Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NB 68178, USA
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Abstract |
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Introduction |
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Levofloxacin, the active L-isomer of racemic ofloxacin, has greater in-vitro activity against S. pneumoniae than ciprofloxacin and its parent compound, ofloxacin. In comparisons with ciprofloxacin and ofloxacin, levofloxacin is generally twice as potent, with MIC90s for S. pneumoniae at or just below the 2 mg/L susceptible breakpoint.2,3,5 In addition to its enhanced anti-pneumococcal potency, levofloxacin has a pharmacokinetic advantage over ciprofloxacin, with higher peak serum levels and a lower rate of elimination. 13,14 The pharmacokinetic advantages of levofloxacin, together with its enhanced activity against pneumococci, suggest that levofloxacin may have a clinical advantage over ciprofloxacin in the treatment of S. pneumoniae respiratory tract infections. This study was designed to compare the in-vitro pharmacodynamics of levofloxacin and ciprofloxacin against S. pneumoniae in an in-vitro pharmacokinetic model of infection when oral doses of levofloxacin 500 mg and ciprofloxacin 750 mg were simulated.
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Materials and methods |
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The experimental strains evaluated in this study included eight clinical isolates of S. pneumoniae, four of which were resistant to penicillin (MICs = 24 mg/L). Each clinical isolate was obtained from a different patient. Logarithmic-phase cultures were prepared by suspending ten colonies from a 14 h culture on trypticase soy agar supplemented with 5% sheep blood (BBL Microbiology Systems, Cockeysville, MD, USA) into 6 mL of ToddHewitt broth (Unipath/Oxoid, Ogdensburg, NY, USA) supplemented with 0.5% yeast extract (THY). 15 Viable bacterial counts after 10 h of incubation at 37°C in 5% CO2 ranged from 1 x 108 cfu/mL to 5 x 108 cfu/mL.
Antibiotic preparations and susceptibility testing
Levofloxacin powder was supplied by R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA. Ciprofloxacin powder was supplied by Bayer Corporation, West Haven, CT, USA. Levofloxacin powder was dissolved in 0.2 mL of 0.1 M NaOH, diluted to final volume with distilled water, and sterilized by passage through an Acrodisc syringe filter membrane (0.20 µm pore size; Gelman Sciences, Ann Arbor, MI, USA). Ciprofloxacin powder was reconstituted with distilled water and filter-sterilized.
Susceptibility tests with levofloxacin and ciprofloxacin were performed by broth microdilution according to the procedure recommended by the National Committee for Clinical Laboratory Standards.16
In-vitro pharmacokinetic model
The basics of the in-vitro pharmacokinetic model used in this study have been described in detail previously, 15,17 and a schematic representation of the model is presented in Figure 1. A hollow-fibre cartridge (Unisyn Fibertech, San Diego, CA, USA) was connected by a continuous loop of silicone tubing to a central reservoir. At the start of each experiment, peak antibiotic concentrations in THY in the central reservoir were pumped through the hollow fibres of the cartridge and back into the central reservoir. As drug-containing THY passed through the hollow fibres, pores in the fibre walls allowed antibiotic and nutrients to diffuse freely from the lumen of the fibres into the space surrounding the hollow fibres within the cartridge (peripheral compartment) and back into the lumen of the hollow fibres. The exclusion size of the pores in the fibre walls (mol. wt cut-off 30,000) prohibited bacteria introduced into the peripheral compartment from entering the lumen of the hollow fibres. Thus, the drug concentration within the peripheral compartment space could be altered without disrupting bacterial growth. The bacterial culture within the peripheral compartment was continuously circulated through a loop of silicone tubing attached to two ports entering and exiting the peripheral compartment, and samples were removed from the peripheral compartment through a three-way stopcock connected within the loop of silicone tubing. The initial volume of culture circulated through the peripheral compartment and silicone tubing was 30 35 mL.
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Pharmacokinetics of levofloxacin and ciprofloxacin in the in-vitro pharmacokinetic model
Peak levels of levofloxacin and ciprofloxacin achieved in human serum after oral doses of levofloxacin 500 mg 13,18 and ciprofloxacin 750 mg 14,19 were targeted in these studies. To evaluate the pharmacokinetics of levofloxacin and ciprofloxacin, peak concentrations of levofloxacin and ciprofloxacin were dosed into the central reservoir of the in-vitro pharmacokinetic model and samples were removed from the peripheral compartment at 0, 0.5, 1, 2, 4, 8, 12, 12.5, 13, 14, 16, 20 and 24 h. Drug concentrations in each sample were measured by disc diffusion bioassay using a susceptible strain of Escherichia coli. The linear range of the bioassay was 0.10.9 mg/L. The area under the concentration curve over 24 h (AUC24) for levofloxacin and ciprofloxacin was calculated using the trapezoidal rule. The AUIC24 for levofloxacin and ciprofloxacin against specific strains was calculated by dividing the AUC24 by the MIC of the antibiotic for the target strain of S. pneumoniae.20
Pharmacodynamic experiments
Logarithmic-phase cultures were diluted into fresh THY (prewarmed to 37°C) for a final inoculum of 1 x 106 to 1 x 107 cfu/mL, introduced into the peripheral compartment of the in-vitro pharmacokinetic model and exposed to levofloxacin or ciprofloxacin as described above. Pharmacodynamic experiments were performed in ambient air at 37°C. At 0, 1, 2, 4, 6, 8, 12, 24 and 36 h, samples were removed from the peripheral compartment and viable bacterial counts were measured by plating serial ten-fold dilutions of each sample into ToddHewitt agar (THA; BBL) and incubating plates overnight at 37°C in 5% CO2. The lowest dilution plated was 0.1 mL of undiluted sample from the peripheral compartment. Since 30 colonies is the lower limit of accurate quantification with pour-plate methodology, the lowest number of bacteria that could be accurately counted was 300 cfu/mL. The lowest level of detection, although actual counts were inaccurate, was 10 cfu/mL.
