Comparison of Efficacies of Oral Levofloxacin and Oral Ciprofloxacin in a Rabbit Model of a Staphylococcal Abscess

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1999, p Vol. 43, No /99/$ Copyright 1999, American Society for Microbiology. All Rights Reserved. Comparison of Efficacies of Oral Levofloxacin and Oral Ciprofloxacin in a Rabbit Model of a Staphylococcal Abscess JEFFREY FERNANDEZ,* JOHN F. BARRETT, LISA LICATA, DHAMMIKA AMARATUNGA, AND MARYBETH FROSCO The R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey Received 10 February 1998/Returned for modification 25 May 1998/Accepted 23 December 1998 Oral levofloxacin was compared to oral ciprofloxacin in a Staphylococcus aureus subcutaneous abscess model in rabbits. Rabbits were surgically prepared with subcutaneous wiffle balls (43 mm in diameter) and allowed to recover for 4 to 6 weeks. Rabbits were infected by direct injection into the capsule with S. aureus ATCC ( CFU) and were allowed to remain infected for 8 days before the initiation of anti-infective treatment. Efficacy was determined by assessing the bacterial load within the capsule over a 10-day treatment period. In single-dose pharmacokinetic studies in infected rabbits, similar area under the concentration-time curve/mic ratios were obtained in the plasma and abscess fluid for levofloxacin at 45 mg/kg of body weight and ciprofloxacin at 200 mg/kg of body weight. Similar efficacies were seen with levofloxacin at 45 mg/kg/day and ciprofloxacin 400 mg/kg/day by day 10. In this model, levofloxacin was significantly more efficacious than ciprofloxacin (P < 0.01). Treatment of deep, nondraining abscesses clinically is often met with difficulty and sometimes with clinical failure. Some of the reasons for treatment failure are low concentrations of anti-infective agents at the infection site, large numbers of bacteria in the stationary phase, low ph, and high protein content within the abscess (12). It has been shown in an uninfected peritoneal capsule model that drug penetration rate, extravascular drug elimination rate, and free (not protein bound) drug molecules are related to the agent s ability to penetrate into capsular fluid (9). Also, slow capsule kinetics (penetration and elimination) have been shown to be important factors contributing to the drug s ability to cure an abscess infection (7). In effect, if the drug can penetrate the abscess, its efficacy will be aided by the slower elimination kinetics. In an acute subcutaneous rabbit abscess model, ciprofloxacin was shown to be effective against gram-negative bacilli (4). More recently, Bamberger et al. have shown that ciprofloxacin (1) was not effective, while cefazolin (1), cefmetazole (1), and ofloxacin (2) were moderately effective, and azithromycin (3) and combinations of ciprofloxacin plus rifampin (1) and ofloxacin plus rifampin (2) were highly effictive in killing Staphylococcus aureus in a peritoneal abscess model. To date, there has been no report of a fluoroquinolone alone being highly effective in curing a staphylococcal infection in a rabbit abscess model. Levofloxacin, the active L-isomer of the racemate ofloxacin, has broad-spectrum activity against both gram-positive and gram-negative bacteria (6). Because levofloxacin is nearly 100% orally bioavailable (5), it has an advantage over other fluoroquinolones that have a broad spectrum of activity but limited oral bioavailability. In this study, we compared the in * Corresponding author. Mailing address: The R. W. Johnson Pharmaceutical Research Institute, 1000 Rt. 202 South, Raritan, NJ Phone: (908) Fax: (908) jfernand@prius.jnj.com. This paper is dedicated to the memory of our friend Richard Rick Schwalbe, who offered many valuable suggestions throughout this project. Present address: Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT vivo efficacies of orally administered levofloxacin and ciprofloxacin in a rabbit model of a subcutaneous staphylococcal abscess. (This paper was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997 [abstr. B-105].) MATERIALS AND METHODS Surgical preparation and animal care. Female New Zealand White rabbits (Covance, Inc., Denver, Pa.), weighing between 3.0 and 4.1 kg, were anesthetized, and a single, sterile golf practice wiffle ball (43 mm in diameter; Wilson Sporting Goods, Chicago, Ill.) was