ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1999, p. 672677 0066-4804/99/$04.000 Copyright 1999, American Society for Microbiology.

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Vol. 43, No. 3

Pharmacodynamic Comparisons of Levooxacin, Ciprooxacin, and Ampicillin against Streptococcus pneumoniae in an In Vitro Model of Infection
MELINDA K. LACY,1* WEN LU,2 XIAOWEI XU,2 PAMELA R. TESSIER,2 DAVID P. NICOLAU,2,3 RICHARD QUINTILIANI,4 AND CHARLES H. NIGHTINGALE4 Department of Pharmacy Practice, The University of Kansas Medical Center, Kansas City, Kansas 66160-7231,1 and Department of Pharmacy Research,2 Division of Infectious Diseases,3 and Ofce of Research Administration,4 Hartford Hospital, Hartford, Connecticut 06102-5037
Received 23 January 1998/Returned for modication 7 June 1998/Accepted 8 December 1998

The increasing frequency of penicillin-resistant pneumococcus continues to be of concern throughout the world. Newer uoroquinolone antibiotics, such as levooxacin, have shown enhanced in vitro activity against Streptococcus pneumoniae. In this study, the bactericidal characteristics and pharmacodynamic proles of levooxacin, ciprooxacin, and ampicillin against four isolates of S. pneumoniae were compared by using an in vitro model of infection. Standard antibiotic dosing regimens which simulated the pharmacokinetic prole observed in humans were used. Control and treatment models were sampled for bacterial CFU per milliliter over the duration of each 24- or 48-h experiment. In addition, treatment models were sampled for MIC determinations and drug concentration. Regrowth of all isolates as well as an increase in MICs throughout the study period was observed in the ciprooxacin experiments. A limited amount of regrowth was noted during levooxacin therapy for one isolate; however, no change in MIC was detected for any isolate. Ampicillin showed rapid and sustained bactericidal activity against all isolates. In this study, ratios of effective uoroquinolone area under the concentration-time curve (AUC):MIC values ranged from 30 to 55. Levooxacin, owing to its larger AUC024 values, has excellent and sustained activity against different pneumococcal strains superior to that of ciprooxacin. Streptococcus pneumoniae continues to be the leading cause of community-acquired pneumonia, acute sinusitis, and bacterial meningitis worldwide (1, 10, 15, 30). Penicillin resistance for pneumococcus has been reported to be widespread since it was rst noted in 1967 (17), especially over the last 6 years (6, 14, 31). Recent surveillance studies have shown that the frequency of S. pneumoniae with reduced susceptibility to penicillin (intermediate and resistant strains) in the United States is currently around 24 to 34% (6, 32). Therefore, the continuing trend toward penicillin resistance for S. pneumoniae is leading clinicians to consider non--lactam alternatives for coverage of this important community-acquired pathogen. It is well established that uoroquinolone (FQ) antibiotics have good activity against gram-negative and atypical pathogens. However, a review of the literature shows that treatment failures have been reported for older FQ agents when used in patients infected with S. pneumoniae (3, 5, 12, 18, 20, 28, 33). Since those reports appeared, newer agents, such as levooxacin, have been approved for use in the United States. To further investigate the activity of FQ antibiotics against S. pneumoniae, we evaluated the bactericidal and pharmacodynamic proles of levooxacin and ciprooxacin against four isolates with various susceptibilities. For comparative purposes, the -lactam ampicillin was also tested. (This work was presented in part at the 20th International Congress of Chemotherapy, Sydney, Australia, 29 June 1997 to 3 July 1997.)
MATERIALS AND METHODS Bacterial strains and susceptibility testing. Thirty-ve S. pneumoniae clinical isolates from Hartford Hospital were screened for susceptibility to penicillin, ciprooxacin, and levooxacin. MICs and minimum bactericidal concentrations (MBC) were determined according to method of the National Committee for Clinical Laboratory Standards by using a microdilution technique (23). Four isolates which displayed a range of susceptibility proles for penicillin, ciprooxacin, and levooxacin were selected. Antibiotics. The following antibiotics were used: sterile ampicillin sodium, 500 mg of powder for injection (lot F5V04A; expiration date, June 1999; Apothecon); ciprooxacin for intravenous injection, 40 mg/ml (lot 5GFC; expiration date, July 1998; Bayer Corporation); and levooxacin standard powder (RWJ02513-097-AQ; potency, 969.5 mg/g; lot CRO38; expiration date 19 January 1997; R. W. Johnson). Bacterial growth media. Cation-adjusted Mueller-Hinton broth (CAMHB) (Becton Dickinson, Cockeysville, Md.) supplemented with 2.5 to 5% lysed horse blood (LHB) (Remel, Lenexa, Kans.) was used as the bacterial growth medium in all in vitro model experiments and susceptibility determinations. Trypticase soy agar plates (100-mm diameter) with 5% sheep blood and Mueller-Hinton agar plates (150-mm diameter) with 5% sheep blood (Becton Dickinson) were used for the quantitative determinations. In vitro model. The in vitro model used in this study has previously been described (13). By using a central compartment model, bacteria were exposed to changing concentrations of antibiotics to simulate human pharmacokinetic parameters. Each experiment consisted of three independent models (two antibiotic-treated models and one growth control model) which were run simultaneously for all organisms and treatment regimens. The models were placed in a 37C temperature-controlled circulating water bath for optimal temperature control, and magnetic stir bars were used in each model to ensure adequate mixing of all contents. Fresh CAMHB supplemented with LHB was continuously pumped into each of the models by a peristaltic pump at rates which simulated the elimination half-lives of the test antibiotics (for ampicillin, ciprooxacin, and levooxacin the half-lives are 1, 4, and 7 h, respectively). A starting inoculum of 106 CFU/ml was prepared from an overnight culture of the test isolate for all model experiments. To ensure that bacteria were in logarithmic growth phase prior to antimicrobial exposure, experiments were started 1 h after inoculation of bacteria into the models. Twenty-four-hour studies were initially conducted for each antibiotic against all S. pneumoniae isolates. In the cases where bacterial regrowth was detected at 24 h, separate 48-h experiments were performed to further characterize antibiotic activity over time. Bacterial regrowth was assessed with quantitative cultures

