Laura Lawrence

Rχ-01, a New Family of Oxazolidinones That Overcome Ribosome-Based Linezolid Resistance

ABSTRACT New and improved antibiotics are urgently needed to combat the ever-increasing number of multidrug-resistant bacteria. In this study, we characterized several members of a new oxazolidinone family, Rχ-01. This antibiotic family is distinguished by having in vitro and in vivo activity against hospital-acquired, as well as community-acquired, pathogens. We compared the 50S ribosome binding affinity of this family to that of the only marketed oxazolidinone antibiotic, linezolid, using chloramphenicol and puromycin competition binding assays. The competition assays demonstrated that several members of the Rχ-01 family displace, more effectively than linezolid, compounds known to bind to the ribosomal A site. We also monitored binding by assessing whether Rχ-01 compounds protect U2585 (Escherichia coli numbering), a nucleotide that influences peptide bond formation and peptide release, from chemical modification by carbodiimide. The Rχ-01 oxazolidinones were able to inhibit translation of ribosomes isolated from linezolid-resistant Staphylococcus aureus at submicromolar concentrations. This improved binding corresponds to greater antibacterial activity against linezolid-resistant enterococci. Consistent with their ribosomal A-site targeting and greater potency, the Rχ-01 compounds promote nonsense suppression and frameshifting to a greater extent than linezolid. Importantly, the gain in potency does not impact prokaryotic specificity as, like linezolid, the members of the Rχ-01 family show translation 50% inhibitory concentrations that are at least 100-fold higher for eukaryotic than for prokaryotic ribosomes. This new family of oxazolidinones distinguishes itself from linezolid by having greater intrinsic activity against linezolid-resistant isolates and may therefore offer clinicians an alternative to overcome linezolid resistance. A member of the Rχ-01 family of compounds is currently undergoing clinical trials.

In Vitro Activities of the Rx-01 Oxazolidinones against Hospital and Community Pathogens

ABSTRACT Rx-01_423 and Rx-01_667 are two members of the family of oxazolidinones that were designed using a combination of computational and medicinal chemistry and conventional biological techniques. The compounds have a two- to eightfold-improved potency over linezolid against serious gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant streptococci, and vancomycin-resistant enterococci. This enhanced potency extends to the coverage of linezolid-resistant gram-positive microbes, especially multidrug-resistant enterococci and pneumococci. Compounds from this series expand the spectrum compared with linezolid to include fastidious gram-negative organisms like Haemophilus influenzae and Moraxella catarrhalis. Like linezolid, the Rx-01 compounds are bacteriostatic against MRSA and enterococci but are generally bactericidal against S. pneumoniae and H. influenzae.

A randomized phase 2 study comparing two doses of delafloxacin with tigecycline in adults with complicated skin and skin-structure infections

BACKGROUND A randomized, double-blind, multicenter trial was done to compare two doses of delafloxacin with tigecycline in patients with various complicated skin and skin-structure infections (wound infections following surgery, trauma, burns, or animal/insect bites, abscesses, and cellulitis). METHODS Patients were randomized 1:1:1 to receive delafloxacin 300mg intravenous (IV) every 12h, delafloxacin 450mg IV every 12h, or tigecycline 100mg IV×1, followed by 50mg IV every 12h; randomization was stratified by infection type. Duration of therapy was 5-14 days. The primary efficacy analysis, performed on the clinically evaluable (CE) population at the test-of-cure (TOC) visit (14-21 days after the final dose of study drug), compared clinical response rates in the delafloxacin and tigecycline arms. Clinical response rates in the two delafloxacin arms were also compared. RESULTS Among CE patients, clinical cure rates at TOC visit were similar in the delafloxacin and tigecycline arms (94.3%, 92.5%, and 91.2%, respectively in delafloxacin 300-mg, delafloxacin 450-mg, and tigecycline arms). Overall, the most frequent adverse events were nausea, vomiting, and diarrhea; the 300-mg delafloxacin arm was the best-tolerated regimen. CONCLUSIONS Delafloxacin was similarly effective as tigecycline for a variety of complicated skin and skin-structure infections and was well tolerated. (Clinicaltrials.gov NCT 0719810).

Staphylococcus aureus Mutants Selected by BMS-284756

ABSTRACT BMS-284756, a novel des-fluoro(6)-quinolone, was used to select for in vitro mutants of Staphylococcus aureusISP794. Step mutants were obtained, and the quinolone resistance-determining regions of four target genes, gyrA, gyrB, grlA, and grlB, were sequenced. The data suggest that DNA gyrase is the primary target for BMS-284756 in S. aureus.

Efflux pumps in bacteria: overview, clinical relevance, and potential pharmaceutical target.

Trends in microbial resistance suggest a dramatic increase in the frequency of reports of multi-drug efflux pumps in bacteria and fungi. Although it is difficult to determine whether this increase is due to the increased attention given to this resistance mechanism, or an increase in frequency, efflux pumps are becoming an important consideration in resistance emergence. These efflux pumps comprise at least four different classes in Gram-positive and Gram-negative bacteria, as well as in Streptomyces and fungi. As more efflux pumps are characterised and studied, both biochemically and structurally, the opportunity for intervention may arise.