surgically implanted in the dorsal cervical area. The rabbits were given a single 0.1-mg/kg (body weight) dose of butorphanol (Torbugesic; Fort Dodge Laboratories, Fort Dodge, Iowa) intravenously for the reduction of pain and also a single 84,000-IU/kg dose of penicillin G- procaine-benzathine (PenG; Phoenix Pharmaceutical, Inc., St. Joseph, Mo.) subcutaneously for the prevention of infection following surgery. The rabbits were given a 4- to 6-week recovery period before the initiation of any studies. During this recovery period, the rabbits were observed daily, and the Wiffle ball (capsule) became walled off and filled with sterile fluid. All protocols for animal use were approved by the R. W. Johnson Pharmaceutical Research Institute Institutional Animal Care and Use Committee, and the animals were housed in accordance with the Guide for the Care and Use of Laboratory Animals (11). Fluoroquinolone concentrations in uninfected rabbits. Prior to studies with infected rabbits, the maximum concentration of fluoroquinolone (C max )/MIC ratio and the time to achieve the maximum concentration (T max ) were determined with the plasma and abscess fluid of uninfected rabbits following a single oral dose of fluoroquinolone. The area under the concentration-time curve (AUC)/MIC ratio was calculated from single-dose pharmacokinetic studies over a 24-h period. The time above the MIC () was also calculated. Uninfected rabbits were given a single oral dose of levofloxacin (The R. W. Johnson Pharmaceutical Research Institute, Raritan, N.J.) at 22.5, 45, or 90 mg/kg or ciprofloxacin (Bayer-Pentex, Kankakee, Ill.) at 45, 90, or 200 mg/kg. Three rabbits were used at each dose. At 0, 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4, 6, and 24 h after administration, blood was collected via the marginal ear vein into lithium heparin tubes (LH/1.3; Sarstedt, Newton, N.C.) and centrifuged at 7,400 g for 5 min. The supernatant was removed and frozen at 80 C until analyzed. A small volume of abscess fluid was removed from the capsule with a syringe and collected in nontreated tubes at 0, 0.75, 1, 1.5, 2, 3, 4, 6, and 24 h after administration of fluoroquinolone. The samples were centrifuged at 7,400 g for 5 min, and the supernatant was frozen at 80 C until analyzed. Standard curves for levofloxacin and ciprofloxacin were prepared by spiking the plasma or abscess fluid with a known concentration of fluoroquinolone. Serial twofold dilutions were performed in either plasma or abscess fluid, and the fluoroquinolone was extracted with the addition of 2 volumes of acetonitrile. The precipitated proteins were removed by centrifugation at 10,000 g for 10 min. The resulting supernatant was analyzed by high-performance liquid chromatography (series 1100; Hewlett-Packard, Wilmington, Del.) by using a C 18 reversed- 667

2 668 FERNANDEZ ET AL. ANTIMICROB. AGENTS CHEMOTHER. Treatment TABLE 1. Single-dose pharmacodynamics of levofloxacin and ciprofloxacin in plasma of uninfected and infected rabbits Dose (mg/kg) Uninfected Result for rabbit group: Infected Levofloxacin ND a ND ND ND ND ND Ciprofloxacin ND ND ND ND ND ND a ND, not done. phase column (YMC, Wilmington, N.C.) with a gradient of 0.1% trifluoroacetic acid in acetonitrile. A standard curve of absorbance versus fluoroquinolone concentration was constructed. All plasma and abscess fluid samples taken from the rabbits were prepared in the same manner as the standards, and the fluoroquinolone concentrations in the samples were quantified from the standard curve. Fluoroquinolone concentrations in infected rabbits. The fluoroquinolone concentrations in infected rabbits were studied to relate efficacy to the concentration of fluoroquinolone in the plasma and abscess fluid. The infection was established according to the method outlined below in the infection studies section. Levofloxacin at 45 mg/kg and ciprofloxacin at 200 mg/kg were given once orally to rabbits with an infection that had been established and maintained for 8 days. Blood and abscess fluid were collected and analyzed as described above in order to determine the fluoroquinolone concentrations in uninfected rabbits. In addition, the was calculated. Three rabbits were tested at each dose. Fluoroquinolone concentrations during the infection study. Blood and abscess fluid were removed two to