Timothy W. Morris

Besifloxacin, a Novel Fluoroquinolone, Has Broad-Spectrum In Vitro Activity against Aerobic and Anaerobic Bacteria

ABSTRACT The antibacterial spectrum of besifloxacin, a novel fluoroquinolone recently approved for treatment of ocular infections, was studied using 2,690 clinical isolates representing 40 species. Overall, besifloxacin was the most potent agent tested against gram-positive pathogens and anaerobes and was generally equivalent to comparator fluoroquinolones in activity against most gram-negative pathogens. Besifloxacin demonstrated potent, broad-spectrum activity, which was particularly notable against gram-positive and gram-negative isolates that were resistant to other fluoroquinolones and classes of antibacterial agents.

In vitro evaluation of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic: studies of the mode of action in Staphylococcus aureus.

ABSTRACT Rifamycins have proven efficacy in the treatment of persistent bacterial infections. However, the frequency with which bacteria develop resistance to rifamycin agents restricts their clinical use to antibiotic combination regimens. In a program directed toward the synthesis of rifamycins with a lower propensity to elicit resistance development, a series of compounds were prepared that covalently combine rifamycin and quinolone pharmacophores to form stable hybrid antibacterial agents. We describe mode-of-action studies with Staphylococcus aureus of CBR-2092, a novel hybrid that combines the rifamycin SV and 4H-4-oxo-quinolizine pharmacophores. In biochemical studies, CBR-2092 exhibited rifampin-like potency as an inhibitor of RNA polymerase, was an equipotent (balanced) inhibitor of DNA gyrase and DNA topoisomerase IV, and retained activity against a prevalent quinolone-resistant variant. Macromolecular biosynthesis studies confirmed that CBR-2092 has rifampin-like effects on RNA synthesis in rifampin-susceptible strains and quinolone-like effects on DNA synthesis in rifampin-resistant strains. Studies of mutant strains that exhibited reduced susceptibility to CBR-2092 further substantiated RNA polymerase as the primary cellular target of CBR-2092, with DNA gyrase and DNA topoisomerase IV being secondary and tertiary targets, respectively, in strains exhibiting preexisting rifampin resistance. In contrast to quinolone comparator agents, no strains with altered susceptibility to CBR-2092 were found to exhibit changes consistent with altered efflux properties. The combined data indicate that CBR-2092 may have potential utility in monotherapy for the treatment of persistent S. aureus infections.

In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Microbiology Profiling Studies with Staphylococci and Streptococci

ABSTRACT We present data from antimicrobial assays performed in vitro that pertain to the potential clinical utility of a novel rifamycin-quinolone hybrid antibiotic, CBR-2092, for the treatment of infections mediated by gram-positive cocci. The MIC90s for CBR-2092 against 300 clinical isolates of staphylococci and streptococci ranged from 0.008 to 0.5 μg/ml. Against Staphylococcus aureus, CBR-2092 exhibited prolonged postantibiotic effects (PAEs) and sub-MIC effects (SMEs), with values of 3.2, 6.5, and >8.5 h determined for the PAE (3× MIC), SME (0.12× MIC), and PAE-SME (3× MIC/0.12× MIC) periods, respectively. Studies of genetically defined mutants of S. aureus indicate that CBR-2092 is not a substrate for the NorA or MepA efflux pumps. In minimal bactericidal concentration and time-kill studies, CBR-2092 exhibited bactericidal activity against staphylococci that was retained against rifampin- or intermediate quinolone-resistant strains, with apparent paradoxical cidal characteristics against rifampin-resistant strains. In spontaneous resistance studies, CBR-2092 exhibited activity consistent with balanced contributions from its composite pharmacophores, with a mutant prevention concentration of 0.12 μg/ml and a resistance frequency of <10−12 determined at 1 μg/ml in agar for S. aureus. Similarly, CBR-2092 suppressed the emergence of preexisting rifamycin resistance in time-kill studies undertaken at a high cell density. In studies of the intracellular killing of S. aureus, CBR-2092 exhibited prolonged bactericidal activity that was superior to the activities of moxifloxacin, rifampin, and a cocktail of moxifloxacin and rifampin. Overall, CBR-2092 exhibited promising activity in a range of antimicrobial assays performed in vitro that pertain to properties relevant to the effective treatment of serious infections mediated by gram-positive cocci.