* Corresponding author. Mailing address: Department of Pharmacy Practice, The University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7231. Phone: (913) 588-5314. Fax: (913) 588-2355. E-mail: mlacy@kumc.edu. 672

VOL. 43, 1999

QUINOLONE PHARMACODYNAMIC COMPARISONS FOR PNEUMOCOCCUS TABLE 1. Observed MICs over time in treatment models

673

Antibiotic and isolate

MIC (g/ml) at: Screening 0h 6h 12 h 24 h 36 h 48 h

Ciprofloxacin SP28 SP34 SP12 SP4 Levooxacin SP28 SP34 SP12 SP4 Ampicillin SP28 SP34 SP12 SP4
a b

1 2 48 4 1 2 2 4 0.5 816 0.125 0.25

0.5 2 4 8 1 24 2 2 NDa ND ND ND

12 4 48 4 2 24 4 24 NG NG NG NG

2 8 4 4 12 12 NG 4

4 8 816 816 NGb NG NG, 2 24

4 8 8 16

832 816 832 816

ND, not determined due to lack of growth at 6 h. NG, no growth.

as outlined below. Additionally, since MIC and MBC tests were performed on samples obtained throughout the duration of each experiment, any bacteria present at the 24- or 48-h time point were therefore detected. Antibiotics were added to the models to simulate intravenous (IV) bolus dosing for the following regimens: for ampicillin, 500 mg (with test isolate SP4) or 1,000 mg (with test isolates SP28, SP34, and SP12) IV every 6 h (q6h), with peak concentrations of 50 and 25 g/ml, respectively; for ciprooxacin, 400 mg IV q12h, with a peak concentration of 4.6 g/ml; and for levooxacin, 500 mg IV q24h, with a peak concentration of 6.4 g/ml. To conrm the simulation of human pharmacokinetic parameters, samples were taken throughout the entire duration of the model experiment and samples were stored at 80C until they were assayed for drug concentration. To assess bacterial density over time, samples were obtained from each model and serially diluted in saline. Aliquots of each diluted sample were plated in triplicate for quantitative culture. To minimize any effect of antibiotic carryover on the less-diluted samples, larger plates (150-mm-diameter agar plates) were used for detection of bacterial growth. After 24-h incubation at 37C, the change in log10 CFU/ml at 24-h intervals was calculated and time-kill curves were constructed by plotting log10 CFU/ml against time. In addition, preliminary experiments were conducted to assess the inuence of antibiotic carryover with each of the test agents. As a result of these data, the limit of quantication for ampicillin was determined to be 102 CFU/ml. No antibiotic carryover was observed for the FQ; thus, the limit of quantication was 101 CFU/ml. Development of resistance was assessed by performing MIC and MBC determinations on S. pneumoniae recovered from experimental models at 0, 6, 12, and 24 h (and at 36 and 48 h when longer experiments were performed). Antibiotic concentration determinations. Samples of CAMHB supplemented with LHB taken from each of the treatment models were assayed for ampicillin, levooxacin, and ciprooxacin. Samples containing the FQ were analyzed by an ion-paired validated high-performance liquid chromatography method as previously described (21), with modications. CAMHB with LHB was used to prepare standards, check samples, and dilute samples as required. Briey, 50 l of pipemidic acid (Sigma Chemical Co., St. Louis, Mo.) used as an internal standard was added to 200 l of sample or standard and mixed. After the addition of 3.5 ml of methylene chloride (Mallinckrodt Baker, Paris, Ky.) for ciprooxacincontaining samples or chloroform (Mallinckrodt Baker) for levooxacin-containing samples, all samples were shaken for 10 min and then centrifuged at 3,000 g for 10 min. To the organic layer, 200 l of 0.1-mol/liter sodium hydroxide (Sigma Chemical Co.) was