three times during the second week (days 13 to 18) of the treatment phase to determine the concentration of fluoroquinolone in the plasma or abscess fluid. Blood was removed 1 h after and abscess fluid was removed 4 h after the first dose of the day. During the infection study, all fluoroquinolone-treated groups had peak fluoroquinolone concentrations within the capsule that were greater than the MICs, except for the rabbits dosed with levofloxacin at 22.5 mg/kg (data not shown). Infection studies. On day 1 of the infection study, a 0.2-ml sample of abscess fluid was removed from the capsule to determine if the abscess fluid was sterile. Rabbits with bacteria present in the abscess fluid before inoculation with S. aureus would be excluded from the study. For this report, no rabbits were excluded due to a prior infection within the capsule. The capsules were infected by injection of S. aureus ATCC ( CFU suspended in 3 ml of 5% mucin [Sigma Chemical Company, St. Louis, Mo.]) directly into the capsule. The MIC of levofloxacin and ciprofloxacin for S. aureus ATCC was 0.5 g/ml. The rabbits remained infected without antibiotic treatment for 8 days. On day 9, the rabbits received either oral levofloxacin (22.5, 45, or 90 mg/kg once a day) or oral ciprofloxacin (45, 90, or 200 mg/kg twice a day) for 10 days. All treatments were delivered through a no. 16 French urethral catheter (Davol, Inc., Cranston, R.I.) passed orally into the stomach. Treatment groups were randomized before the start of the study. Each group consisted of three rabbits. Periodically during the infection (days 1 to 8) and treatment (days 9 to 18) phases of the study, a small volume of abscess fluid was collected to determine the number of bacteria present within the capsule. Abscess fluid was diluted by 10-fold serial dilutions (1:10 1 to 1:10 8 ) in saline, and 100 l of each dilution was added to plates containing 20 ml of Todd-Hewitt agar according to the pour plate technique. The cultures were incubated at 37 C for 24 h. The lower limit of detection was CFU/ml. Rate of change of viable bacterial count analysis. The relative efficacies of the two drugs were assessed in the following manner. For each rabbit, the rate at which the log 10 CFU/ml decreased over the 10-day dosing period was regarded as its response and was estimated as the slope of a linear fit to the plot of log 10 CFU/ml versus time. These responses were modeled against dose by using a slope-ratio type of model, i.e., a linear model with three parameters: an intercept term, the dose-response effect for levofloxacin, and the dose-response effect for ciprofloxacin. Statistical significances for the levofloxacin and ciprofloxacin terms were considered indicative of individual drug effects. If the ratio of the levofloxacin and ciprofloxacin dose-response effects was significantly different from 1, this was considered indicative of a difference between drugs. Susceptibility of recovered organisms. Twenty-four hours following the last dose of each study, abscess fluid was removed to evaluate the susceptibilities of any surviving S. aureus organisms. The MIC of each fluoroquinolone was determined by the broth microdilution method according to the guidelines established by the National Committee for Clinical Laboratory Standards (10). RESULTS Fluoroquinolone concentrations in uninfected rabbits. Single-dose plasma and abscess pharmacokinetic values for uninfected rabbits are summarized in Tables 1 and 2. Single doses of levofloxacin at 22.5, 45, and 90 mg/kg produced levels in plasma of 5.1, 12.4, and 39.9 g/ml, respectively, in uninfected rabbits. Single doses of ciprofloxacin at 45, 90 and 200 mg/kg achieved s in plasma of 6.3, 4.8, and 8.1 g/ml, respectively. The T max in plasma of both fluoroquinolones was approximately 1 to 2 h. A single dose of levofloxacin at 22.5, 45, and 90 mg/kg had s in abscess fluid of 2.2, 4.7, and 20.2 g/ml, respectively, in uninfected rabbits. By comparison, a single dose of ciprofloxacin at 45, 90, and 200 mg/kg achieved a of 2.8, 2.7, and 2.6 g/ml, respec- Treatment TABLE 2. Single-dose pharmacodynamics of levofloxacin and ciprofloxacin in abscess fluid of uninfected and infected rabbits Dose (mg/kg) Uninfected Result for rabbit group Infected Levofloxacin ND ND ND ND ND ND Ciprofloxacin ND ND ND ND ND ND a ND, not done.

