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.

The relationship between the chemical structure and epileptogenic activity of quinolones was investigated. When the quinolones were administered intravenously to mice concomitantly with oral biphenylacetic acid, a major metabolite of the nonsteroidal antiinflammatory drug fenbufen, enoxacin, norfloxacin, ciprofloxacin, and pipemidic acid, which have an unsubstituted piperazine moiety at the 7 position of their parent nuclei, provoked clonic convulsions and subsequent death at doses of 6.25 mg/kg or more in a dose-dependent manner. AM-1091 and T-3262, which have an unsubstituted aminopyrrolidine moiety at their 7 positions, were less epileptogenic than the compounds listed above were. In contrast, ofloxacin, AT-4140, and nalidixic acid, which have piperazine substituted with methyl group(s) or no piperazine moiety at their 7 positions, never induced convulsions, even at doses of 100 mg/kg. Lomefloxacin, which has a 3-methyl piperazine, however, provoked convulsions at doses of 6.25 mg/kg or more. In the presence of biphenylacetic acid, all the test quinolones except nalidixic acid competitively inhibited [3H]muscimol binding to receptor sites for y-aminobutyric acid (GABA) in vitro. Nalidixic acid did not inhibit the binding at all, even at the highest concentration tested, i.e., 10-4 M. The 50% inhibition doses for [3H]muscimol binding varied within 4 orders of magnitude or more, between 10-8 to more than 10-4 M for various compounds, and there was a close correlation between the epileptogenic activities of quinolones and their inhibitory potencies for [3H]muscimol binding to GABA receptor sites. These results indicate that the epileptogenic activity of quinolones possibly relates to the GABA-like structures of substituents at their 7 positions, which act as antagonists of GABA receptors.

The abilities of Yersinia pestis to undergo restriction in Ca2+-deficient medium with concomitant production of V and W antigens (Vwa+) and to absorb exogenous pigments (Pgm+) are established virulence factors. Mutation of Y. pestis to Pgm-is known to promote resistance to pesticin (Pstr) and reduced lethality by peripheral routes of injection. Vwa+ Pgm-isolates of Y. pestis were shown in this study to retain virulence in mice when injected intravenously. Although Pgm-in appearance, wild-type cells of Yersinia pseudotuberculosis and Yersinia enterocolitica may also be sensitive to pesticin. Pstr mutants of Vwa+ strains of these species were similarly of reduced virulence, especially by peripheral routes of injection. The consequences of mutation to Vwa-and Pgmor Pstr on growth and persistence in vivo were determined. After intravenous injection, Vwa+ yersiniae of all species exhibited sustained growth in mouse spleen, liver, and lung and accumulated in blood. Septicemia was not observed after similar injection of Vwamutants which were unable to maintain comparable rates of net increase in tissues. Mutation to Pgmor Pstr did not influence proliferation but resulted in enhanced clearance from organs. It is known that reticuloendothelial cells serve as favored sites of replication for all wild-type yersiniae. Our results are consistent with the hypothesis that the Vwa+ phenotype favors growth within macrophages and that the Pgm+ and pesticin-sensitive phenotypes permit long-term, probably extracellular, retention within organs. Virulence in standard animal models (mice, rats, and guinea pigs) was not correlated with resistance to the bactericidal action of serum.

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.During the past decade, the development of fluorinated quinolones such as norfloxacin (6), ofloxacin (10), ciprofloxacin (12), fleroxacin (3), and NY-198 (4) has been aimed mainly at enhancing activity against gram-negative and gram-positive pathogens. Among these compounds, ofloxacin (10) is characterized chemically by a tricyclic structure with a methyl group at the C-3 position in the oxazine ring, thus providing an asymmetric center at this position (Fig. 1). In the course of studies on the structure-activity relationships between stereochemistry and activity, two optically active isomers of ofloxacin, i.e., DR-3355 [S-(-)-ofloxacin] and DR-3354 [R-(+)-ofloxacin], were prepared successfully by use of their optically resolved synthetic intermediates ( Fig. 1) (2). A preliminary study of the spectrum of activity of these isomers against selected laboratory strains revealed that DR-3355 was 8 to 128 times more potent than DR-3354 and approximately twice as active as ofloxacin (2). In this paper we describe the in vitro activity of DR-3355, relative to other fluorinated quinolones, against a large number of clinical isolates. MATERIALS AND METHODSCompounds. DR-3355 and ofloxacin were synthesized at the Research Institute, Daiichi Seiyaku Co., Ltd., Tokyo, Japan, as were ciprofloxacin, fleroxacin, and NY-198. Methicillin sodium (Banyu Pharmaceutical Co., Ltd., Tokyo, Japan) and clindamycin phosphate (Japan Upjohn Ltd., Tokyo, Japan) were commercial products.Organisms Ofloxacin: R = -CH3

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.DNA topoisomerases are a class of enzymes that alter the topology of DNA (3,18,28). These enzymes catalyze the reactions called supercoiling, relaxing, knotting, or catenating. Among these enzymes, bacterial topoisomerase II (DNA gyrase) has been purified from various bacterial species such as Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii, and Bacteroides fragilis and has been verified to be a major target of quinolones (1,15,22,23). Eucaryotic topoisomerase II also has been purified from various sources (2,4,8,9, 19,20,25), including bovine calf thymus. Hussy et al. (13) have reported that quinolones such as ciprofloxacin, norfloxacin, and ofloxacin interfere slightly with the function of mammalian topoisomerase II as well. We purified DNA gyrase and mammalian topoisomerase II from E. coli and fetal calf thymus, respectively, and examined the selectivities of various quinolones in terms of their inhibitory effects on both enzymes by the use of plasmid DNA as substrates.Ofloxacin (24), ciprofloxacin (29), enoxacin (16), fleroxacin (10), CI-934 (12), T-3262 (5), lomefloxacin (11), and nalidixic acid were synthesized in our laboratory. All other agents were purchased from their respective manufacturers and were at least of analytical grade. Supercoiled pBR322 plasmid DNA was prepared by the cesium chloride density gradient method (17) and was relaxed by topoisomerase I (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). The A and B subunits of DNA gyrase were purified separately from E. coli KL-16 by using novobiocin-epoxy-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) and heparin-Sepharose CL-6B (Pharmacia) column chromatographies, as described previously (6, 26). Topoisomerase II was purified from the isolated nuclei of calf thymus by using BioRex 70 ion-exchange (Bio-Rad Laboratories, Richmond, Calif.), hydroxyapatite (Bio-Rad), Affigel Blue (Bio-Rad), and double-stranded DNA cellulose (Sigma Chemical Co., St. Louis, Mo.) column chromatographies by the method of Halligan et al. (9).The activity of the reconstituted DNA gyrase in the presence and absence of quinolones was determined by previously described methods (6, 7). One unit of enzyme activity was defined as the amount that brought 50% of relaxed pBR322 plasmid DNA to the supercoiled form, as detected following electrophoresis in agarose. The reaction mixture (20 ,lI) (Polaroid). The negatives were traced with a c...