added, and all tubes were shaken for 20 min and then centrifuged at 3,000 g for 15 min. Twenty microliters of the aqueous layer was injected onto a 10-m-particle-diameter C18 column (4.6 mm [outside diameter] by 250 mm [height]) (Nucleosil; Alltech Associates, Inc., Deereld, Ill.), and uorescence was monitored at excitation wavelengths of 278 nm for ciprooxacin and 282 nm for levooxacin by using a uorescence detector (model 980; Applied Biosystems Inc., Foster City, Calif.) with a lter with an emission cutoff of 418 nm. The mobile phase consisted of 0.01 M phosphate buffer (pH 2.5) (Sigma Chemical Co.) with 0.01 M tetrabutylammonium hydrogen sulfate (Sigma Chemical Co.) and acetonitrile (Mallinckrodt Baker) in an 87:13 ratio (vol/vol) pumped at ow rates of 1.8 ml/min for ciprooxacin and 1.4 ml/min for levooxacin by an isocratic pump (model 510; Waters Associates, Milford, Mass.). The assay of ciprooxacin was linear over the range from 0.1 to 6 g/ml. Intrarun coefcients of variation (CVs) were 1.98% (n 10) and 2.40% (n 10) for the

check samples with low (1 g/ml) and high (5 g/ml) concentrations, and interrun CVs were 2.56% (n 9), 2.17% (n 16), and 1.92% (n 25) for the check samples with low (0.5 and 1 g/ml) and high (5 g/ml) concentrations, respectively. The assay of levooxacin was linear over the range from 0.1 to 10 g/ml. Intrarun CVs were 1.24% (n 10) and 0.57% (n 10), and interrun CVs were 1.90% (n 17) and 1.19% (n 17), for the low (1 g/ml) and high (8 g/ml) check samples, respectively. The limit of sensitivity for both assays was 0.1 g/ml. Ampicillin concentrations in CAMHB with LHB were determined by a validated bioassay method. CAMHB supplemented with LHB was used to prepare standards and check samples and was used to dilute samples as required. Antibiotic medium 11 (Difco Laboratories, Detroit, Mich.) was seeded with a spore suspension of Bacillus subtilis ATCC 6633 (Difco) and poured into sterile, square petri dishes (245 mm by 245 mm; 20-mm depth; Nalge Nunc International, Rochester, N.Y.). After the agar cooled and hardened, four 1/4-inch paper discs (Schleicher and Schuell, Inc., Keene, N.H.) spotted with 20 l of each standard or sample were placed on the agar in a random, Latin square pattern. The bioassay plates were incubated for 16 to 18 h at 37C in ambient air. Zones of inhibition around the paper discs were measured and concentrations in samples were extrapolated by using the line equation from the standard curve. The limit of sensitivity of the ampicillin assay was 0.5 g/ml, and the assay was linear over the range from 0.5 to 10 g/ml. Intrarun CVs were 5.40% (n 9) and 3.63% (n 9) for the check samples with low (1.0 g/ml) and high (7.5 g/ml) concentrations, respectively. Interrun CVs were 5.7% (n 29) and 5.6% (n 29) for the check samples with low and high concentrations, respectively. Pharmacokinetic and pharmacodynamic analysis. Target human pharmacokinetic parameters were selected prior to initiation of the study. By using actual drug concentration data from each set of experiments, the following parameters were determined for each antibiotic by noncompartmental methods: peak concentration (peak), elimination rate constant, half-life, and area under the curve (AUC). The AUC values were calculated by the trapezoidal method. By using experimental pharmacokinetic and screening MIC data the following pharmacodynamic parameters were determined: peak:MIC ratio, AUC024:MIC ratio (for ciprooxacin and levooxacin), and time above the MIC (for ampicillin). The higher screening MIC was used for these calculations when the MICs were one dilution apart.

