INTRODUCTION

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Considerable controversy exists regarding the most appropriate way to administer antibiotics to maximize bacterial killing and minimize toxicity. In the case of the fluoroquinolones, debate exists as to not only the best pharmacodynamic parameter (i.e., peak serum drug concentration/MIC [peak/MIC] ratio or area under the curve [AUC]/MIC ratio) to characterize bactericidal activity but also the magnitude of the required interaction to optimize therapeutic outcome (10, 13).

The current study was undertaken to investigate the pharmacodynamic relationship between gatifloxacin exposure and outcome as assessed by bacterial density and survival in a murine thigh model of pneumococcal infection. Additionally, the influence of the host on these outcomes was assessed in an immunocompetent model.

	MATERIALS AND METHODS

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Antimicrobial test agents. Gatifloxacin analytical grade standard was obtained for in vitro testing from Bristol-Myers Squibb, Princeton, N.J. For all in vivo studies, laboratory standard grade gatifloxacin (lot no. 9D07329; expiration date, Sept. 2000) was obtained from the manufacturer, reconstituted based on weight, and administered via oral gavage, using a feeding tube.

Bacterial isolates and susceptibilities. Eight clinical isolates of Streptococcus pneumoniae were included in this study. Organisms were selected to represent a range of susceptibilities to gatifloxacin, thus allowing the opportunity for pharmacodynamic modeling over a larger range for the parameters of AUC/MIC ratio, peak/MIC ratio, and duration of time that serum drug concentration remained above the MIC (T > MIC). The MIC of gatifloxacin was determined in duplicate using the microdilution method according to NCCLS guidelines (9). The MICs were determined in cation-adjusted Mueller-Hinton broth (20 to 25 mg of calcium/liter, 10 to 12.5 mg of magnesium/liter) with 5% lysed horse blood in ambient air. Trypticase soy agar with 5% sheep blood was used as the growth medium.

Protein binding studies. The protein binding studies were done in triplicate using an ultrafiltration method. An aqueous stock solution of gatifloxacin was prepared, and dilutions were made in fresh ICR mouse serum to yield final concentrations of 1, 10, and 100 µg/ml. All spiked serum samples were placed in a 37°C shaking water bath for 10 min. Then, 0.9-ml aliguots of the serum samples were transferred to Centrifree Micropartition devices (Millipore, Bedford, Mass.; 30,000 molecular weight cut-off filter). These filters were spun at 1,000 × g at 10°C for 15 min. Protein binding studies were conducted in triplicate at each concentration. Concentrations of gatifloxacin in both initial serum solutions and filtrates were determined by a validated high-performance liquid chromatography (HPLC) method.

Thigh infection model. Specific-pathogen-free ICR mice weighing approximately 25 g were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.). Mice were rendered transiently neutropenic by injecting cyclophosphamide intraperitoneally (i.p.) on days 4 (150 mg/kg of body weight) and 1 (100 mg/kg) before treatment began. This regimen has been shown to induce neutropenia in the animals for 5 days (1, 5, 12). In addition, renal impairment was produced by a single i.p. injection of uranyl nitrate on day 3 prior to the initiation of antimicrobial therapy (1, 12).

Pharmacokinetic studies. Studies were undertaken to determine the pharmacokinetic profile of gatifloxacin in infected ICR mice. The mice received i.p. injections of cyclophosphamide, as described above. Three days prior to the pharmacokinetic study, ICR mice received a single i.p. injection of 1, 2.5, or 5 mg of uranyl nitrate/kg, resulting in various degrees of renal impairment. Mice were administered a single oral gatifloxacin dose of 10, 25, or 40 mg/kg, utilizing a method previously described. Animals were euthanatized by CO₂ exposure followed by cervical dislocation, and blood was obtained through intracardiac puncture in groups containing three to five mice at 0.25, 0.5, 1, 2, 4, 6, 12, and 24 h following drug administration. The blood was centrifuged at 10,000 × g for 10 min; the serum was transferred into a polypropylene tube and stored at 80°C until analyzed. Abbreviated pharmacokinetic studies were completed using the CBA/J species with gatifloxacin (25 mg/kg) and uranyl nitrate (2.5 mg/kg).

Efficacy as assessed by bacterial density. Six S. pneumoniae isolates were used in these studies: 53, 84, 59, 21, 77, and 75. Once the animals had been prepared as above and inoculated, treatment with gatifloxacin was initiated at 2 h post-thigh infection in a method previously described. The concentration and treatment regimen of gatifloxacin as well as the concentration of uranyl nitrate were varied to simulate a range of drug exposures. Final dosage range of gatifloxacin was varied from 10 to 80 mg/kg every 6 to 24 h. Control animals received water orally at the same volume and schedule as gatifloxacin-treated animals. Groups of untreated control mice were sacrificed at the initiation of therapy and after 24 h. Treated mice were sacrificed after 24 h of therapy.