A randomized, double-blind, Phase 2 study to evaluate subjective and objective outcomes in patients with acute bacterial skin and skin structure infections treated with delafloxacin, linezolid or vancomycin

Objectives Delafloxacin is an investigational anionic fluoroquinolone being developed to treat infections caused by Gram-positive and -negative organisms. This clinical trial evaluated the efficacy and safety of delafloxacin in the treatment of acute bacterial skin and skin structure infections (ABSSSIs). Methods In a double-blind, Phase 2 trial, 256 patients were randomized (1 : 1 : 1) to 300 mg of delafloxacin, 600 mg of linezolid or 15 mg/kg vancomycin (actual body weight), each administered intravenously twice daily for 5–14 days. Randomization was stratified by infection category. The primary endpoint was the investigators assessment of cure, defined as complete resolution of baseline signs and symptoms at follow-up. Secondary endpoints included reductions in the total areas of erythema and induration and assessments of bacterial eradication. This trial has been registered at ClinicalTrials.gov under registration number NCT01283581. Results Cure rates were significantly greater with delafloxacin versus vancomycin (mean difference: −16.3%; 95% CI, −30.3% to −2.3%; P = 0.031); differences were significant for obese patients (BMI ≥30 kg/m2; mean difference: −30.0%; 95% CI, −50.7% to −9.3%; P = 0.009), but not for non-obese patients. Cure rates with delafloxacin and linezolid were similar. Using digital measurement, the percentage decrease in total erythema area was significantly greater with delafloxacin versus vancomycin at follow-up (−96.4% versus −84.5%; P = 0.028). There were no differences in bacterial eradication among the treatment groups. The most frequently reported treatment-emergent adverse events were nausea, diarrhoea and vomiting. Conclusions These data show that delafloxacin is effective in the treatment of ABSSSIs and is well tolerated.

Bactericidal Activities of BMS-284756, a Novel Des-F(6)-Quinolone, against Staphylococcus aureus Strains with Topoisomerase Mutations

ABSTRACT The antistaphylococcal activities of BMS-284756 (T-3811ME), levofloxacin, moxifloxacin, and ciprofloxacin were compared against wild-type and grlA and grlA/gyrA mutant strains of Staphylococcus aureus. BMS-284756 was the most active quinolone tested, with MICs and minimal bactericidal concentrations against S. aureus wild-type strain MT5, grlA mutant MT5224c4, and grlA/gyrA mutant EN8 of 0.03 and 0.06, 0.125 and 0.125, and 4 and 4 μg/ml, respectively. In the time-kill studies, BMS-284756 and levofloxacin exhibited rapid killing against all strains. Ciprofloxacin, however, was not bactericidal for the double mutant, EN8. BMS-284756 and levofloxacin were bactericidal (3 log10 decrease in CFU/ml) against the MT5 and MT5224c4 strains at two and four times the MIC within 2 to 4 h. Against EN8, BMS-284756 was bactericidal within 4 h at two and four times the MIC, and levofloxacin achieved similar results within 4 to 6 h. Both the wild-type strain MT5 and grlA mutant MT5224c4 should be considered susceptible to both BMS-284756 and levofloxacin, and both quinolones are predicted to have clinical efficacy. The in vivo efficacy of BMS-284756, levofloxacin, and moxifloxacin against S. aureus strain ISP794 and its single mutant 2C6(1)-1 directly reflected the in vitro activity: increased MICs correlated with decreased in vivo efficacy. The 50% protective doses of BMS-284756 against wild-type and mutant strains were 2.2 and 1.6 mg/kg of body weight/day, respectively, compared to the levofloxacin values of 16 and 71 mg/kg/day and moxifloxacin values of 4.7 and 61.6 mg/kg/day. BMS-284756 was more potent than levofloxacin and equipotent with moxifloxacin against ISP794 both in vitro and in vivo, while BMS-284756 was more potent than levofloxacin and moxifloxacin against 2C6(1)-1.

A Thorough QT Study To Evaluate the Effects of Therapeutic and Supratherapeutic Doses of Delafloxacin on Cardiac Repolarization

ABSTRACT A randomized, double-blind, placebo-controlled, 4-period crossover study was conducted in 52 healthy adults to assess the effect of delafloxacin on the corrected QT (QTc) interval. The QT interval, corrected for heart rate using Fridericias formula (QTcF), was determined predose and at 0.5, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 12, 18, and 24 h after dosing with delafloxacin at 300 mg intravenously (i.v.; therapeutic), delafloxacin at 900 mg i.v. (supratherapeutic), moxifloxacin at 400 mg orally (p.o.; positive control), and placebo. The pharmacokinetic profile of delafloxacin was also evaluated. At each time point after delafloxacin administration, the upper limit of the 90% confidence interval (CI) for the placebo-corrected change from the predose baseline in QTcF (ΔΔQTcF) was less than 10 ms (maximum, 3.9 ms at 18 h after dosing), indicating an absence of a clinically meaningful increase in the QTc interval. The lower limit of the 90% CI of ΔΔQTcF for moxifloxacin versus placebo was longer than 5 ms at all 5 time points selected for assay sensitivity analysis, demonstrating that the study was adequately sensitive to assess QTc prolongation. There was no positive relationship between delafloxacin plasma concentrations and ΔΔQTcF. Treatment-emergent adverse events (AEs) were more frequent among subjects receiving a single supratherapeutic dose of 900 mg delafloxacin. There were no deaths, serious AEs, or AEs leading to study discontinuation and no clinically meaningful abnormalities in laboratory values or vital signs observed at any time point after any dose of the study drug.

