tested, including ofloxacin, ciprofloxacin, fleroxacin, and NY-198. The activity of DR-3355 was not affected by different media, inoculum size, or human serum, but decreased under acidic conditions at pH 5.0 or in human urine.

Inhibitory effects of quinolones on DNA gyrase of Escherichia coli and topoisomerase II of fetal calf thymus.

The in vitro inhibitory effects of quinolones on the bacterial DNA gyrase of Escherichia coli KL-16 and topoisomerase II of fetal calf thymus were compared. All the quinolones tested required higher concentrations to inhibit the topoisomerase II than to inhibit the DNA gyrase, and no correlation existed among their inhibitory activities against both enzymes. However, there was a large difference among the quinolones in their selectivities between the bacterial enzyme and its eucaryotic counterpart. The selectivity of ofloxacin was highest, and the selectivities of CI-934 and nalidixic acid were lowest.

Significance of the methyl group on the oxazine ring of ofloxacin derivatives in the inhibition of bacterial and mammalian type II topoisomerases.

A study was made of the correlation between the in vitro inhibitory effects of several quinolones, including four ofloxacin derivatives, on bacterial DNA gyrase from Escherichia coli KL-16 and on topoisomerase II from fetal calf thymus. No correlation was observed between the inhibitions of DNA gyrase activity and topoisomerase II activity. On the other hand, the inhibitory effects of these quinolones against topoisomerase II were closely correlated with their inhibition of cell growth. Furthermore, among the oxazine derivatives tested, the derivative with a methyl group at position 3 in an S configuration showed the highest activity against DNA gyrase and derivatives without a methyl group on the oxazine ring were more potent against topoisomerase II than those with a methyl group. Among these derivatives, DR-3355, the S isomer of ofloxacin, showed the highest activity against DNA gyrase and low activity against topoisomerase II. These results indicate that the methyl group on the oxazine ring plays an important role in the inhibitory activities of ofloxacin derivatives for these enzymes.

Antimicrobial activity of DV-7751a, a new fluoroquinolone.

We compared the in vitro antibacterial activity of DV-7751a against gram-positive and -negative bacteria with those of quinolones currently available. MICs for 90% of the strains tested (MIC90s) against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis were 0.20, 0.39, 0.20, and 0.78 micrograms/ml, respectively. Moreover, MIC50s for DV-7751a against ofloxacin-resistant methicillin-resistant S. aureus were 4-, 8-, 16-, 32-, and 64-fold lower than those for tosufloxacin and sparfloxacin, levofloxacin, ofloxacin and fleroxacin, ciprofloxacin, and lomefloxacin, respectively. DV-7751a inhibited the growth of all strains of Streptococcus pneumoniae, Streptococcus pyogenes, and Peptostreptococcus spp. at 0.39, 0.39, and 0.78 micrograms/ml, respectively, and was 4- to > 16-fold more active against enterococci at the MIC90 level than the other quinolones tested. The activity of DV-7751a against Pseudomonas aeruginosa was roughly comparable to those of levofloxacin and sparfloxacin at the MIC90 level and was two- to fourfold less than that of ciprofloxacin. DV-7751a showed activity comparable to those of levofloxacin and ciprofloxacin against the other glucose-nonfermenting bacteria Haemophilus influenzae, Neisseria gonorrhoeae, and Moraxella catarrhalis (MIC90s of 0.025, 0.20, and 0.10 micrograms/ml, respectively). DV-7751a activity was not affected by medium, inoculum size, or the addition of human serum but was decreased under acidic conditions and in human urine, as were the other quinolones tested. Time-kill curve studies demonstrated the rapid bactericidal action of DV-7751a against S. aureus, S. pneumoniae, Escherichia coli, and P. aeruginosa. The frequency of spontaneous resistance to DV-7751a was less than or equal to those of the reference drugs. DV-7751a inhibited the supercoiling activity of DNA gyrases from S. aureus, E. coli, and P. aeruginosa at concentrations comparable to those of levofloxacin and sparfloxacin.

Enhancement of non-specific resistance to Pseudomonas pneumonia by a synthetic derivative of muramoyl dipeptide in immunosuppressed guinea pigs.

A synthetic derivative of muramoyl dipeptide, 6-O-stearoyl-N-acetylmuramoyl-L-alanyl-D-isoglutamine [L18-MDP(A)], showed a protective effect against bacteraemic and non-bacteraemic pneumonia caused by Pseudomonas aeruginosa in immunosuppressed guinea pigs. In about half of the animals treated with the compound before infection, death from bacteraemic pneumonia produced by intratracheal inoculation of P. aeruginosa was delayed for 7 d, although all of the animals infected without prior treatment with the compound died within 4 d of infection. Multiplication of the organisms in the lung was also suppressed for at least 10 d by treatment with the compound when the animals inhaled an aerosol of P. aeruginosa. In contrast, in untreated animals the numbers of bacteria in the lung gradually increased from 10(6) to 10(9) c.f.u. g-1, and a few animals in which the organism increased to 10(9) c.f.u. g-1 had died by 6 and 10 d after infection. In both healthy and immunosuppressed animals, the accumulation of polymorphonuclear leukocytes (PMNs) in a subcutaneous air-pouch injected with heat-killed organisms was augmented by subcutaneous treatment with L18-MDP(A) 1 d before bacterial injection. The phagocytic activity of peritoneal PMNs was also increased by treatment with this compound. The augmentation of protective mechanisms against pseudomonas pneumonia by L18-MDP(A) may be attributed at least partly to the increased chemotactic and phagocytic activity of PMNs.

