Effective antimicrobial regimens for use in humans for therapy of Bacillus anthracis infections and postexposure prophylaxis

Abstract

Expanded options for treatments directed against pathogens that can be used for bioterrorism are urgently needed. Treatment regimens directed against such pathogens can be identified only by using data derived from in vitro and animal studies. It is crucial that these studies reliably predict the efficacy of proposed treatments in humans. The objective of this study was to identify a levofloxacin treatment regimen that will serve as an effective therapy for Bacillus anthracis infections and postexposure prophylaxis. An in vitro hollow-fiber infection model that replicates the pharmacokinetic profile of levofloxacin observed in humans (half-life [t(1/2)], 7.5 h) or in animals, such as the mouse or the rhesus monkey (t(1/2), approximately 2 h), was used to evaluate a proposed indication for levofloxacin (500 mg once daily) for the treatment of Bacillus anthracis infections. The results obtained with the in vitro model served as the basis for the doses and the dose schedules that were evaluated in the mouse inhalational anthrax model. The effects of levofloxacin and ciprofloxacin treatment were compared to those of no treatment (untreated controls). The main outcome measure in the in vitro hollow-fiber infection model was a persistent reduction of culture density (> or = 4 log10 reduction) and prevention of the emergence of levofloxacin-resistant organisms. In the mouse inhalational anthrax model the main outcome measure was survival. The results indicated that levofloxacin given once daily with simulated human pharmacokinetics effectively sterilized Bacillus anthracis cultures. By using a simulated animal pharmacokinetic profile, a once-daily dosing regimen that provided a human-equivalent exposure failed to sterilize the cultures. Dosing regimens that "partially humanized" levofloxacin exposures within the constraints of animal pharmacokinetics reproduced the antimicrobial efficacy seen with human pharmacokinetics. In a mouse inhalational anthrax model, once-daily dosing was significantly inferior (survival end point) to regimens of dosing every 12 h or every 6 h with identical total daily levofloxacin doses. These results demonstrate the predictive value of the in vitro hollow-fiber infection model with respect to the success or the failure of treatment regimens in animals. Furthermore, the model permits the evaluation of treatment regimens that "humanize" antibiotic exposures in animal models, enhancing the confidence with which animal models may be used to reliably predict the efficacies of proposed antibiotic treatments in humans in situations (e.g., the release of pathogens as agents of bioterrorism or emerging infectious diseases) where human trials cannot be performed. A treatment regimen effective in rhesus monkeys was identified.

FIG. 1.

A pharmacokinetic profile that approximates free (non-protein-bound) drug concentrations that result from once-daily administration of 500 mg levofloxacin to human subjects (dashed line) is depicted. The solid line describes an equivalent levofloxacin exposure (the same AUC₂₄) under conditions of monkey and murine pharmacokinetics. The datum points (circles and squares) represent the experimentally attained levofloxacin concentrations in the hollow-fiber infection model.

FIG. 2.

Antimicrobial activity of levofloxacin (once-daily dosing) against Bacillus anthracis in the hollow-fiber infection model under conditions of human and monkey-murine pharmacokinetics. (Upper panel) Effects on the total bacterial population. An untreated culture served as a control. The simulated pharmacokinetic profile (human or animal) and AUC₂₄/MIC ratio of each treatment regimen are shown. (Lower panel) Emergence of levofloxacin-resistant organisms during the experiment. Samples were plated on medium containing 3× the MIC of levofloxacin to enumerate resistant organisms. With animal pharmacokinetics, resistant organisms emerged and replaced the susceptible organisms with an AUC₂₄/MIC ratio of ≤300. No resistant organisms were observed in the control, the human pharmacokinetics, or the animal pharmacokinetics with AUC₂₄/MIC ratios of 500 and 1,000. The datum points for those treatment regimens are superimposed along the x axis.

FIG. 3.

Bacterial killing and regrowth and the role of sporulation in treatment regimens simulating human and animal pharmacokinetics. (Upper panel) Results obtained with daily treatment regimens with an AUC₂₄/MIC of 250. The simulated human exposure sterilized the culture within 72 h. With animal pharmacokinetics, a cycle of killing and regrowth was seen during each 24-h dosing interval. Similar results were seen with wild-type and sporulation-negative organisms. (Lower panel) A simulated animal exposure at an AUC₂₄/MIC of 1,000 rapidly sterilized the cultures of the spore-negative [spore (−)] organisms but not cultures of spore-positive [spore (+)] bacteria.

FIG. 4.

Efficacy of levofloxacin against B. anthracis under “partially humanized” animal pharmacokinetic profiles. (Upper panels) Treatment regimens in which levofloxacin was administered at the beginning of each 24-h dosing interval (AUC = 23 mg · h/liter) and a smaller dose at 12 h (AUC = 6.1 mg · h/liter; partially humanized [A]) or in which levofloxacin was administered in three decreasing doses at 8-h intervals (AUCs = 22, 7.5, and 4.5 mg · h/liter, respectively; partially humanized [B]). For each regimen, the solid line depicts the theoretical pharmacokinetic profile of the partially humanized regimens with datum points for the measured levofloxacin concentrations. The broken line shows an equivalent human exposure (AUC₂₄ = 36 mg · h/liter; AUC₂₄/MIC = 300). (Lower panel) Effect of the human exposure and “partially humanized” animal exposures on B. anthracis cultures.

FIG. 5.

Effect of dose schedule on efficacy of ciprofloxacin against B. anthracis. A ciprofloxacin exposure of an AUC₂₄ of mg · h/liter (AUC₂₄/MIC = 256) was given as two equal doses at 12-h intervals or as a single dose at 24-h intervals. The twice-daily regimen substantially reduced the total culture density and no resistant organisms were observed. Daily dosing prevented culture growth for 2 days, but the culture density increased to near control levels thereafter. Resistant organisms emerged after 1 day and replaced the susceptible organisms in the culture by day 2. The growth of an untreated control is also shown.

FIG. 6.

Effect of dose schedule on efficacy of levofloxacin in an in vivo mouse model of inhalational anthrax. Dosing at 12- and 6-h intervals (triangles and diamonds, respectively) conferred virtually complete protection (one death in each treatment group [the data curves overlap]), whereas 30% of the animals treated once daily (circles) died. All untreated control animals (squares) died within 3 days. A stratified Kaplan-Meier analysis demonstrated that the schedule of administration significantly influenced survivorship (P < 0.000001).