To prevent antibiotic carry-over, 2.5 mM ferric chloride was added to the THA for the least diluted sample.21 Antibiotic carry-over was not a problem with the other dilutions since each antibiotic was diluted to at least 200-fold below the MICs of the drugs against the most susceptible strains. To evaluate the selection of mutants with decreased susceptibility to quinolones, samples removed from the peripheral compartment at 36 h were also plated into THA containing levofloxacin or ciprofloxacin at a concentration of 4 x MIC.
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Results |
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Levofloxacin and ciprofloxacin MICs were similar or within one two-fold dilution of each other against all eight strains of S. pneumoniae, ranging from 0.5 to 2 mg/L for levofloxacin and from 1 to 2 mg/L for ciprofloxacin. When a difference in MIC was observed, the MIC of ciprofloxacin was always twice that of levofloxacin.
The pharmacokinetic profiles of levofloxacin and ciprofloxacin within the peripheral compartment of the in-vitro pharmacokinetic model are shown in Figure 2. Peak concentrations (mean ± S.D.) of levofloxacin and ciprofloxacin in the peripheral compartment at 0.5 h were 6.6 ± 0.2 mg/L and 4.6 ± 0.1 mg/L, respectively. The AUC24 for levofloxacin was 64 mgh/L and that for ciprofloxacin was 44 mgh/L. The AUIC24 for levofloxacin was 32 SIT 1h against one strain, 64 SIT 1h against five strains, and 128 SIT 1h against two strains. In comparison, the AUIC24 for ciprofloxacin was 44 SIT 1h against five strains and 22 SIT 1h against three strains.
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The pharmacodynamics of levofloxacin and ciprofloxacin against representative penicillin-susceptible strains, S. pneumoniae 212 and S. pneumoniae 213, are shown in Figure 3. Levofloxacin was rapidly bactericidal against all four penicillin-susceptible S. pneumoniae, with viable counts falling 5 6 logs to undetectable levels and remaining below this limit for the remainder of the 36 h experimental period (Figure 3). The time required to achieve a significant 3-log kill of the initial inoculum (time to 99.9% kill) ranged from 2 to 4.5 h. The time required to decrease viable counts below the 10 cfu/mL limit of detection ranged from 6 8 h in studies with S. pneumoniae 212 (Figure 3a) and S. pneumoniae 257 (data not shown), to 12 24 h in studies with S. pneumoniae 213 (Figure 3b).
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Pharmacodynamics against penicillin-resistant S. pneumoniae
The pharmacodynamics of levofloxacin and ciprofloxacin against representative penicillin-resistant strains S. pneumoniae 3956 and 3938 are shown in Figure 4. Levofloxacin was rapidly bactericidal against all four strains of penicillin-resistant S. pneumoniae, with viable counts falling 56 logs to undetectable levels and remaining below this limit for the remainder of the 36 h experimental period (Figure 4). The time to 99.9% kill ranged from 1.5 to 4 h, and the time required to decrease viable counts below the 10 cfu/mL limit of detection ranged from 46 h in studies with S. pneumoniae 3956 (Figure 4a) to 1224 h in studies with S. pneumoniae 3935 (data not shown).
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Discussion |
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Levofloxacin was significantly and rapidly bactericidal against all eight strains of S. pneumoniae evaluated in this study. To our knowledge, no other data have been published on the pharmacodynamics of levofloxacin in a pharmacokinetically based model similar to this one. However, using static time kill methodologies other investigators have also demonstrated significant killing of S. pneumoniae with levofloxacin. 22,23,24 The eradication of six isolates of S. pneumoniae from the in-vitro pharmacokinetic model in this study, despite simulated AUICs of only 3264 SIT -1h, suggests that an AUIC of 125 SIT-1h may not be the minimum required for levofloxacin efficacy against S. pneumoniae. This conclusion is supported by clinical experience with levofloxacin in the treatment of community-acquired pneumonia.25 File and colleagues reported that a once-daily dose of 500 mg of levofloxacin was successful in eradicating all of 30 S. pneumoniae from the lungs and all of nine S. pneumoniae from the bloodstream of patients with pneumococcal pneumonia.25 Furthermore, all 30 patients treated with levofloxacin were either clinically cured (23/30) or clinically improved (7/30) 57 days after therapy. These clinical data, combined with the pharmacodynamics observed in the current study simulating the same clinical dose, suggest that a once-daily dose of levofloxacin 500 mg should be effective in treating lower respiratory tract infections and bacteraemias caused by susceptible strains of S. pneumoniae. Although the minimum AUIC required for clinical efficacy appears to be well below 125 SIT 1h, more systematic studies comparing the pharmacodynamics of levofloxacin over a range of AUICs are required to determine the true minimum which must be achieved.