3 VOL. 43, 1999 RABBIT MODEL OF SUBCUTANEOUS S. AUREUS ABSCESS 669 tively in abscess fluid. The T max in abscess fluid of both fluoroquinolones was approximately 4 to 6 h. Levofloxacin at 22.5, 45, and 90 mg/kg produced s in plasma and abscess fluid of 42 and 47, 109 and 89, and 273 and 177 g h/ml/mic, respectively. Ciprofloxacin at 45, 90, and 200 mg/kg produced s in plasma and abscess fluid of 88 and 52, 55 and 60, and 74 and 60 g h/ml/mic, respectively. In uninfected rabbits, the in plasma for levofloxacin ranged from 17.1 to 23.6 h, whereas the for ciprofloxacin in plasma was 24 h for all doses tested. The in abscess fluid for both levofloxacin and ciprofloxacin was 24 h in uninfected rabbits. We have observed in pharmacokinetic studies with uninfected rabbits a dose-response relationship with levofloxacin, but not with ciprofloxacin, for the and parameters. Some side effects (lethargy, head swaying, and red coloration in the ears) were observed with infected rabbits dosed with ciprofloxacin at 400 mg/kg, and this prevented us from further evaluating the rabbits at this dose. Fluoroquinolone concentrations in infected rabbits. Singledose plasma and abscess pharmacokinetic values for infected rabbits are summarized in Tables 1 and 2. A single dose of levofloxacin at 45 mg/kg produced levels in plasma and abscess fluid of 28.9 and 5.7 g/ml, respectively, in infected rabbits. A single dose of ciprofloxacin at 200 mg/kg produced comparable concentrations, with levels in plasma and abscess fluid of 45.4 and 8.8 g/ml, respectively, in infected rabbits. Levofloxacin at 45 mg/kg produced s in plasma and abscess fluid of 268 and 101 g h/ml/mic, respectively. Ciprofloxacin at 200 mg/kg produced comparable ratios of s in plasma and abscess fluid of 236 and 121 g h/ml/mic, respectively. In infected rabbits, the in plasma and abscess fluid for ciprofloxacin at 200 mg/kg was 24 h, whereas the was 24 h only in the abscess fluid for levofloxacin at 45 mg/kg. In infected rabbits, the in plasma for levofloxacin at 45 mg/kg was 22.9 h. Infection studies. The efficacy of each fluoroquinolone was assessed by evaluating the number of viable bacteria in the abscess over the 10-day dosing period (Fig. 1). The bacterial load within all capsules was between and CFU/ml by the start of treatment. After 10 days of treatment, levofloxacin at 22.5, 45, and 90 mg/kg once a day had reduced the log 10 CFU/ml in abscess fluid by 4.5, 7.1, and 10.4, respectively. Ciprofloxacin at 45, 90, and 200 mg/kg twice a day reduced the log 10 CFU per milliliter by 0.1, 5.7, and 7.2, respectively, after the same time period. Rate of change of viable bacterial counts. Levofloxacin at 22.5, 45, and 90 mg/kg once a day affected the rate of change of viable S. aureus by 0.44, 0.69, and 1.04 log 10 CFU/ml/ day, respectively (Fig. 2). The rates of change of viable S. aureus cells treated with ciprofloxacin at 45, 90, and 200 mg/kg twice a day were 0.03, 0.54, and 0.71 log 10 CFU/ml/day, respectively. The bacteria in the control group had a rate of change of viable bacteria of 0.09 log 10 CFU/ml/day. The results shown in Fig. 2 indicate that each drug had a statistically significant dose-response effect (P 0.01), indicating that the rates of decrease of log 10 CFU per milliliter over time increased with increasing dose for both drugs. Levofloxacin, having a statistically significant higher rate of decrease, was superior to ciprofloxacin (P 0.01). Bacterial eradication within the capsule. The proportions of rabbits having eradication of bacteria ( CFU/ml) increased significantly (P 0.01; Cochran-Armitage dose-response trend test) with increasing levofloxacin dose (0 of 3 for nontreated controls, 0 of 3 for 22.5 mg/kg, 2 of 3 for 45 mg/kg, and 3 of 3 for 90 mg/kg). All ciprofloxacin-treated rabbits, FIG. 1. Efficacy of