Paul Robert Mcguirk

Use of in vitro topoisomerase II assays for studying quinolone antibacterial agents.

Several quinolones and antitumor compounds were tested as inhibitors of purified calf thymus topoisomerase II in unknotting, catenation, radiolabeled DNA cleavage, and quantitative nonradiolabeled cleavage assays. The antitumor agents VP-16 (demethylepipodophyllotoxin ethylio-beta-D-glucoside) and ellipticine demonstrated drug-enhanced topoisomerase II DNA cleavage (the concentration of drug that induced 50% of the maximal DNA cleavage in the test system [CC50]) at levels of less than or equal to 5 micrograms/ml. Nalidixic acid, norfloxacin, and oxolinic acid did not induce significant topoisomerase II DNA cleavage, whereas ciprofloxacin did induce some cleavage above background levels. CP-67,015, a new 6,8-difluoro-7-pyridyl 4-quinolone which possesses potent antibacterial activity, inhibited bacterial DNA gyrase at 0.125 micrograms/ml in a nonradioactive DNA cleavage assay. Unlike other quinolones characterized to date, CP-67,015 was shown to strongly enhance topoisomerase II-induced radiolabeled DNA cleavage with a CC50 of 33 micrograms/ml and demonstrated cleavage in a nonradiolabeled DNA cleavage assay with a CC50 of 73 micrograms/ml. The topoisomerase II-mediated cleavage of DNA by CP-67,015 is consistent with its reported clastogenic effect on DNA in cell culture and its positive mutagenic response in mouse lymphoma cells. In vitro topoisomerase II catalytic and cleavage assays are useful for gaining preliminary information concerning the possible interaction(s) of some quinolones with eucaryotic topoisomerase II which may relate directly to their safety (mutagenicity, clastogenicity, or both) in human and veterinary medicinal usage. Images

New quinolones in development

Fluoroquinolones, such as ciprofloxacin and ofloxacin have recently gained wide acceptance for use in the treatment of respiratory tract, skin/soft tissue, sexually transmitted diseases and urinary tract infections. The broad spectrum activity and good oral absorption characteristics of these antimicrobials promotes their use in both community and hospital settings. Despite these favourable properties, ciprofloxacin and ofloxacin have limited potency against some clinically important organsims, such as Streptococcus pneumoniae, enterococci and anaerobes including Bacteroides fragilis and many methicillin-resistant staphylococci. In addition, occasional clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa have emerged resistant to these compounds following their introduction. In an effort to expand upon the clinical utility of the existing fluoroquinolones, several new agents of this class have been identified and are in various stages of development. Some of these newer fluoroquinolones have...

In vitro activity of CP-99,219, a novel 7-(3-azabicyclo[3.1.0]hexyl) naphthyridone antimicrobial

The in vitro activity of CP-99,219 was compared with that of ciprofloxacin and sparfloxacin against 814 clinical bacterial isolates using a microdilution method with brain-heart infusion broth. CP-99,219 was the most potent agent tested against methicillin-resistant, ciprofloxacin-susceptible staphylocci (minimum inhibitory concentration [MIC]90 < or = 0.25 microgram/ml). CP-99,219 was 32-fold and fourfold more potent than ciprofloxacin and sparfloxacin, respectively, against Streptococcus pneumoniae, including strains resistant to penicillin G and erythromycin (MIC90 < or = 0.25 microgram/ml). CP-99,219 was also the most potent agent tested against S. pyogenes and Enterococcus faecalis (MIC90 < or = 0.5 microgram/ml). The activity of CP-99,219 against Enterobacteriaceae was comparable to that of sparfloxacin, with 90% of Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Citrobacter freundii, C. diversus, Helicobacter pylori, and K. oxytoca being inhibited by < or = 0.5 microgram/ml. Serratia marcescens, Morganella morganii, and Pseudomonas aeruginosa were less susceptible, with MIC90 values to CP-99,219 of 4, 2, and 2 micrograms/ml, respectively. The MIC90 for Bacteroides fragilis was 0.39 microgram/ml for CP-99,219 compared with 12.5 micrograms/ml for ciprofloxacin. CP-99,219 was highly bactericidal at 1 x to 4 x MIC against both Gram-positive and Gram-negative organisms; its activity was similar in nutrient, trypticase soy, and cation-supplemented Mueller-Hinton broths. The spectrum and potency observed with CP-99,219 warrant further testing with this novel quinolone.