RESULTS Susceptibility testing. Table 1 shows the preexperimental MICs of ampicillin, ciprooxacin, and levooxacin against the four clinical isolates utilized in this study. The screening MICs of penicillin for each isolate were as follows: for SP28, 0.06 g/ml; for SP34, 4 g/ml; for SP12, 0.06 g/ml; and for SP4, 0.125 g/ml. Pharmacokinetic analysis. Target pharmacokinetic parameters and experimental pharmacokinetic data are summarized in Table 2. The FQ pharmacokinetic proles observed in the model were similar to that of the target values. In addition, the variation of these proles over the several months required to

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LACY ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Simulated dosing regimens, target human pharmacokinetic parameters, and resultant pharmacokinetic data
Antibiotic Simulated regimen Peak (g/ml) Elimination half-life (h) AUC (g h/ml)e

Ciprooxacin Target parametersa Actual (mean SD) Levooxacin Target parametersb Actual (mean SD) Ampicillin (isolates SP28, SP34, and SP12) Target parametersc Actual (mean SD) Ampicillin (isolate SP4) Target parametersd Actual (mean SD)
a b c

400 mg IV q12h

4.6 4.0 0.2 6.4 5.7 0.4 50 40.9 6.6 25 10.2 0.8

4 4.2 0.4 7 7.0 0.3 1 0.9 0.1 1 1.3 0.2

24 30.2 2.6 54.6 57.7 5.2 60 65.1 20.5 30 32.7 4.2

500 mg IV q24h

1 g IV q6h

500 mg IV q6h

See reference 2. See reference 24. See references 16, 22, 27, and 29. d See reference 16. e AUC is AUC024 for ciprooxacin and levooxacin and AUC06 for ampicillin.

conduct all studies of drug-isolate combinations was minimal. While more variability was observed with the pharmacokinetic data for ampicillin relative to targeted values, the elimination half-life values were very similar and thus produced similar values for time above the MIC, the important correlate related to -lactam activity. We are unsure why the peaks for ampicillin in the SP4 experiments were much lower than the target values. Although unlikely, it is possible that these samples might have been drawn late. In spite of the lower peaks, the resultant AUCs and elimination half-lives were close to the target values. Therefore, since time above the MIC and not magnitude of peak concentrations is the important pharmacodynamic parameter for ampicillin, the effect of the low measured peak values in this set of experiments on the results is not thought to be of extreme importance. Bactericidal activity. The starting inocula were all within one dilution of the target (106 CFU/ml) except for the two cases noted below. Against SP12, the starting inoculum for levooxacin was slightly smaller than projected, and for ampicillin against SP4, a slightly larger starting inoculum was used. However, neither change appeared to have a profound inuence on the bactericidal proles of these agents. Figures 1 and 2 summarize the resultant kill curves for two of the test isolates, SP34 and SP4. Plotted data are the means for the two treatment models and the growth control model for each experiment. Rapid bactericidal effect was observed for ampicillin against all isolates; the concentrations declined to the limit of detection (102 CFU/ml) during the initial 6 h, and regrowth was not observed over the remaining 18 h, of the experiments with ampicillin. Furthermore, this was also evident in the experiment that used 500-mg doses of ampicillin against the isolate with intermediate response to penicillin, SP4. Rapid and sustained bactericidal activity was observed for levooxacin against all isolates except SP4, which showed regrowth at the end of each dosing interval. The rates of kill for levooxacin, as shown by the slopes of the resultant kill curves, were comparable to those of ampicillin against SP34 and SP12. In contrast, the rate and extent of ciprooxacin bactericidal

