Faculties of 1 Pharmacy and 2 Medicine, University of Manitoba; 3 Pharmacy, 4 Infectious Diseases and 5 Microbiology Laboratory, St Boniface General Hospital, Winnipeg, MB, Canada
Received 27 August 2002; returned 26 November 2002; revised 8 January 2003; accepted 11 January 2003
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Abstract |
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Methods: In dose escalation studies, single doses with peak concentrations equivalent to 1 x, 2 x, 4 x, 8 x, 16 x and 32 x MIC against two isolates of S. pneumoniae were studied over 24 h. Traditional pharmacodynamic indices, including peak concentration divided by MIC (peak/MIC), time of concentration above MIC (T > MIC) and AUC24/MIC, were estimated for all regimens. As a continuous index of fluoroquinolone exposure, AUC0t/MIC was also calculated, as AUC from time 0 to 1, 2 and 6 h divided by MIC. Correlations between pharmacodynamic indices and antibacterial effects were examined using linear and non-linear methods. In validation experiments, the pharmacodynamic model was used to predict bacterial kill curves, produced by simulated clinical doses of moxifloxacin and levofloxacin against two other S. pneumoniae isolates.
Results: Peak/MIC was most predictive of early bacterial kill, whereas T > MIC was significantly associated with final bacterial counts at 24 h. Antibacterial effects were bacteriostatic when T > MIC was 48% and bactericidal when values exceeded 55%. AUC0t/MIC was strongly associated with bacterial kill throughout the dosing interval. Bactericidal activity and bacterial eradication were associated with AUC0t/MICs of 28 and 135, respectively. AUC0t/MIC was also highly predictive of bacterial kill curves produced by simulated clinical doses of moxifloxacin and levofloxacin (precision 0.36 log10 cfu/mL, bias 0.02 log10 cfu/mL).
Conclusion: This study demonstrated the novel application of AUC0t/MIC as a continuous index of antibiotic activity, and provided extensive characterization of fluoroquinolone pharmacodynamics against S. pneumoniae.
Keywords: moxifloxacin, levofloxacin, pharmacodynamics, pneumococcus
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Introduction |
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Increases in penicillin-resistant Streptococcus pneumoniae and fluoroquinolone use for respiratory tract infections have stimulated significant interest in fluoroquinolone PDs against Gram-positive bacteria. IPDMs have demonstrated the generally rapid and potent activities of simulated clinical doses of agents such as gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin and trovafloxacin against susceptible S. pneumoniae isolates.111 More comprehensive PD characterizations have shown concentration-dependent antibiotic activity, and further characterized relationships between peak/MIC or AUC24/MIC and microbiological response.1214 AUC/MIC in vitro has been primarily investigated as an index of final or overall measures of bacterial kill. Most often, it has been associated with bacterial kill at the end of dosing intervals, or with areas related to the bacterial kill curve, such as the area between the control growth and timekill curves (ABBC), the area above the curve (AAC) and the area under the timekill curve (AUBC). Innovative work by Firsov et al.15 found that measures of response that do not consider effect duration (i.e. time at which response is achieved) resulted in different AUC/MIC relationships. The investigators overcame this issue by using a measure of intensity of antibiotic effect (IE), or the area between control growth and timekill curves from time zero to the time of maximal regrowth, even if the time was greater than the dosing interval.16
Our goal was to conduct a comprehensive PD analysis based on dose escalations of moxifloxacin and levofloxacin against S. pneumoniae in an IPDM. In addition to traditional PD indices (i.e. peak/MIC, T > MIC, AUC24/MIC), we were interested in the application of the latter as a continuous index of fluoroquinolone exposure, and the potential association between AUC0t/MIC during the dosing interval and bacterial kill at the relative times.
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Materials and methods |
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Four strains of S. pneumoniae, including an ATCC (49619) isolate and three clinical (81, 63, 26) isolates from blood, were used. MICs were determined using the broth microdilution method described by the NCCLS.17 Isolates ATCC 49619 and 63 were penicillin sensitive (MICs 0.03, 0.03 mg/L), and isolates 81 and 26 were penicillin resistant (MICs 2, 2 mg/L). Moxifloxacin and levofloxacin MICs were 0.125 and 1 mg/L for isolates ATCC 49619 and 81, and 0.25 and 0.5 mg/L for isolates 63 and 26. Isolates ATCC 49619 and 81 were used in dose escalation studies, whereas isolates 63 and 26 were used in validation experiments.