Efficacy as assessed by survival. Six S. pneumoniae isolates were used in these studies: 59, 84, 21, 77, 85, and 63. Groups of mice were similarly infected with each test strain for evaluation of survival during 96 h of therapy. Gatifloxacin therapy was initiated at a time corresponding to 2 h post-thigh inoculations. Similarly to density studies, gatifloxacin was administered in various regimens, from 10 to 80 mg/kg every 6 to 24 h for 96 h of therapy. Control animals received water orally at the same volume and schedule as gatifloxacin. The cumulative mortality was calculated during 96 h of therapy. Although death has historically been used as an end point for studies of this type, this end point is no longer suitable in the current era of animal research. Therefore, our study methodology has been modified to contemporary standards, which have been recently employed in studies conducted at our institution (4). The term mortality has been used as an end point for this study; however, it should be clearly understood that when possible every attempt was made to minimize pain and suffering of the animals. Animals were euthanatized prior to naturally succumbing to infection if symptoms of impending death were observed, including substantial alterations in posture (e.g., abnormal posture or head tucked into abdomen), coat, exudate around eyes and/or nose, and breathing or movement. For the purposes of this study, whether an animal died due to the natural process or was euthanatized, both were considered the same end point for experimental and statistical purposes.

Data analysis. Spearman's rank correlation coefficient was used to evaluate the relationship between mortality and T > MIC, the peak/MIC ratio, and the AUC/MIC ratio, for gatifloxacin. This test was also used to evaluate the relationship between change in CFU after 24 h of therapy and the three pharmacodynamic parameters listed above. Additionally, the goodness of fit for each of these relationships was characterized and evaluated using the sigmoid E_max model (WinNonlin Pro, version 3.0; Pharsight Corporation).

	RESULTS

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Eight clinical isolates of S. pneumoniae were utilized; the MICs of gatifloxacin for these isolates are displayed in Table 1.

Protein binding and pharmacokinetic studies. The percent protein bound (mean ± standard deviation) for ICR mice measured at concentrations of 1, 10, and 100 µg/ml was 22.5% ± 1.8%, 26.6% ± 5.3%, and 17.2% ± 0.7%, respectively. An abbreviated study was initiated for the CBA/J strain, and a concentration of 10 µg/ml demonstrated 11.1% ± 2.2% protein binding, which was less than half that observed with the ICR mice. Published data on human protein binding in serum in volunteers indicate results of approximately 20% binding (Tequin [gatifloxacin] tablets/injection prescribing information, doc. no. E5-A002, Bristol-Myers Squibb, Dec. 1999).

Bacterial density studies. ICR mice received 0.1 ml of a 10⁵ to 10⁷ CFU/ml suspension of S. pneumoniae in each thigh. Time zero cultures at 2 h post-thigh inoculation (prior to initiation of therapy) revealed good bacterial recovery from tissue, as the median log CFU was 5.08 (range, 4.04 to 5.67). Organisms also grew well in untreated control animals over the 24-h study period, as the median log change in CFU for control animals was 2.21 (range, 0.4 to 3.03) (Fig. 1). Isolate 84 was utilized in two experimental runs using differing gatifloxacin exposures, and growth controls were similar in each run, demonstrating reproducibility of growth kinetics within the strain (Fig. 1).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Growth of S. pneumoniae in the thighs of infected mice not receiving gatifloxacin (controls). Values represent means ± standard deviations. *, isolate 84 was utilized twice.

View larger version (8K): [in this window] [in a new window]   FIG. 2.   Decrease in log CFU at 24 h versus AUC/MIC ratio for neutropenic (ICR) mice. Each data point represents the mean of six to eight thighs. r2 = 0.8647.

View larger version (8K): [in this window] [in a new window]   FIG. 3.   Decrease in log CFU at 24 h versus percent T > MIC of neutropenic (ICR) mice. Each data point represents the mean of six to eight thighs. r2 = 0.8445.

View larger version (8K): [in this window] [in a new window]   FIG. 4.   Decrease in log CFU at 24 h versus peak/MIC ratio for neutropenic (ICR) mice. Each data point represents the mean of six to eight thighs. r2 = 0.6428.

View larger version (30K): [in this window] [in a new window]   FIG. 5.   Comparison of ICR (neutropenic) and CBA/J (nonneutropenic) species for the relationship between log change CFU and AUC/MIC ratio. Each group contains data from eight thighs. Data were derived using one isolate, 21.