The pharmacodynamics of ciprofloxacin in this study were much more variable than those of levofloxacin. In contrast to levofloxacin, ciprofloxacin failed to decrease viable counts of three strains below the 10 cfu/mL limit of detection. Although these strains were all from the penicillin-susceptible group, there is no evidence in the literature to suggest that this trend is biologically significant. In studies with two of these strains, gradual net increases in viable counts were observed over the second and third dose intervals. Since no resistant mutants were detected on drug-selection plates at 36 h, the decreased antibacterial activity observed after the first dose of ciprofloxacin probably represents adaptive resistance, or the reversible decrease in susceptibility after first exposure to an antibiotic. 26,27 In direct comparisons between the quinolones against individual strains, the rates of killing observed with ciprofloxacin were consistently lower than those observed with levofloxacin. The decreased rate of killing observed with ciprofloxacin in this study may be related to the lower peak/MIC ratios in the in-vitro pharmacokinetic model since fluoroquinolones have been shown to exhibit dose-response bactericidal activity. 2829Although the pharmacodynamics of ciprofloxacin and levofloxacin may have been more comparable in this study if their pharmacokinetics were similar in the in-vitro pharmacokinetic model (adjusting peak levels to attain similar peak/MIC ratios), the purpose of this investigation was to determine if the enhanced in-vitro potency of levofloxacin and its more favourable pharmacokinetic profile would translate into enhanced pharmacodynamic activity. Data from this investigation do suggest that levofloxacin is pharmacodynamically superior to ciprofloxacin against S. pneumoniae when their highest recommended oral doses are simulated.
Similar to the pharmacodynamics observed with levofloxacin, the ability of ciprofloxacin to eradicate five S. pneumoniae from the in-vitro pharmacokinetic model in this study, despite simulated AUICs of only 44 SIT-1h, suggests that an AUIC of 125 SIT-1h may not be the minimum required for clinical efficacy with ciprofloxacin against pneumococci. In fact, a comparison of the pharmacodynamics of ciprofloxacin against the five strains for which the MIC was 1 mg/L (AUIC = 44 SIT -1h) with the pharmacodynamics observed against the three strains for which the MIC of ciprofloxacin was 2 mg/L (AUIC = 22 SIT-1h) suggest that the minimum AUIC required for eradication of S. pneumoniae from the in-vitro pharmacokinetic model lies somewhere between 22 and 44 SIT-1h. Once again, the precise minimum AUIC required for bacterial eradication with ciprofloxacin in this model would require examination of pharmacodynamics over a range of AUICs.
A comparison of the pharmacokinetics of ciprofloxacin in the in-vitro pharmacokinetic model with the MICs of ciprofloxacin against the pneumococcal strains in this study indicates that ciprofloxacin concentrations fell below the 1 mg/L MIC for five strains at 8 h after dosing and below the 2 mg/L MIC for the other three strains at 5 h after dosing. The lack of inoculum regrowth during the first dose interval in these experiments, despite 47 h of subinhibitory concentrations of ciprofloxacin, suggests that a substantial post-antibiotic sub-MIC effect (PA-SME) was occurring. The PA-SME is an extension of the post-antibiotic effect period when sub-inhibitory concentrations of antibiotic remain in the environment, in contrast to when antibiotic is virtually eliminated,30 and this pharmacodynamic phenomenon has been reported with both levofloxacin and ciprofloxacin against S. pneumoniae.31 In the current study, the PA-SME intervals were longer than the 1.253.25 h reported by Licata and colleagues. 31 However, this is not surprising since antibiotic was removed from the bacterial environment more gradually and by more natural processes in the in-vitro pharmacokinetic model, as opposed to the centrifugation and washing techniques used by Licata and colleagues. In contrast to ciprofloxacin, PA-SME interactions could not be evaluated for levofloxacin since drug levels either remained above the MIC during the entire 24 h dose interval or fell below the MIC only after viable counts fell below detectable levels.
In summary, levofloxacin was pharmacodynamically superior to ciprofloxacin in this study. The increased rates of killing and higher rates of bacterial eradication observed with levofloxacin were probably the result of the increased potency of levofloxacin against many pneumococcal strains in combination with its more favourable pharmacokinetic profile. Data from this study and recent clinical data support the use of a once-daily dose of 500 mg of levofloxacin for the treatment of pneumococcal pneumonia caused by susceptible strains, and suggest that the minimum AUIC required for clinical efficacy with both levofloxacin and ciprofloxacin may be well below the 125 SIT-1h breakpoint suggested by other studies. Additional studies will be required to address this issue.
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Acknowledgments |
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Notes |
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References |
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Received 8 April 1998; returned 15 June 1998; revised 15 July 1998; accepted 12 August 1998