Pharmacodynamics of Moxifloxacin and Levofloxacin against Staphylococcus aureus and Staphylococcus epidermidis in an In Vitro Pharmacodynamic Model

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1 SUPPLEMENT ARTICLE PHARMACOLOGY Pharmacodynamics of Moxifloxacin and Levofloxacin against Staphylococcus aureus and Staphylococcus epidermidis in an In Vitro Pharmacodynamic Model Philip D. Lister Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha An in vitro pharmacokinetic model was used to compare the pharmacodynamics of moxifloxacin and levofloxacin against 3 Staphylococcus aureus and 3 Staphylococcus epidermidis strains. Logarithmic-phase cultures were inoculated into the peripheral compartment of hollow-fiber cartridges and exposed to the peak serum concentrations achieved in humans with oral doses of moxifloxacin (400 mg) and levofloxacin (500 mg). Drugs were added at 0 and 24 h, elimination kinetics were simulated, and changes in viable bacterial counts were evaluated over the course of 36 h. Moxifloxacin was bactericidal against all 6 staphylococci (times to 99.9% kill, 1 3 h). Against most strains, bacterial killing continued through 36 h, with total kills exceeding 5.5 logs. Levofloxacin was bactericidal against 5 of the strains, with similar times to 99.9% kill. In contrast to moxifloxacin, however, resistant subpopulations emerged in 4 strains during therapy with levofloxacin, and this could have important implications for treatment of staphylococcal infections. These in vitro observations warrant the clinical evaluation of moxifloxacin in the treatment of staphylococcal infections. Staphylococci are only marginally susceptible to the older fluoroquinolone antibiotics, with ciprofloxacin 90% MICs (MIC 90 ) against methicillin-susceptible strains in the range of mg/ml [1]. As a result of this marginal susceptibility, staphylococci have rapidly developed fluoroquinolone resistance [2]. As fluoroquinolone resistance among staphylococci continues to increase, the need for more effective agents becomes essential. Moxifloxacin is a new fluoroquinolone that exhibits enhanced potency against gram-positive bacterial pathogens [1]. In comparisons with ciprofloxacin and levofloxacin, moxifloxacin is 4- to 8-fold more potent against Financial support: Bayer Corporation. Reprints or correspondence: Dr. Philip D. Lister, Center for Research in Anti- Infectives and Biotechnology, Dept. of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE (pdlister@creighton.edu). Clinical Infectious Diseases 2001; 32(Suppl 1):S by the Infectious Diseases Society of America. All rights reserved /2001/3206S1-0006$03.00 methicillin-susceptible and -resistant isolates of Staphylococcus aureus and Staphylococcus epidermidis. Furthermore, moxifloxacin retains its activity against many staphylococcal isolates that develop resistance to ciprofloxacin [3]. The purpose of this study was to compare the pharmacodynamics of moxifloxacin and levofloxacin against 6 clinical isolates of S. aureus and S. epidermidis in an in vitro pharmacokinetic model (IVPM). MATERIALS AND METHODS Bacterial strains and culture conditions. The experimental strains evaluated in this study included 3 clinical isolates of S. aureus and 3 of S. epidermidis. Two isolates from each group were resistant to methicillin and ciprofloxacin. The MICs of moxifloxacin, levofloxacin, and ciprofloxacin against these isolates are shown in table 1. Stocks of all strains were frozen at 70 C in 50% brain-heart infusion broth (Becton- Dickinson) and 50% sterile horse serum (Colorado Pharmacodynamics of Moxifloxacin and Levofloxacin CID 2001:32 (Suppl 1) S33