Antibiotics and medium
Moxifloxacin was supplied by Bayer Corporation (West Haven, CT, USA) and levofloxacin by Ortho-McNeil Pharmaceutical (Raritan, NJ, USA). MuellerHinton broth (Difco Laboratories, Detroit, MI, USA) supplemented with calcium 25 mg/L and magnesium 12.5 mg/L, with 4% lysed horse blood (Quelab, QC, Canada) was used in all experiments (CSMHB4%LHB).
In vitro pharmacodynamic model
A one-compartment IPDM was used to simulate S. pneumoniae bacteraemia in an immunocompromised host. Four central compartments consisting of 250 mL glass flasks were stirred with magnetic bars and maintained at 37°C in a heated water bath. A computerized pump (Masterflex, Cole-Parmer Instrument Company, Chicago, IL, USA) was used to program the flow of sterile CSMHB4%LHB through the central compartments, and produce 11 and 7 h elimination half-lives for moxifloxacin and levofloxacin, respectively.18 Flow rates were calibrated prior to experiments and were measured again at 6 and 24 h. Experiments were excluded if the variation exceeded 5% of the original flow rate.
Bacterial suspensions were prepared with the inoculation of 1520 colonies from solid blood agar into 100 mL of broth. Suspensions were incubated for 4 h, adjusted to a 0.5 McFarlands standard density and injected into the central compartments to yield initial inocula of 1 x 106 cfu/mL at time zero. Dose escalation studies were performed by injecting single bolus doses of moxifloxacin and levofloxacin, with peak concentrations equivalent to 1 x, 2 x, 4 x, 8 x, 16 x and 32 x MIC, against isolates ATCC 49619 and 81. Samples were collected at 0, 0.5, 1, 2, 6 and 24 h, serially diluted in normal saline at 4°C and aliquotted (10 and 100 µL) onto solid blood agar in duplicate. Plates were incubated for 24 h and then viable colonies between 10 and 100 per plate were counted. The lower limit of detection was 1 x 102 cfu/mL. To identify potential antibiotic carry-over, concurrent samples underwent two cycles of washing to remove antibiotic. Washing was performed by centrifuging at 4000g for 10 min, decanting the supernatant and suspending the remaining pellet in broth to the initial volume. Colony counts from washed and unwashed samples were compared. All experiments were performed with a growth control and in triplicate on separate occasions.
Pharmacodynamic analysis
Initial inocula were standardized to 1 x 106 cfu/mL, and bacterial kill curves were constructed using mean log10 cfu/mL (for experiments performed in triplicate) versus time. Twenty-four bacterial kill curves were produced from dose escalation studies of six concentration profiles of moxifloxacin and levofloxacin against two isolates. Colony count variation at each time point within and among experiments performed in triplicate was <10%, and antibiotic carry-over effects were not observed. Bacterial kill was measured by subtracting bacterial counts at 1, 2, 6 and 24 h from initial inocula, with positive values indicating net kill and negative values representing net growth. Bactericidal activity was defined as bacterial kill >3 log10 cfu/mL, and bacterial eradication was defined as bacterial counts below the limit of detection, or bacterial kill >4 log10 cfu/mL.
Using a one-compartment, linear pharmacokinetic model, PD indices were estimated for the six concentration profiles of moxifloxacin and levofloxacin against isolates ATCC 49619 and 81. Peak/MIC was calculated as the peak concentration divided by MIC, T > MIC was the percentage of time of concentration above MIC for the 24 h dosing interval, and AUC24/MIC was the area under the concentrationtime curve from 0 to 24 h divided by MIC. For moxifloxacin, peak/MICs of 1, 2, 4, 8, 16 and 32 produced T > MICs of 0, 46%, 92%, 100%, 100% and 100%, and AUC24/MICs of 12, 25, 50, 99, 198 and 396, respectively. For levofloxacin, the same peak/MICs produced T > MICs of 0, 29%, 58%, 88%, 100% and 100%, and AUC24/MICs of 9, 18, 37, 73,147 and 293. As a continuous index of antibiotic exposure, AUC0t/MIC was also calculated as AUC from 0 to 1, 2 and 6 h divided by MIC (Figure 1). AUC0t/MICs, including AUC01/MIC, AUC02/MIC, AUC06/MIC and AUC024/MIC, ranged from 0.97 to 396 for moxifloxacin and 0.95 to 293 for levofloxacin. Relationships between the PD indices and bacterial kill were analysed using linear and non-linear (e.g. sigmoidal Emax) methods where appropriate. Goodness of fit for non-linear models was assessed using root mean squares, residual plots and Akaikes Information Criteria.