Placement of alkyl substituents on the C-7 piperazine ring of fluoroquinolones: dramatic differential effects on mammalian topoisomerase II and DNA gyrase.

Several substituted analogs of 7-(cis-3,5-dimethylpiperazinyl)-6,8-difluoro-5-amino-1-cyclopropyl quinolone were prepared and tested in a DNA cleavage assay with calf thymus topoisomerase II. Positioning of the methyl groups on the C-7 piperazine ring influenced potency against the mammalian enzyme; the cis-3,5-dimethyl configuration did not stimulate cleavage at drug concentrations less than or equal to 2,000 microM, while the trans configuration was active at drug levels as low as 36 microM. Removal of the cis-methyl groups produced a compound that was only sixfold less potent than the antitumor agent etoposide in stimulating enzyme-mediated DNA cleavage. The cis- and trans-methyl substitutions on the piperazine that conferred potency against the mammalian type II enzyme had little effect on bacterial DNA gyrase cleavage activity, suggesting that an asymmetric barrier exists with the mammalian enzyme which influences productive quinolone interaction, favoring the less bulky trans-3,5-dimethylpiperazine substituent at C-7. Images

Chapter 13. Antibacterial Agents

Publisher Summary Antibacterial research has focused on the discovery of agents that exhibit improved activity, particularly against resistant organisms, and have a decreased incidence of side effects. This chapter discusses the mechanisms of resistance and transport for various classes of antibacterial agents. The development of new quinolone compounds has been characterized with an emphasis on structural novelty, improvement of potency versus anaerobic and gram-positive pathogens, incorporation of activity against quinolone-resistant strains, and early assessment of the potential for adverse drug reactions. New β-lactam quinolone hybrids have been reported; the carbamate-linked Ro 24-4383 shows broad spectrum activity and exhibits aqueous solubility much greater than that of the quinolone component alone. A new fluoromethoxyimino parenteral cephalosporin, E1077, shows a two-fold greater activity than cefpirome against methicillin-resistant Staphylococcus aureus (MRSA) and P. aeruginosa. The development of new macrolide antibacterial agents has focused on agents with a broad spectrum of activity, including gram-negative (Haemophilus influenzae) and macrolide-lincosamide-streptogramin (MLS)-resistant bacteria, high oral absorption, acid stability, and minimal gastrointestinal side effects. Magainins composed entirely of d -amino acids have been found to retain antibacterial potency but to resist proteolytic cleavage. RP59500 is a synergistic, water-soluble combination of RP57669 and RP54476, which are semisynthetic derivatives of the two main components of the naturally occurring streptogramins.

Novel 1-8-bridged chiral quinolones with activity against topoisomerase II: stereospecificity of the eukaryotic enzyme.

A series of novel C-7 quinolyl-substituted enantiomers of ofloxacin were used to determine the stereospecificity of topoisomerase II for the C-11 methyl group in tricyclic quinolones. In all cases, the S isomer was the most active compound against the eukaryotic enzyme. It was approximately 2.2-fold more potent than the R isomer at inhibiting the overall catalytic activity of topoisomerase II (as monitored by DNA relaxation assays). A markedly greater difference in quinolone activity was observed in enzyme-mediated DNA cleavage reactions. While the S enantiomer stimulated nucleic acid breakage approximately 3.5-fold, the R compound did not enhance and, in fact, decreased initial DNA cleavage levels by approximately 50%. The activity of the racemic mixture more closely resembled that of the R enantiomer. In competition experiments, the DNA cleavage-enhancing effects of the S isomer were attenuated by the R compound. Taken together, these latter results indicate that the R enantiomer is an antagonist of S isomer-promoted topoisomerase II-mediated DNA cleavage. Finally, the cytotoxic potential of quinolyl-substituted ofloxacin analogs correlated with the ability to stimulate enzyme-mediated DNA cleavage. Thus, stereochemistry appears to be a governing factor for the potential development of tricyclic quinolones as topoisomerase II-targeted drugs with antineoplastic activity. Images