Restoration by MDP-Lys(L18) of Resistance to Pseudomonas Pneumonia in Immunosuppressed Guinea Pigs

Infections caused by gram-negative bacteria are quite frequent in patients with marginal or defective host defenses caused by antineoplastic chemotherapy or various forms of immunosuppressive treatment (12, 13). One of the most common infection sites in these patients is the lower respiratory tract including the lungs (2), and these infections are usually difficult to treat in spite of the development of potent antibiotics (14). In particular, pneumonia induced by Pseudomonas aeruginosa has attracted a great deal of attention because of the high rate of mortality involved

Protective Role of Complement in the Development of Experimental Pneumococcal Pneumonia in Mice

To evaluate the role of complement in the lung defense against Streptococcus pneumoniae, mice decomplemented with multiple injections of cobra venom factor were challenged with type 3 pneumococci by inhalation. Without injection of cobra venom factor, the organisms were eliminated rapidly from the lungs in the majority of mice, accompanied by a significant but transient decrease in the serum C3 level. Focal pneumonia developed occasionally in some mice retaining the organisms in the lungs. By decomplementation with cobra venom factor, on the other hand, pneumococci were not eliminated completely from the lungs during the early stage of infection and afterward proliferated extensively. Consequently, the mice developed typical pneumococcal pneumonia with attendant bacteremia, while the serum C3 level has recovered compensatory during the course of infection. Thus, the complement was indicated to play an important role in the lung defense against pneumococci in mice, especially during the early stage of infection.

In vitro activity of DQ-2556, a new cephalosporin.

The in vitro antibacterial activity of DQ-2556, a new semisynthetic cephalosporin, was compared with that of ceftazidime and cefotaxime. The activity of DQ-2556 against members of the family Enterobacteriaceae was roughly comparable to that of cefotaxime. Against Pseudomonas aeruginosa, DQ-2556 was slightly less active than ceftazidime. DQ-2556 was more active than the reference cephalosporins against staphylococci. Haemophilus influenzae and Neisseria gonorrhoeae were also highly susceptible to DQ-2556.

Phagocytosis stimulation by an extracellular product of virulent Shigella flexneri 2a.

It has been made clear that the cell infectivity of virulent Shigella flexneri 2a was stimulated by divalent cations such as Mg++ or Ca++ added to the growth medium (16, 17). The stimulative effect of the divalent cations was confirmed by the fact that addition of EDTA to the growth medium gave a reduction of the increased cell infectivity of the bacilli. Furthermore, the reductive effect of EDTA was noticed in the keratoconjunctival infection system in guinea pigs, i.e., the development and severity of lesions were suppressed by treatment with lotions of EDTA (17). These facts caused us to speculate that cell invasiveness of virulent Shigella might be mediated by a certain extracellular product specifically activated, stabilized, or mediated in its formation by Mg++ and/or Ca++. From this point of view, we examined the effect of crude extracellular products of a virulent strain of S. flexneri 2a on the cell infectivity of a few strains of Shigella. The bacteria used in this study were S. flexneri 2a, strain 5503, and Shigella sonnei, strains 74-306 and 74-487. Strain 5503 used mainly was isolated from the colonal specimens of a cynomolgus monkey which died of natural bacillary dysentery (8). It has been maintained in the lyophilized state as a standard strain in our laboratory. Strains 74-306 and 74-487 obtained from Dr. Zen-Yoji, The First Department of Bacteriology, Tokyo Metropolitan Research Laboratories of Public Health, were isolated from patients with sporadic bacillary dysentery in 1974 in Tokyo, and have also been maintained in the lyophilized state. Strain 5503 was subcultured in Luria broth (4) supplemented with MgSO4 at a final concentration of 5 mm at 37 C for 18 hr. One milliliter of the broth culture was then inoculated into 400 ml of the same broth, and incubated at 37 C for a further 18 hr with shaking. The shaking culture was centrifuged at 10,000 rpm for 15 min, and the supernatant fluid was sterilized by millipore filtration. The filtrate was then lyophilized after sufficient dialysis against distilled water and named as LB ( +). As controls, the organisms were cultivated in the broth without MgSO4, and the lyophilized sample named LB (-) was prepared by the same procedure as mentioned above. These