In Vivo Bioluminescent Monitoring of Therapeutic Efficacy and Pharmacodynamic Target Assessment of Antofloxacin against Escherichia coli in a Neutropenic Murine Thigh Infection Model

Abstract

Antimicrobial resistance among uropathogens has increased the rates of infection-related morbidity and mortality. Antofloxacin is a novel fluoroquinolone with broad-spectrum antibacterial activity against urinary Gram-negative bacilli, such as Escherichia coli This study monitored the in vivo efficacy of antofloxacin using bioluminescent imaging and determined pharmacokinetic (PK)/pharmacodynamic (PD) targets against E. coli isolates in a neutropenic murine thigh infection model. The PK properties were determined after subcutaneous administration of antofloxacin at 2.5, 10, 40, and 160 mg/kg of body weight. Following thigh infection, the mice were treated with 2-fold-increasing doses of antofloxacin from 2.5 to 80 mg/kg administered every 12 h. Efficacy was assessed by quantitative determination of the bacterial burdens in thigh homogenates and was compared with the bioluminescent density. Antofloxacin demonstrated both static and killing endpoints in relation to the initial burden against all study strains. The PK/PD index area under the concentration-time curve (AUC)/MIC correlated well with efficacy (R² = 0.92), and the dose-response relationship was relatively steep, as observed with escalating doses of antofloxacin. The mean free drug AUC/MIC targets necessary to produce net bacterial stasis and 1-log₁₀ and 2-log₁₀ kill for each isolate were 38.7, 66.1, and 147.0 h, respectively. In vivo bioluminescent imaging showed a rapid decrease in the bioluminescent density at free drug AUC/MIC exposures that exceeded the stasis targets. The integration of these PD targets combined with the results of PK studies with humans will be useful in setting optimal dosing regimens for the treatment of urinary tract infections due to E. coli.

Keywords: Escherichia coli; PK/PD; antofloxacin; bioluminescence; murine thigh infection.

FIG 1

Plasma drug concentrations after administration of single subcutaneous doses of antofloxacin in infected neutropenic mice. Error bars represent the standard deviations of the concentrations measured in six mice. Pharmacokinetic parameters listed in the box include the peak (maximum) concentration (C_max) in plasma, the AUC from time zero to infinity (AUC), and the elimination half-life (t_1/2) for each dose.

FIG 2

In vivo dose-response curves for antofloxacin against E. coli using a neutropenic murine thigh infection model. Mice received one of a series of 2-fold increasing doses of antofloxacin every 12 h over a 24-h treatment period. Each symbol represents the mean organism burden for four thighs. The horizontal dashed line represents the net stasis of the burden from the start of therapy. Data points below the line represent bactericidal activity, and points above the line represent net growth.

FIG 3

Relationship between the antofloxacin plasma 24-h fAUC/MIC ratios and the in vivo microbiological effect against multiple isolates of E. coli. Each symbol represents the mean organism burden for four thighs. Each of six drug dose levels was fractionated into a regimen administered every 12 h. The horizontal dashed line represents the net stasis of the burden from the start of therapy. Data points below the line represent killing, and points above the line represent growth. The line drawn through the data points is the best-fit line based upon the sigmoidal E_max formula.

FIG 4

(A) Bacterial loads in mice infected with E. coli 161549 containing pAKlux2 plated on MHA alone (gray circles) and MHA with ampicillin (white circles). (B) Bacterial loads in mice infected with wild-type E. coli 161549 (gray circles) or E. coli 161549 carrying pAKlux2 (white circles). Each point represents a value from a single thigh, and the horizontal lines represent the mean for the group. Animals were infected with ∼10⁶ CFU of bacteria in each posterior thigh. Tissues were homogenized and plated for determination of bacterial counts at the indicated time points.

FIG 5

Monitoring of the therapeutic efficacy of antofloxacin in a murine thigh infection model by use of in vivo bioluminescent imaging data. (A) Bioluminescence in mice infected with E. coli 161549 containing pAKlux2 at 10⁶ or 10⁸ CFU/thigh. Mice were administered antofloxacin at 20 mg/kg subcutaneously every 12 h for 2 days. Images were taken at 2 h after thigh infection (0 h) and then at 6, 12, 24, and 48 h after treatment. Mice were anesthetized with isoflurane during imaging procedures. Bioluminescence is reported as radiance (number of photons per second per square centimeter per steradian [p/sec/cm²/sr]) on a scale paired with a color bar shown next to the images. (B) Quantification of time course-dependent bioluminescent imaging data in mice. Data are expressed as the mean ± standard deviation (SD). (C) Comparison of the bacterial burdens in the untreated control and antofloxacin treatment groups. Bacterial visible counts were conducted 48 h after treatment. Antofloxacin produced 3.93-log₁₀ and 3.72-log₁₀ reductions compared to the initial burdens achieved with 10⁶ and 10⁸ CFU/thigh, respectively.