Bristol Centre for Antimicrobial Research and Evaluation, North Bristol NHS Trust and University of Bristol, Department of Medical Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, UK
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Abstract |
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Introduction |
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Previous work using in-vitro models has concentrated on studying the antibacterial effect of moxifloxacin on respiratory tract pathogens such as Streptococcus pneumoniae5,6,7 or Haemophilus influenzae and Moraxella cattarhalis.8 This study focused on pathogens likely to be associated with skin and soft tissue infection, i.e. S. aureus or ß-haemolytic streptococci. The antibacterial effect of moxifloxacin was assessed by simulating changing concentrations of antibiotic in an in-vitro pharmacodynamic model of infection. As in previous studies the serum concentrations of moxifloxacin associated with doses of 400 mg administered once a day were modelled, as this is the dose of moxifloxacin used most commonly in human clinical trials.
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Materials and methods |
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An in-vitro model (New Brunswick Bioflo 1000; Hatfield, UK) was used to simulate oral administration of moxifloxacin 400 mg every 24 h over 48 h; i.e. two simulated doses. The apparatus consists of a single central culture chamber connected via aluminium and silicone tubing first to a dosing chamber (which is in turn connected to a reservoir containing broth) and secondly to a vessel collecting outflow broth from the central chamber. The dosing chamber and central culture chamber were diluted with broth from the reservoir using a peristaltic pump (Ismatec Bennett & Co, Weston-super-Mare, UK) at a flow rate of 66 mL/h, to simulate the decline in antibiotic concentration. The temperature was maintained at 37°C and the broth in the dosing and central chambers was agitated by a magnetic stirrer.
Media
Brain Heart Infusion (BHI) broth (75%) (Unipath, Basingstoke, UK), supplemented with haemin 10 mg/L and ß-nicotinamide adenine dinucleotide 10 mg/L and L-histidine 10 mg/L, was used for the experiment with ß-haemolytic streptococci, and 6% IsoSensitest broth (ISB) (Unipath) was used for S. aureus. Preliminary investigations indicated that these broths supported a growth density of 5 x 1075 x 108 cfu/mL 18 h after inoculation into the model. Magnesium chloride at 1% (BDH, Poole, UK) was incorporated into nutrient agar plates (Merck, Poole, UK) containing 5% whole horse blood (TCS Microbiology, Buckingham, UK) to neutralize moxifloxacin before viable counts were determined.
Strains
S. aureus SMH 13942, methicillin and ciprofloxacin sensitive; S. aureus SMH 251, methicillin resistant, ciprofloxacin sensitive; Group A streptococcus SMH 14200 and Group G streptococcus SMH 14230 were used. Strains 13942M and 14200M were laboratory-generated mutants of strains 13942 and 14200 that were produced by the method of Dalhoff et al.1 Briefly this method involves serial passage through antibiotic-containing broth, using bacterial isolates that grow at or around the MIC value.
Antibiotic
Moxifloxacin was obtained from Bayer AG, Wuppertal, Germany. Stock solutions were prepared according to The British Society of Antimicrobial Chemotherapy (BSAC) guidelines9 and stored at 70°C.
MICs
MICs were determined by the BSAC-defined standard broth dilution method9 modified to use moxifloxacin concentrations that decreased by 0.2 mg/L or 0.02 mg/L steps rather than doubling dilutions.
Pharmacokinetics and bacterial killing curves
The in-vitro activity of changing concentrations of moxifloxacin against the six strains was tested in the model described above. Concentrations were chosen to simulate serum concentrations in man after a 400 mg oral dose every 24 h (to give a target peak serum concentration of 2.5 mg/L at 2 h post dose and a 24 h post-dose concentration of 0.4 mg/L10). The area under the curve (AUC) was 24.4 mg/L.
For all the experiments, 100 µL of an overnight broth suspension of the test organism was inoculated into the central culture chamber via an entry port (initial inoculum of about 106 cfu/mL) and the model run for 18 h to allow equilibrium to be attained at a density of about 5 x 1075 x 108 cfu/mL. Moxifloxacin (1332 µL of a 1 g/L solution) was then added to the dosing chamber at time 0, and at 24 h. Samples were taken from the central chamber via a port throughout the 48 h period (at times 0, 1, 2, 3, 4, 5, 6, 7, 10, 12, 22, 24, 25, 26, 27, 28, 29, 30, 31, 34, 36, 46 and 48 h) for assessment of viable bacterial count. The bacteria were counted without dilution and after 1/100 dilution using a spiral plater (Don Whitley Spiral Systems, Shipley, UK). The minimum detection level was 2 x 102 cfu/mL. In addition, aliquots were taken at the same time intervals and stored at 70°C for measurement of moxifloxacin concentration. Samples were assayed by bioassay using Escherichia coli NCTC 10418 as indicator organism.11 All standards and samples were prepared and diluted as necessary in the same concentration of BHI or ISB as that used in the model simulations. The limit of detection was 0.03 mg/L. All pharmacokinetic simulations and bacterial killing experiments were performed at least three times.