Integrated analysis of three bacterial conjunctivitis trials of besifloxacin ophthalmic suspension, 0.6%: etiology of bacterial conjunctivitis and antibacterial susceptibility profile

Background The purpose of this paper is to report on the bacterial species isolated from patients with bacterial conjunctivitis participating in three clinical trials of besifloxacin ophthalmic suspension, 0.6%, and their in vitro antibacterial susceptibility profiles. Methods Microbial data from three clinical studies, conducted at multiple clinical sites in the US and Asia were integrated. Species were identified at a central laboratory, and minimum inhibitory concentrations were determined for various antibiotics, including β-lactams, fluoroquinolones, and macrolides. Results A total of 1324 bacterial pathogens representing more than 70 species were isolated. The most common species were Haemophilus influenzae (26.0%), Streptococcus pneumoniae (22.8%), Staphylococcus aureus (14.4%), and Staphylococcus epidermidis (8.4%). H. influenzae was most frequently isolated among patients aged 1–18 years, while S. aureus was most prevalent among those >65 years. Drug resistance was prevalent: Of H. influenzae isolates, 25.3% were β-lactamase positive and 27.2% of S. pneumoniae isolates were penicillin-intermediate/ resistant; of S. aureus isolates, 13.7% were methicillin-resistant (MRSA), and of these, 65.4% were ciprofloxacin-resistant, while 45.9% of S. epidermidis isolates were methicillin-resistant (MRSE), and, of these, 47.1% were ciprofloxacin-resistant. Besifloxacin was more potent than comparator fluoroquinolones overall, and particularly against Gram-positive bacteria. Against ciprofloxacin-resistant MRSA and MRSE, besifloxacin was four-fold to ≥ 128-fold more potent than other fluoroquinolones. Conclusions While the pathogen distribution in bacterial conjunctivitis has not changed, drug resistance is increasing. Patient age and local antibiotic resistance trends should be considered in the treatment of this ocular infection. Besifloxacin showed broad-spectrum in vitro activity and was particularly potent against multidrug-resistant staphylococcal isolates.

Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations

ABSTRACT The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

The identification and characterization of hydrazinyl urea-based antibacterial agents through combinatorial chemistry

An effort to identify novel inhibitors of peptidoglycan synthesis with antibacterial activity resulted in the discovery of a series of biaryl urea-based antibacterial agents through isolation of a by-product from a mixture-based combinatorial library of semi-carbazones and subsequent parallel synthesis efforts. The compounds were shown to possess broad spectrum antibacterial activity against gram-positive drug resistant pathogens, and showed apparent specificity for disruption of the bacterial cell wall biosynthesis pathway.

Besifloxacin ophthalmic suspension 0.6% in the treatment of bacterial conjunctivitis patients with Pseudomonas aeruginosa infections

Background The purpose of this study was to determine the efficacy of besifloxacin ophthalmic suspension 0.6% when used in the treatment of bacterial conjunctivitis infections due to Pseudomonas aeruginosa. Methods We undertook a post hoc analysis of clinical outcomes in patients with bacterial conjunctivitis due to P. aeruginosa across four prospective, multicenter, double-masked, randomized, controlled, clinical studies of besifloxacin ophthalmic suspension 0.6%. Efficacy outcomes included bacterial eradication and clinical resolution of the baseline infection at follow-up visits. Bacterial eradication was defined as the absence of ocular bacterial species present at or above threshold at baseline, while clinical resolution was defined as grade 0 ocular discharge and bulbar conjunctival injection. Safety outcomes included the incidence of adverse events, changes in visual acuity, and biomicroscopy and ophthalmoscopy findings. Patient outcomes were summarized and bacterial eradication and clinical resolution rates integrated. Results Of 1317 patients with culture-confirmed bacterial conjunctivitis across four clinical studies, nine (0.7%) were infected with P. aeruginosa at baseline, and of these, five were randomized to treatment with besifloxacin ophthalmic suspension 0.6%. Bacterial eradication of the baseline infection was observed at both follow-up visits in all five patients. Clinical resolution was achieved in two of five patients by the first follow-up visit and four of five patients by the second follow-up visit. There were no adverse events reported in these patients. There were no clinically meaningful biomicroscopy findings or changes in ophthalmoscopy or visual acuity. Conclusion The incidence of bacterial conjunctivitis due to P. aeruginosa was low. Treatment of patients with P. aeruginosa infections with besifloxacin ophthalmic suspension 0.6% led to bacterial eradication of P. aeruginosa by the first follow-up visit and high rates of clinical resolution.

Contribution of the R8 substituent to the in vitro antibacterial potency of besifloxacin and comparator ophthalmic fluoroquinolones

Introduction Previous work has shown that besifloxacin, an 8-chloro-fluoroquinolone, has more potent activity against gram-positive pathogens than moxifloxacin and gatifloxacin, which carry an 8-methoxy group. This study was conducted to determine the contribution of the R7 and R8 substituent to fluoroquinolone antibacterial activity. Materials and methods Besifloxacin, moxifloxacin, gatifloxacin, their R8 structural analogs, and ciprofloxacin were tested against representative isolates of various gram-positive and gram-negative species and previously characterized fluoroquinolone-resistant mutants of Staphylococcus aureus. Minimum inhibitory and minimum bactericidal concentrations were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Reserpine was used to determine the effect of efflux pumps on antibacterial activity. Results In general, exchanging the R8 residue in besifloxacin slightly reduced the molecule’s potency, while introducing an 8-chloro group in moxifloxacin increased its potency. A similar change in gatifloxacin had little to no effect. Substituting the R8 residues did not increase the susceptibility to the efflux pump inhibitor reserpine or result in a loss of bactericidal activity. In contrast, the positive control, ciprofloxacin, was shown to be a substrate for reserpine and lost bactericidal activity against some fluoroquinolone-resistant isolates of S. aureus. Conclusion The data presented here show that, depending on the R7 substituent, replacing an 8-methoxy group with an 8-chloro substituent can improve potency or can have little-to-no effect. These findings highlight the importance of the interplay between the R7 and R8 substituents in determining antibacterial potency.