Selection and Genetic Characterization of Streptococcus pneumoniae Mutants Resistant to the Des-F(6) Quinolone BMS-284756

ABSTRACT Existing quinolones are known to target the type II topoisomerases in bacteria. In order to determine which of these targets are of key importance in Streptococcus pneumoniae treated with BMS-284756 (T-3811ME), a novel des-F(6) quinolone, resistant mutants were selected in several steps of increasing resistance by plating pneumococci on a series of blood agar plates containing serial twofold-increasing concentrations of drug. After incubation, colonies that arose were selected and passaged twice on antibiotic-containing media at the selection level. Mutants generally showed increases in resistance of four- to eightfold over the prior level of susceptibility. Mutants in the next-higher level of resistance were selected from the previous round of resistant mutants. Subsequently, chromosomal DNA was prepared from parental (R6) pneumococci and from at least three clones from each of four levels of increasing antibiotic resistance. Using PCR primers, 500- to 700-bp amplicons surrounding the quinolone resistance determining regions (QRDR) ofgyrA, gyrB, parC, andparE genes were prepared from each strain. Internal primers were used to sequence both DNA strands in the regions of approximately 400 bp centered on the QRDR. Mutations identified with increasing levels of resistance included changes in GyrA at Ser-81 and Glu-85 and changes in ParC at Ser-79 and Asp-83. Changes in GyrB and ParE were not observed at the levels of resistance obtained in this selection. The resistance to comparator quinolones (levofloxacin, ciprofloxacin, and moxifloxacin) also increased in four- to eightfold steps with these mutations. The intrinsically greater level of antibacterial activity and thus lower MICs of BMS-284756 observed at all resistance levels in this study may translate to coverage of these resistant pneumococcal strains in the clinic.

In Vitro Activity of Delafloxacin and Microbiological Response against Fluoroquinolone-Susceptible and Nonsusceptible Staphylococcus aureus Isolates from Two Phase 3 Studies of Acute Bacterial Skin and Skin Structure Infections

ABSTRACT Delafloxacin is an investigational anionic fluoroquinolone antibiotic with broad-spectrum in vitro activity, including activity against Gram-positive organisms, Gram-negative organisms, atypical organisms, and anaerobes. The in vitro activity of delafloxacin and the percent microbiological response in subjects infected with fluoroquinolone-susceptible and nonsusceptible Staphylococcus aureus isolates were determined from two global phase 3 studies of delafloxacin versus vancomycin plus aztreonam in patients with acute bacterial skin and skin structure infections (ABSSSI). Patients from 23 countries, predominately the United States but also Europe, South America, and Asia, were enrolled. The microbiological intent-to-treat (MITT) population included 1,042 patients from which 685 S. aureus isolates were submitted for identification and susceptibility testing per CLSI guidelines at the central laboratory (JMI Laboratories, North Liberty, IA). The comparator fluoroquinolone antibiotics included levofloxacin and ciprofloxacin. Nonsusceptibility to these antibiotics was determined using CLSI breakpoints. S. aureus isolates were 33.7% levofloxacin nonsusceptible (LVX-NS). The delafloxacin MIC90 values against levofloxacin-nonsusceptible S. aureus, methicillin-resistant S. aureus (MRSA), and methicillin-susceptible S. aureus isolates were all 0.25 μg/ml. Delafloxacin demonstrated high rates of microbiological response against LVX-NS isolates as well as isolates with documented mutations in the quinolone resistance-determining region (QRDR). S. aureus was eradicated or presumed eradicated in 98.4% (245/249) of delafloxacin-treated patients. Similar eradication rates were observed for delafloxacin-treated subjects with levofloxacin-nonsusceptible S. aureus isolates (80/81; 98.8%) and MRSA isolates (70/71; 98.6%). Microbiological response rates of 98.6% were observed with delafloxacin-treated subjects with S. aureus isolates with the S84L mutation in gyrA and the S80Y mutation in parC, the most commonly observed mutations in global phase 3 studies. The data suggest that delafloxacin could be a good option for the treatment of infections caused by S. aureus isolates causing ABSSSI, including MRSA isolates, where high rates of ciprofloxacin and levofloxacin nonsusceptibility are observed. (The phase 3 studies described in this paper have been registered at ClinicalTrials.gov under identifiers NCT01984684 and NCT01811732.)