Pharmacokinetics, pharmacodynamic measurement of antibacterial effects and statistical analysis
Antibacterial activity was assessed by calculating the log change in viable count compared
with time 0 at 12 h (12), 24 h (
24), 36 h (
36) and 48 h (
48). In
addition, the maximum reduction in viable count was recorded (
max). The area under the
bacterial killing curve (AUBKC, log cfu/mL.h) was calculated, after the inoculum was
standardized, by the log-linear trapezoidal rule for the time periods 024 h (AUBKC24) and 048 h (AUBKC48). For pharmacodynamic analysis the
AUC/MIC ratio, the percentage of time the simulated concentration exceeded the MIC (T
> MIC) (T = time), the AUC over MIC, MIC ratio ((AUC >
MIC)/MIC) and weighted AUC: WAUC = (AUC/MIC x T > MIC/100)
were also calculated.12
AUC/MIC, T > MIC, (AUC > MIC)/MIC and WAUC were related to AUBKC24 and AUBKC48 using an inhibitory sigmoid Emax model (WinNonlin, Microsoft Corp., USA). Goodness of fit was assessed by r and inspection of the plot of the residuals.
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Results |
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Moxifloxacin was detected at a concentration of 2.3 ± 0.5 mg/L at 2 h and 0.5 ± 0.1 mg/L at 24 h. These levels correlated well with those expected.
Bacterial killing curves
The killing curves for S. aureus after exposure to moxifloxacin are shown in Figures 1 and 2. For the two strains with
moxifloxacin MIC 0.14 mg/L there was a marked reduction
in viable count (6 log) at 36 h with no regrowth occurring up to 48 h. In contrast, killing of the
laboratory-generated mutant (MIC 1.0 mg/L) was reduced (2 log reduction in count), with
marked regrowth between 36 and 48 h. The AUBKC values were less for the more susceptible
strains when compared with the mutant strains, especially AUBKC48. Results for the
Group A streptococci tested showed that the more susceptible strain (MIC 0.16 mg/L) was killed
more rapidly (3.5 log10) with no regrowth. The mutant strain (MIC 1.8 mg/L) showed
marked regrowth (2 log) and was poorly killed between 0 and 24 h (Figure 3). For these
simulations a significant between-experiment variability was exhibited (Figure 3
and Table).
Killing of Group G streptococci (MIC 0.4 mg/L) was poor, with only a 2 log reduction in count
over 48 h (Figure 4 and Table).
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Curve fitting using an inhibitory Emax sigmoidal model indicated that T > MIC was poorly related to AUBKC24 and AUBKC48 (r <0.37). AUC/MIC, WAUC and (AUC > MIC)/MIC were strongly related to AUBKC24 (r = 0.750.79) and AUBKC48 (r = 0.780.84). The plot of AUC/MIC versus AUBKC24 is shown in Figure 5. Inspection of the residual plots was satisfactory. The maximum response, i.e. the lowest plateau AUBKC, occurs at an AUC/MIC ratio of >150.
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Discussion |
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Animal data, in which E. coli, Klebsiella pneumoniae, S. aureus and S. pneumoniae are used in neutropenic mouse models of lung or thigh infection, have indicated that the dose required to produce a net bacteriostatic effect over 24 h is unchanged, whether moxifloxacin is given every 3, 6 or 12 h. In mice, moxifloxacin has a serum half-life of <1 h compared with 1215 h in man. This probably indicates that once-daily dosing will be as effective as twice daily or more frequent doses. Drug AUC correlated with antibacterial efficacy more strongly than did Cmax or T > MIC.14 Furthermore, in an experimental animal model of S. pneumoniae meningitis, the drug AUC in cerebrospinal fluid correlated with the change in viable count.15 Previous studies by this group, using the model system described here, have shown that the antibacterial effect of moxifloxacin correlated best with AUC/MIC for H. influenzae and M. catarrhalis.18 These data also show that AUC parameters correlate best with antimicrobial effect, whether this be AUC/MIC, (AUC > MIC)/MIC or WAUC. T > MIC is not a good predictor using an inhibitory sigmoidal Emax model, and WAUC here was not superior to the other parameters as has been postulated recently.12 This is perhaps not surprising, as T > MIC is an integral part of WAUC. As this study is not of a dose fractionation or escalation design, it was not possible to define the relative roles of Cmax/MIC ratio in comparison with AUC/MIC, despite recent interest in this area.16
In conclusion, moxifloxacin has marked bactericidal action against S. aureus with
MICs 0.14 mg/L but may be less bactericidal against ß-haemolytic streptococci;
AUC-based pharmacodynamic factors predict antibacterial effect better than does T >
MIC.
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Acknowledgments |
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Notes |
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References |
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Received 5 February 1999; returned 5 May 1999; revised 25 May 1999; accepted 9 August 1999