High Proportion of Nontypeable Streptococcus pneumoniae Isolates among Sporadic, Nonoutbreak Cases of Bacterial Conjunctivitis

Purpose: Outbreaks of bacterial conjunctivitis have been linked to nontypeable strains of Streptococcus pneumoniae that lack a capsule, a key virulence factor for invasive infections. In contrast, isolates from sporadic, nonoutbreak cases of conjunctivitis were thought to be similar to invasive or nasopharyngeal isolates with respect to their capsular serotype and antibiotic resistance profile. This hypothesis was tested for 302 strains isolated during three prospective, multicenter clinical studies of bacterial conjunctivitis. Materials and methods: S. pneumoniae capsular serotypes were determined by agglutination assay and confirmed by the Statens Serum Institute. The presence of the cpsAB capsule genes was determined by polymerase chain reaction (PCR). Minimum inhibitory concentrations were measured for 17 antibacterial drugs by the broth microdilution method. Results: Only 25 (8.3%) isolates reacted with the capsule-specific antisera and only one (0.3%) of these serotypes was covered by the capsule-specific PCV7 vaccine. The remaining 277 (91.7%) isolates were nontypeable, suggesting that they did not produce a capsule. PCR analysis indicated the loss of the capsule operon in 24/25 randomly selected nontypeable strains. Resistance rates were highest for azithromycin, trimethoprim, and tetracycline, while no resistance was detected for the fluoroquinolones, linezolid, and vancomycin. Antibiotic resistance rates were generally lower than those reported for invasive isolates, although some highly resistant or multidrug-resistant isolates were identified. Conclusions: The prevalence of nontypeable strains of S. pneumoniae was higher than expected, while the number of isolates responsive to the PCV7 vaccine was surprisingly low. These results highlight the need for new vaccines that can target all S. pneumoniae strains regardless of the presence or nature of a capsule. In addition, resistance to azithromycin, erythromycin, tetracycline, and trimethoprim was greater than 10%, which may be relevant when selecting empiric treatments for ocular surface infections. Trial registration: ClinicalTrials.gov identifier: NCT00622908. Trial registration: ClinicalTrials.gov identifier: NCT00347932. Trial registration: ClinicalTrials.gov identifier: NCT00348348.

Unique Biological Properties and Molecular Mechanism of 5,6-Bridged Quinolones

ABSTRACT We have characterized an early series of 5,6-bridged dioxinoquinolones which behaved strikingly different from typical quinolones. The 5,6-bridged dioxinoquinolones inhibited Escherichia coli DNA gyrase supercoiling activity but, unlike typical quinolones, failed to stimulate gyrase-dependent cleavable complex formation. Analogous unsubstituted compounds stimulated cleavable complex formation but were considerably less potent than the corresponding 5,6-bridged compounds. Consistent with a previous report (M. Antoine et al., Chim. Ther. 7:434-443, 1972) and contrary to established quinolone SAR trends, a compound with an N-1 methyl substitution (PGE-8367769) was more potent than its analog with an N-1 ethyl substitution (PGE-6596491). PGE-8367769 was shown to antagonize ciprofloxacin-mediated cleavable complex formation in a dose-dependent manner, suggesting an interaction with the gyrase-DNA complex that overlaps that of ciprofloxacin. Resistance to PGE-8367769 in E. coli was found to arise through missense mutations in gyrA, implicating DNA gyrase as the primary antibacterial target. Notably, only 1 of 15 distinct mutations selected on PGE-8367769 (D87G) has previously been implicated in quinolone resistance in E. coli. The remaining 14 mutations (E16V, G31V, R38L, G40A, Y50D, V70A, A84V, I89L, M135T, G173S, T180I, F217C, P218T, and F513C) have not been previously reported, and most were located outside of the traditional quinolone resistance-determining region. These novel GyrA mutations decreased sensitivity to 5,6-bridged dioxinoquinolones by four- to eightfold, whereas they did not confer resistance to other quinolones such as ciprofloxacin, clinafloxacin, or nalidixic acid. These results demonstrate that the 5,6-bridged quinolones act via a mechanism that is related to but qualitatively different from that of typical quinolones.