2 Table 1. MICs, peak/mic ratios, and AUC/MIC ratios of moxifloxacin and levofloxacin against strains of Staphylococcus aureus and Staphylococcus epidermidis. Strain Methicillin susceptibility Ciprofloxacin MIC, mg/ml Moxifloxacin Levofloxacin MIC, mg/ml Peak/MIC AUC/MIC MIC, mg/ml Peak/MIC AUC/MIC S. aureus 1 Susceptible S. aureus 230 Resistant S. aureus 213 Resistant S. epidermidis 102 Susceptible S. epidermidis 87 Resistant S. epidermidis 125 Resistant NOTE. AUC, area under the concentration-time curve. Serum Company). Before use in experiments, frozen cultures were subcultured to trypticase soy agar supplemented with 5% sheep blood (BAP; BBL Prepared Media, Becton-Dickinson) and incubated overnight at 37 C to ensure strain purity. For pharmacodynamic experiments, logarithmic-phase cultures were prepared by inoculating colonies from overnight BAP cultures into 70 ml of Mueller-Hinton broth (MHB; Oxoid, Unipath) to equal an optical density at 540 nm of 0.1. The broth cultures were then incubated at 37 C with shaking for 2 h until the optical density at 540 nm increased to 0.4. Logarithmic-phase cultures were diluted 10-fold into fresh MHB at 37 C to give a final inoculum of cfu/ml. Antibiotic preparations and susceptibility testing. Moxifloxacin powder was supplied by Bayer Corporation. Levofloxacin powder was supplied by R. W. Johnson Pharmaceutical Research Institute. Antibiotic powders were dissolved in 0.2 ml of 0.1 M NaOH, diluted to final volume with distilled water, and sterilized by passage through a 0.20-mm pore size Acrodisc syringe filter membrane (Gelman Sciences). Susceptibility tests with moxifloxacin and levofloxacin were performed by broth microdilution according to the procedure recommended by the National Committee for Clinical Laboratory Standards [4]. IVPM. The basics of the IVPM used in this study have been described in detail elsewhere [5, 6]. The hollow-fiber cartridges (Unisyn Fibertech) used in these studies consisted of 2250 cellulose acetate hollow fibers contained within a polycarbonate housing, with each fiber having 30,000 molecular weight pores within its wall. The surface area of exchange between the fibers and the extracapillary space (peripheral compartment) was 0.45 m 2. Medium containing antibiotic was pumped through the lumen of the fibers at a flow rate of 20 ml/min with Masterflex computerized peristaltic pumps and Easy-Load pump heads (Cole-Parmer Instrument). In addition, the bacterial culture within the peripheral compartment was continuously circulated with similar peristaltic pumps at a rate of 20 ml/min through a loop of silicone tubing attached to 2 ports entering and exiting the peripheral compartment. The initial volume of culture circulated through the peripheral compartment and loop of silicone tubing was 45 ml. When samples were required from the peripheral compartment, 0.5-mL volumes were removed through a 4-way sterile stopcock (Medex) positioned within the loop of silicone tubing. The volume of MHB within the central reservoir varied with each drug depending on the elimination half-life, such that the rate of dilution and elimination could be set at the minimum 0.7 ml/min allowed by the peristaltic pumps. Elimination halflives of 13 h for moxifloxacin [7] and 7.5 h for levofloxacin [8] were simulated. The corresponding central reservoir volumes were 800 ml for studies with moxifloxacin and 450 ml for levofloxacin. In drug-free control experiments, the volume of MHB in the central reservoir was 500 ml, and the flow rate for addition of fresh media and elimination from the central reservoir was 2 ml/min. Quinolone pharmacokinetics within the IVPM. The serum pharmacokinetics of oral doses of 400 mg of moxifloxacin [7] and 500 mg of levofloxacin [8] were simulated in these studies. To evaluate the pharmacokinetics of moxifloxacin and levofloxacin in the IVPM, peak concentrations of each drug were dosed into the central reservoir and samples were removed from the peripheral compartment at 0, 0.5, 1, 2, 4, 8, 12, and 24 h. Drug concentrations were measured by disk diffusion bioassay [9] with a susceptible strain of Escherichia coli. Areas under the concentration-time curve (AUC) over 24 h (AUC 0 24 ) for moxifloxacin and levofloxacin were calculated with the trapezoidal rule. The AUC/MIC ratios for moxifloxacin and levofloxacin were calculated by dividing the AUC 0 24 by the MICs for specific strains of S. pneumoniae [10]. Pharmacodynamic experiments. Logarithmic-phase cultures were diluted into fresh MHB at 37 C for a final inoculum of cfu/ml, introduced into the peripheral compartment of the IVPM, and exposed to the fluoroquinolones as described above. Pharmacodynamic experiments were performed in ambient air at 37 C. At 0, 1, 2, 4, 6, 8, 24, and 36 h, samples were removed from the peripheral compartment and viable bacterial counts were measured by plating serial 10-fold dilutions of each sample into Mueller-Hinton agar and incubating plates over- S34 CID 2001:32 (Suppl 1) Lister