activity against all isolates tested were related to in vitro susceptibilities over the rst 24 h. However, when experiments were continued for an additional 24-h period, diminished bactericidal activity was noted for subsequent doses and regrowth was evident for all isolates at the conclusion of the 48-h experiment. Detection of resistance. Table 1 shows the MIC values observed throughout the study in the treatment models, indicating that penicillin susceptibility had no impact on FQ susceptibility. Between a 1- to 2- and an 8- to 16-fold increase in ciprooxacin MIC was observed for all experiments, as shown in Table 1. The resultant kill curves clearly reect the observed changes in MIC since regrowth was noted for each isolate. The regrowth was more pronounced during the 24- to 48-h period. Pharmacodynamic analysis. The pharmacodynamic results are summarized in Table 3. All FQ peak to MIC ratios were less than 10. Bacterial regrowth was noted when the FQ AUC: MIC ratios were 28 or less. In the ampicillin experiments, the proportion of the time that the drug concentrations exceeded the MIC for the organism during the dosing interval was less than 50% only for the penicillin-resistant isolate, SP34. However, complete bactericidal activity against this isolate was noted, as shown by the lack of bacterial growth in the 6-h sample after 24-h incubation. DISCUSSION The pharmacodynamics of the FQ are well described. It has previously been shown that these antibiotics display concentration-dependent bactericidal effect (4, 7, 8, 25). A correlation of clinical and microbiologic outcomes to the AUC:MIC ratio indicated that values of 125 or higher were predictive of clinical cures against nosocomial pathogens when ciprooxacin was used in hospitalized patients (11). While peak:MIC ratios of 10:1 or greater appear to be associated with optimal bactericidal activity, the AUC:MIC ratio may better correlate with microbiologic effects when the peak:MIC ratio cannot be optimized (25). The optimal AUC:MIC ratio for FQ against S. pneumoniae has not previously been determined. In this study we showed,

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FIG. 1. Bactericidal activity for SP34. AMP, ampicillin; CIP, ciprooxacin; CTL, control; LEV, levooxacin.

using a wide range of MIC values, that the lower threshold of the AUC:MIC ratio for this pathogen appears to be around 30, as demonstrated by a lack of regrowth when values higher than this were achieved. Raddatz and colleagues evaluated the pharmacodynamics of trovaoxacin and ciprooxacin against four penicillin-resistant isolates over 24 h in an in vitro model

of infection (26). These investigators showed that AUC:MIC values of 187.1 for trovaoxacin resulted in superior bactericidal activity as compared with ciprooxacin, which resulted in regrowth at 24 h for all isolates. Resultant values for ciprooxacin AUC:MIC ratios were either 30.4 or 60.8. However, as only one ciprooxacin dose was administered over the 24-h

FIG. 2. Bactericidal activity for SP4. AMP, ampicillin; CIP, ciprooxacin; CTL, control; LEV, levooxacin.

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LACY ET AL. TABLE 3. Pharmacodynamic analysis

Ciprooxacin Levooxacin Log10 CFU/ml at: 24 h 48 h Peak:MIC ratio AUC:MIC ratio

ANTIMICROB. AGENTS CHEMOTHER.

Ampicillin Log10 CFU/ml at: 24 h 48 h % time above MIC Log10 CFU/ ml at: 24 h 48 h

Isolate

Peak:MIC ratio

AUC:MIC ratio

SP28 SP34 SP12 SP4

4.2 1.9 0.5 1.0

28.4 15.5 3.8 7.7

4.9 1.8 1.5 1.6

0.8 1.2 1.1 2.3

5.2 2.9 3.1 1.4

55.1 29.3 30.9 14.3

4.7 4.2 3.4 3.1

3.8

100 39.2 100 100

5.5 3.7 3.6 3.7

evaluation period in this study, the condition does not simulate actual human pharmacokinetics when dosing q12h is utilized in patients with normal renal function. In this study, the development of resistance to ciprooxacin for all study isolates was apparent, especially after 24 h. The induction of resistance of S. pneumoniae to FQ in vitro has been previously reported (19). After repeated transfer in ciprooxacin, temaoxacin, and noroxacin there was an 8- to 16-fold decrease in susceptibility, while after transfer in levooxacin only minimal decreases in susceptibility were observed. Additionally, active efux has been demonstrated as a mechanism of resistance to ciprooxacin for pneumococcus (34). In our study, the actual mechanism of resistance to ciprooxacin was not evaluated. Our results seem to correlate well with published reports of levooxacin clinical efcacy against S. pneumoniae (9). In summary, we have shown that complete bactericidal activity and decreased regrowth or resistance of pneumococcus was noted when FQ AUC:MIC values were around 30.
ACKNOWLEDGMENTS This work was supported by a grant from Ortho-McNeil Pharmaceuticals. We thank Christina Turley for technical support and Matthew Charnas for development of a diagram of the in vitro model of infection.
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