levofloxacin or ciprofloxacin in the rabbit abscess model. Control animals received no antibiotic treatment. Data points represent average values for three rabbits. Data are represented as the decrease in log 10 CFU per milliliter over the 10-day treatment period. The bacterial load for each treatment group was compared to the control group. }, control;, levofloxacin at 90 mg/kg once a day; F, levofloxacin at 45 mg/kg once a day; Œ, levofloxacin at 22.5 mg/kg once a day;, ciprofloxacin at 200 mg/kg twice a day;, ciprofloxacin at 90 mg/kg twice a day;, ciprofloxacin at 45 mg/kg twice a day. except for one rabbit treated with 200 mg/kg twice a day, had bacteria remaining within the capsule at the end of the study. The concentrations of the remaining bacteria ranged from to CFU/ml and correlated with dose. Susceptibility of recovered organisms. All of the bacteria remained susceptible ( 1 twofold dilution) to the fluoroquinolone that was used for the treatment of that group, except for the isolates from one of the ciprofloxacin-treated rabbits. In one rabbit treated with 90 mg of ciprofloxacin per kg for 10 days, the MIC increased from 0.5 g/ml to 2 g/ml for the S. aureus recovered. When tested against levofloxacin, the MIC FIG. 2. Effect of levofloxacin or ciprofloxacin on the rate of growth of S. aureus in the rabbit abscess model. F, levofloxacin;, ciprofloxacin.

4 670 FERNANDEZ ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 3. Reduction in bacterial growth rate of S. aureus for a 10-day treatment period with levofloxacin or ciprofloxacin correlated to ratios in plasma. F, levofloxacin uninfected ratio;, ciprofloxacin uninfected ratio;, levofloxacin infected ratio;, ciprofloxacin infected ratio. was 1 g/ml, compared to a levofloxacin MIC of 0.5 g/ml for the infecting strain. Pharmacokinetics and pharmacodynamics. The pharmacokinetic and pharmacodynamic results are shown in Fig. 3. There was a linear and proportional dose response with levofloxacin with respect to and bacterial growth rate. No dose response was seen with ciprofloxacin with respect to and C max ; however, a dose response was seen with respect to growth rate. Further pharmacokinetic and pharmacodynamic studies with this dose need to be performed with this model. Toxic side effects were seen with a single 400-mg/kg oral dose of ciprofloxacin which prevented us from studying doses higher than 200 mg/kg. The dose-response relationship that was observed with ciprofloxacin with respect to bacterial killing was due to the better pharmacokinetic parameter ( and C max ) values in infected rabbits. In fact, both fluoroquinolones had higher pharmacokinetic parameter ( and C max ) values in infected rabbits. Levofloxacin plasma pharmacokinetic parameter ( and C max ) values were 2.3 to 2.4 times higher in infected rabbits than in noninfected rabbits. In infected rabbits, ciprofloxacin showed increases in plasma pharmacokinetic parameters ( and C max ) of 3.2 to 5.6 times greater than the plasma pharmacokinetics attained in uninfected rabbits. In infected rabbits dosed with levofloxacin, there was an approximately twofold increase in the abscess pharmacokinetic parameter (AUC/ MIC and C max ) values compared to those in uninfected rabbits. Ciprofloxacin abscess pharmacokinetic parameters (AUC/ MIC and C max ) were 2 to 3.4 times higher in the infected rabbits. DISCUSSION Levofloxacin and ciprofloxacin are similar with respect to their pharmacodynamic properties in this infection model. In infected rabbits, we have shown that similar s for the levofloxacin 45-mg/kg and the ciprofloxacin 200-mg/kg treatment regimens (Fig. 3, open symbols) result in similar declines in bacterial growth rate. This also occurs in vitro with equal MICs of both drugs for the S. aureus strain used in this study. As shown by the comparable