3 night at 37 C in5%co 2. The lowest level of detection was 10 cfu/ml. To prevent antibiotic carryover, samples removed from the peripheral compartment were first incubated for 15 min with 0.2 g of nonionic polymeric adsorbent beads (Amberlite XAD- 4; Sigma Chemical) [11]. To evaluate the selection of mutants with decreased susceptibility to quinolones, samples removed from the peripheral compartment at 36 h were also plated into Mueller-Hinton agar containing antibiotic at a concentration 4 times the MIC. RESULTS Characterization of S. pneumoniae isolates and the IVPM. Moxifloxacin was 4- to 8-fold more potent than levofloxacin against these staphylococci, with MICs of mg/ml for moxifloxacin and mg/ml for levofloxacin (table 1). The MICs of moxifloxacin and levofloxacin against the 4 ciprofloxacin-resistant strains were mg/ml for moxifloxacin and 2 8 mg/ml for levofloxacin. The pharmacokinetic profiles of moxifloxacin and levofloxacin within the peripheral compartment of the IVPM are shown in figure 1. Peak concentrations (mean SD) were achieved in the peripheral compartment within 0.5 h after dosing into the central reservoir and were mg/ml for moxifloxacin and mg/ml for levofloxacin. Calculated peak/ MIC ratios were 5:1 to 150:1 for moxifloxacin and 1:1 to 30:1 for levofloxacin (table 1). The AUC 0 24 values were 54 mg/ h/ml for moxifloxacin and 64 mg/h/ml for levofloxacin. Calculated AUC/MIC ratios were for moxifloxacin and for levofloxacin (table 1). Pharmacodynamic studies with S. aureus. Moxifloxacin exhibited bactericidal activity against all 3 isolates of S. aureus (figure 2). Times to achieve 99.9% kill of each strain were 1 3 h. Viable counts of S. aureus 1 and S. aureus 230 continued to decrease throughout the 36-h experimental period, with total kills exceeding 5.5 logs. In studies with S. aureus 213, a gradual increase in viable counts was observed between 8 and 36 h. Nevertheless, viable counts remained 5 logs below the initial inoculum, and no resistant subpopulations were detected. Levofloxacin also exhibited bactericidal activity against all 3 isolates of S. aureus (figure 2). Differences in times to 99.9% kill between levofloxacin and moxifloxacin were 1horless. In contrast to moxifloxacin, however, significant increases in the viable counts of S. aureus 213 and S. aureus 230 were observed between 8 and 36 h as a result of the outgrowth of resistant subpopulations. The MIC of levofloxacin against the mutant populations from both strains was 16 mg/ml. Pharmacodynamic studies with S. epidermidis. Moxifloxacin exhibited significant bactericidal activity against all 3 isolates of S. epidermidis (figure 3). Times to achieve 99.9% kill Figure 1. Pharmacokinetics of moxifloxacin and levofloxacin within the peripheral compartment of the in vitro pharmacokinetic model after dosing into the central reservoir. Each point represents the mean drug concentrations (mg/ml) in the peripheral compartment for 3 experimental runs. Error bars show standard deviations. of each strain were h. Viable counts of S. epidermidis 102 and S. epidermidis 87 continued to decrease throughout the 36-h experimental period, with the total kill exceeding 5.5 logs. In studies with S. epidermidis 125, gradual increases in viable counts were observed between 8 and 36 h. Nevertheless, viable counts remained 2 logs below the initial inoculum, and no resistant subpopulations were detected. Levofloxacin also exhibited significant bactericidal activity against S. epidermidis 102 and S. epidermidis 87 (figure 2). Differences in times to 99.9% kill between levofloxacin and