Survival studies. Overall mortality in untreated control mice injected with the pneumococcal isolates was 97%. As noted for bacterial density, a significant correlation (P  0.001) was noted for the relationships between percent survival and AUC/MIC ratio (r² = 0.9640); percent survival and peak/MIC ratio (r² = 0.9265); and percent survival versus percent T > MIC (r² = 0.9199) for gatifloxacin. Although all three pharmacodynamic parameters examined were correlated, the strongest correlation was found to exist between the percent survival and AUC/MIC ratio (Fig. 6). In the neutropenic model, the maximum effect on survival appeared to occur at AUC/MIC ratios between 30 and 40. 

View larger version (8K): [in this window] [in a new window]   FIG. 6.   Percent survival versus AUC/MIC ratio for neutropenic (ICR) mice. Each data point represents 10 to 15 mice. r2 = 0.9640.

	DISCUSSION

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Since their introduction in clinical practice, numerous studies have investigated the most appropriate way to administer fluoroquinolone antibiotics to maximize bacterial killing and minimize toxicity. Currently available pharmacodynamic data on this class of compounds suggest that the AUC/MIC ratio is the best predictor of success. However, while this pharmacodynamic parameter is generally accepted, considerable controversy still exists as to the required magnitude of exposure to obtain maximum effectiveness (2, 3, 10, 13, 15).

In the current study, we evaluated the pharmacodynamic relationship between gatifloxacin exposure and outcome as assessed by the change in bacterial density and survival in the pneumococcal thigh infection model over 24 and 96 h of therapy, respectively. S. pneumoniae isolates selected for study included a range of susceptibilities to gatifloxacin and, when combined with the dosage regimens utilized, provided a study strategy which enabled a broad view of the compound's dynamic profile. As demonstrated in Fig. 1, differences in the growth control are inherent in in vivo studies, due to natural variations between isolates, and similar growth differences have been published previously (11). Results obtained through analyses of these current gatifloxacin data reveal that all the pharmacodynamic parameters were correlated; however, the AUC/MIC ratio was the best predictor of success for both outcome parameters studied. Similar results demonstrating multiple correlates, with the strongest correlation associated with the AUC/MIC ratio, have been previously reported (3).

Additionally, our results are in accordance with previously reported in vivo findings which suggested the appropriateness of gatifloxacin AUC/MIC ratios as a dynamic predictor of response using a similar neutropenic infection model (D. Andes and W. A. Craig, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. P-0191, 1999). While the eight pneumococcal isolates utilized in this study were different from those used in the previous report, the 24-h AUC/MIC ratio exposures required to produce both a static and bactericidal effect in the infected thighs were similar. In our study gatifloxacin AUC/MIC ratios of approximately 40 appeared to optimize the dynamic response, as judged by the bactericidal activity of the compound within infected thighs.

Our in vivo findings reflect those obtained in recent in vitro models which have evaluated the influence of drug exposure on both the kill and regrowth profiles of pneumococci (6-8). Each of these in vitro studies determined that an AUC/MIC ratio between 30 and 64 for pneumococci was sufficient to result in rapid and sustained bactericidal activity.

We have also demonstrated a similar significant correlation between data obtained from the survival studies and AUC/MIC exposures. Using this outcome parameter it appears that AUC/MIC ratios between approximately 30 and 40 produce maximum survival in immunocompromised hosts infected with lethal inocula of S. pneumoniae.

In the current study, a comparison of bacterial density determinations between the immunocompromised (ICR mice) and the immunocompetent (CBA/J mice) host using the same pneumococcal strain demonstrates greater treatment efficacy in the immunocompetent species. As might be expected, the most notable contribution of the host defenses was observed when static doses (i.e., AUC/MIC ratios of 15 to 20) were administered. Similar to improved bactericidal activity of gatifloxacin in the presence of functional white blood cells, the percent survival for animals infected with a lethal inoculum was 100% despite AUC/MIC ratios of less than 10. While a slight increase in free drug was noted for the CBA/J mice (90%, versus 80% for ICR species) during the protein binding studies, this profound difference in observed survival is most likely related to the presence of white blood cells, since it is doubtful that this relatively small difference in free drug would account for such dramatic changes in treatment outcome.

In conclusion, our data demonstrate the appropriateness of the gatifloxacin AUC/MIC ratio as a dynamic predictor of response to lethal pneumococcal disease and show that AUC/MIC ratios of at least 40 appear to optimize the dynamic response to infection. Lastly, these data further display the importance of the immune status of the host and the need for maximal effective in vivo drug exposures when these systems are compromised.