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Results |
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Discussion |
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The lower threshold (i.e. 28) in this study is similar to that from previous reports. Optimal antibacterial activity has been associated with AUC24/MICs > 3050,24,7,12 whereas suboptimal effects with resistance or regrowth have been observed with AUC24/MICs < 2030.8,12,14 There is some variability in thresholds for bacterial eradication, which may be due to study isolates, ranges of AUC/MICs or definitions of response. Whereas some reports found bacterial eradication with AUC24/MICs > 30,10,12 other studies, such as this one, demonstrated higher thresholds for complete antibacterial responses.13,14 In a study of simulated clinical doses of six fluoroquinolones against two laboratory-derived ciprofloxacin-resistant isolates of S. pneumoniae, Coyle et al.14 determined that bacterial counts below detection at 24 h were associated with AUC24/MICs > 82. Another investigation, which tested three doses of gemifloxacin against five isolates of S. pneumoniae, found maximal AUBCs at 48 h when AUC/MICs were between 300 and 400.13 As previously discussed, AUC0t/MIC expands the application of these thresholds from associations with final or overall antibacterial response to a PD index, which predicts both the magnitude and time of a desired effect. In other words, AUC0t/MIC considers potential differences between agents that may both achieve an AUC/MIC of 30, but where one reaches it at 2 h and the other at 24 h.
The clinical application of AUC24/MIC has been demonstrated, whereas the relevance of AUC0t/MIC remains to be seen. Although optimal fluoroquinolone AUC24/MICs of 125250 are suggested for Gram-negative pathogens,22 lower thresholds have been found for S. pneumoniae infections. One study of pneumococcal pneumonia in mice found a significant AUC24/MIC threshold of >160,23 whereas a second animal study reported optimal responses, with values of 3040.20 Ambrose et al.21 also found a lower threshold based on the analysis of data from two Phase III trials of levofloxacin versus gatifloxacin for community-acquired pneumonia or acute bronchitis involving S. pneumoniae. The probability of microbiological response was 100% compared with 64% when unbound AUC24/MICs were above and below 33.7, respectively. Although lower thresholds appear effective, there are concerns that such targets may promote bacterial resistance.
The characterization of antibiotic PDs is often complicated by the strong co-variance among indices.18,24 In this study, peak/MIC correlated well with early bacterial kill, but was less indicative of later bacterial counts at 24 h. Peak/MIC is founded on the concentration-dependent activity of fluoroquinolones, but does not consider antibiotic elimination and becomes less predictive of antibacterial response over time. On the other hand, AUC0t/MIC includes components of time and antibiotic elimination, and thereby maintains its predictive performance throughout the dosing interval.
Finally, T > MIC is associated with fluoroquinolone activity, especially when concentrations fall below the MIC.25 In contrast to peak/MIC, T > MIC is most predictive of later bacterial counts at the end of dosing intervals. In this study, maximal antibacterial response at 24 h was associated with T > MICs > 70% (17 in 24 h). Although this threshold is similar to those of other IPDM studies,7,13 it differs from another, in which bacterial eradication was observed when T > MICs exceeded 8 in 24 h (33%).12 As mentioned previously, such variability may be explained by study isolates, ranges of T > MICs or definitions of response.
This study has some important limitations. First, the PD characterizations were based on fluoroquinolone activity against two sensitive S. pneumoniae isolates. Dose escalations produced a wide range of values for most PD indices; however, data were less comprehensive for parameters such as T > MIC. In addition, AUC0t/MIC and the PD model would benefit from further validation using isolates with MICs outside the relatively narrow range of those used in this study. Although moxifloxacin and levofloxacin demonstrated the same PD responses, the study of other fluoroquinolones would also broaden the application. The lack of sample times between 6 and 24 h would not have added significantly to the bacterial kill curves, but would have allowed validation at other times. Finally, the application of this PD model, in the setting of multiple dose regimens, requires further investigation.
In conclusion, this study demonstrated the novel application of AUC0t/MIC as a continuous index of fluoroquinolone activity. It was predictive of bacterial kill until eradication or regrowth, and provided an extensive characterization of antibiotic PDs against S. pneumoniae.
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Acknowledgements |
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Footnotes |
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References |
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