moxifloxacin were!1 h. In contrast to moxifloxacin, however, levofloxacin failed to achieve 99.9% kill of S. epidermidis 125, and significant increases in the viable counts of S. epidermidis 87 and S. epidermidis 125 were observed between 8 and 36 h as a result of the outgrowth of resistant subpopulations. The MICs of levofloxacin against the mutant populations from both strains were mg/ml. DISCUSSION An IVPM was used to simulate the serum pharmacokinetics of maximum oral doses of moxifloxacin and levofloxacin and to compare their pharmacodynamics against ciprofloxacin- Pharmacodynamics of Moxifloxacin and Levofloxacin CID 2001:32 (Suppl 1) S35

4 Figure 2. Time-kill pharmacodynamics of moxifloxacin and levofloxacin against Staphylococcus aureus 1 (A), S. aureus 230 (B), and S. aureus 213 (C). Each point represents the mean number of viable bacteria per milliliter of Mueller-Hinton broth from the peripheral compartment for duplicate experiments. Error bars show standard deviations. S36

5 Figure 3. Time-kill pharmacodynamics of moxifloxacin and levofloxacin against Staphylococcus epidermidis 102 (A), S. epidermidis 87 (B), and S. epidermidis 125 (C). Each point represents the mean number of viable bacteria per milliliter of Mueller-Hinton broth from the peripheral compartment for duplicate experiments. Error bars show standard deviations. S37

6 susceptible and ciprofloxacin-resistant S. aureus and S. epidermidis. Both moxifloxacin and levofloxacin exhibited significant bactericidal activity against all 3 S. aureus and 2 of the S. epidermidis. When significant 99.9% kills were achieved, there was little difference observed between these drugs in the initial rates of killing. This observation was unexpected because fluoroquinolones are concentration-dependent antibiotics [12, 13], and peak/mic ratios achieved with moxifloxacin were up to 10-fold higher than the peak/mic ratios achieved with levofloxacin. Furthermore, peak/mic ratios achieved with levofloxacin against 4 of the isolates were only 1:1 to 4:1. Although no differences were observed in initial killing rates, the pharmacodynamics of these 2 fluoroquinolones differed substantially after the initial 8 h. In studies with moxifloxacin, viable counts either continued to decline until they fell below the 10 cfu/ml limit of detection or remained logs below the initial inoculum. No resistant mutants were detected in experiments with moxifloxacin. In contrast, viable counts of 4 strains treated with levofloxacin increased rapidly between 8 and 24 h, and this rapid regrowth was associated with the outgrowth of a resistant mutant subpopulation. The selection and outgrowth of resistant subpopulations with levofloxacin were not surprising because peak/mic ratios were 4:1 or less. Furthermore, the MICs of levofloxacin against the mutant subpopulations were 2- to 4-fold above the peak levels achieved in the IVPM. In studies with the 2 strains in which peak/mic ratios were 15:1 and 30:1, viable counts fell below detectable levels and no mutants were selected. These data highlight the importance of achieving drug concentrations that exceed the MIC of not only the original isolate but also any resistant subpopulations. In contrast to the rapid selection of resistant mutants with levofloxacin in this study, it was of interest that no mutants were selected with moxifloxacin, even with S. epidermidis 87 and S. epidermidis 125, where moxifloxacin peak/mic ratios were only 5:1. Although it is possible that resistant mutants may have been selected from the viable cells remaining at 36 h if these experiments had been extended for several dosing intervals, the rapidity in which levofloxacin-selected mutants grew suggests that there may indeed be a difference between these 2 fluoroquinolones in their propensity to select resistance among staphylococci. This difference between moxifloxacin and levofloxacin carries important therapeutic implications when selecting a fluoroquinolone for the treatment of infections with S. aureus and S. epidermidis. 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