killing of S. aureus with levofloxacin at 45 mg/kg once a day and ciprofloxacin at 200 mg/kg twice a day, it is obvious that levofloxacin had a pharmacokinetic advantage over ciprofloxacin in this model. The differences are probably due to bioavailability, but drug elimination may be a contributing factor. Levofloxacin also had a pharmacoeconomic advantage in this model, achieving the same result (efficacy) with far less drug administered. Levofloxacin, orally administered at 45 and 90 mg/kg once a day, was highly efficacious, achieving at least a 6-log 10 decrease in CFU per milliliter. In contrast, only the highest dose of ciprofloxacin had comparable efficacy in this model. Ciprofloxacin at 90 mg/kg twice a day and levofloxacin at 22.5 mg/kg once a day decreased the bacterial load 4 to 5 log 10 CFU/ml, whereas ciprofloxacin at 45 mg/kg twice a day was not effective. It is not obvious why the 45-mg/kg ciprofloxacin dose was not efficacious in the infection model, given that the drug exposure in plasma and abscess fluid was above the MIC for the entire dosing interval. Similar reductions in log 10 CFU per milliliter were observed at day 10 for levofloxacin at 45 mg/kg once a day and ciprofloxacin at 200 mg/kg twice a day, even though the levofloxacin-treated rabbits received approximately nine times less fluoroquinolone. The decreases in the rate of bacterial growth (inhibition or death of bacteria) for these two treatment groups were also similar. The s of levofloxacin at 45 mg/kg and ciprofloxacin at 200 mg/kg in the plasma and abscess fluid of infected rabbits were similar. The drug penetration into the abscess fluid [(AUC abscess /AUC plasma ) 100%] was slightly higher for ciprofloxacin at 200 mg/kg (51%) than for levofloxacin at 45 mg/kg (37%) in infected rabbits. In light of the fact that ciprofloxacin has increased penetration into the capsule, the s are roughly the same for both fluoroquinolones, and the is greater than 24 h for both drugs, further studies examining the capsule elimination kinetics to fully understand the efficacy of levofloxacin in this model are warranted. In pharmacokinetic studies with infected rabbits at 24 h, the fluoroquinolone concentration in abscess fluid was higher with levofloxacin (2.9 g/ml) than with ciprofloxacin (1.9 g/ml), suggesting that levofloxacin may have slower capsule elimination kinetics and therefore appears more efficacious. Pharmacokinetic studies covering a range of doses in infected rabbits are necessary to further elucidate the elimination kinetics of both fluoroquinolones. Despite the fact that was delivered more ciprofloxacin than levofloxacin was delivered (200 mg/kg twice a day versus 45 mg/kg once a day), the drug concentrations () in plasma and abscess fluid and efficacies were similar. The data from this study are consistent with human data showing that levofloxacin has enhanced oral bioavailability (nearly 100% [5] versus 78% for ciprofloxacin [8]). Levofloxacin was more efficacious than ciprofloxacin in eradicating S. aureus in this model. ACKNOWLEDGMENTS We thank James Hastings, Robert DeWire, Jane Folden, Leslie Chalecki, Barbara Foleno, Marie Grant, and Robert Schuster for their technical assistance. We thank Elizabeth Dodemaide and Audrey Stewart for their veterinary assistance. We also thank Karen Bush for her advice and comments during the completion of these studies for publication. REFERENCES 1. Bamberger, D. M., M. T. Fields, and B. L. Herndon Efficacies of various antimicrobial agents in treatment of Staphylococcus aureus abscesses and correlation with in vitro tests of antimicrobial activity and neutrophil killing. Antimicrob. Agents Chemother. 35: Bamberger, D. M., B. L. Herndon, M. Dew, R. P. Chern, H. Mitchell, L. E. Summers, R. F. Marcus, S. C. 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