Journal of Antimicrobial Chemotherapy (1999) 43, 811-816
© 1999 The British Society for Antimicrobial Chemotherapy
Pharmacodynamics of trovafloxacin in a mouse model of cephalosporin-resistant Streptococcus pneumoniae pneumonia
The University of Texas Southwestern Medical Center, Dallas, TX 75235, USA
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Abstract |
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Trovafloxacin is a potentially useful agent for treatment of infections caused by cephalosporin-resistant Streptococcus pneumoniae. We studied the effectiveness of trovafloxacin therapy and examined the correlation between pharmacodynamic indices in serum and lung, and bacterial killing. Immunocompetent Balb/c mice were infected by intranasal inoculation of a cephalosporin-resistant S. pneumoniaeisolate (MIC of ceftriaxone and trovafloxacin 2 and 0.06 mg/L, respectively). Trovafloxacin 10–30 mg/kg/day in one or three divided doses was started 15 h after infection. Serum and lung drug concentrations were measured at multiple time points for 24 h. Serum concentrations peaked at 30–60 min and lung concentrations approximately 30 min later. The serum T1/2 was approximately 9 h and lung T1/2 varied from 5 to 9 h. Lung AUC and Cmax values were 2–3 times greater than those in serum. At the start of therapy lung bacterial concentrations were 8.4 ± 0.3 log10 cfu/mL and 24 h later had decreased by 3.5 ± 0.2, 4.0 ± 0.2, 0.8 ± 0.3 and 1.0 ± 1.2 log10 cfu/mL with 30 mg/kg x 1, 10 mg/kg x 3, 10 mg/kg x 1 and 3.3 mg/kg x 3 regimens, respectively. Although the larger dosages were more effective (P < 0.001) the differences between divided and single dosage regimens were not significant. Trovafloxacin serum AUC/MIC ratio correlated best with bacterial killing in the lungs over 24 h. Trovafloxacin is likely to be useful in the treatment of cephalosporin-resistant S. pneumoniae pneumonia.
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Introduction |
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Pneumococcal pneumonia continues to be an important cause of community-acquired pneumonia. Athough ß- lactam antibiotics have been the mainstay of treatment for this pathogen their effectiveness could be compromised by the increasing world-wide resistance of Streptococcus pneumoniaeto the penicillins and cephalosporins. 1,2,3 The third generation cephalosporins, cefotaxime and ceftriaxone, probably remain effective in most cases of cephalosporin-resistant (according to NCCLS guidelines) S. pneumoniae pneumonia. However, bacteriological failure of pneumococcal pneumonia treated with intravenous cefotaxime has been reported. 4 Appropriate antibiotic treatment for cephalosporin-resistant S. pneumoniae pneumonia has not yet been defined. Trovafloxacin, unlike the older quinolones, demonstrates excellent in-vitro activity against S. pneumoniae (MIC90 range 0.06–0.25 mg/L) and trovafloxacin susceptibility is independent of ß-lactam susceptibility.5 Murine pneumococcal pneumonia models have been used to demonstrate the effectiveness of trovafloxacin against penicillin-susceptible6,7and penicillin-resistant isolates of S. pneumoniae.7,8 However, most previous studies did not report pharmacodynamic indices and none have examined treatment of cephalosporin-resistant S. pneumoniae pneumonia. Also, most previous investigators used immunocompromised animal models.
We therefore developed an immunocompetent murine pneumonia model using a cephalosporin-resistant S. pneumoniae isolate (ceftriaxone MIC 2 mg/L) and determined serum and lung pharmacokinetic profiles of single (10 and 30 mg/kg) versus multiple (3.3 and 10 mg/kg given every 8 h) trovafloxacin dosing regimens over a 24 h period. We examined the correlation between pharmacodynamic indices and bacterial killing in the lungs and compared the effectiveness of trovafloxacin with that of ceftriaxone therapy.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Bacterial isolate
A cephalosporin-resistant isolate of S. pneumoniae from a patient with pneumonia was grown overnight on blood agar. Colonies were removed and placed in endotoxin-free phosphate-buffered saline (PBS). Aliquots of the resultant suspension were frozen at -80°C. For each experiment an aliquot was thawed and S. pneumoniae was grown overnight in Todd–Hewitt broth, washed with endotoxin-free PBS, and resuspended in PBS to achieve bacterial concentrations of approximately 108 cfu/mL. The MIC and MBC of trovafloxacin and ceftriaxone were measured by the microtitre dilution according to standard NCCLS methods9 and MICs were confirmed by Etest.
Pneumonia model
A mouse pneumonia model adapted from that described by Tateda et al. 10 was used. Briefly, 7–10 week-old female Balb/c mice, weighing 19–25 g, were used in all experiments. Animals were sedated with metofane and inoculated intranasally with 50 µL of the bacterial suspension (approximately 106 cfu). This was the smallest bacterial inoculum with which we were able to consistently induce infection. The development of pneumonia was confirmed histologically in lung specimens removed and fixed in 10% formalin 15 h after inoculation of S. pneumoniae.10
Treatment
Antibacterial therapy was started 15 h after inoculation. Trovafloxacin (Pfizer, Groton, CT, USA) was given subcutaneously in one of four dosing regimens; 3.3 or 10 mg/kg 8-hourly and 10 or 30 mg/kg as a single dose. Animals were studied over a 24 h study period. As a comparator ceftriaxone (Roche Laboratories, Nutley, NJ, USA) 50 mg/kg was given 12-hourly over a 24 h period. Dosages of trovafloxacin (3.3 mg/kg x 3, 10 mg/kg x 1 and 10 mg/kg x 3) were chosen to simulate human serum pharmacokinetic values and to give similar Cmax, T1/2 and AUC.11,12,13
Bacterial counts
Four mice were killed at each time point except for the 24 h endpoint when 10 mice per group were used. Blood was collected by cardiac puncture after an intraperitoneal injection of ketamine and acepromazine. Lungs were removed aseptically, washed with sterile water, weighed and homogenized in 0.5 mL of sterile PBS (tissue homogenizer, Omni International Inc., Waterbury, CT, USA). Serial 100-fold dilutions of the lung homogenate were plated on sheep blood agar containing 2.5 mg/L of gentamicin. The lower limit of detection was 100 cfu/mL. Bacterial killing in the lungs was calculated as the change in cfu/mL over the 24 h study period.
Antibiotic assay
Serum and supernatant from lung homogenates were stored at -80°C. Equal volumes of lung and serum specimens obtained at each time point were pooled together in groups of three or four. Trovafloxacin concentrations in serum and lung homogenates were determined by disc diffusion bioassay using Bacillus subtilisATCC 6633. Standard curves for serum determinations were prepared using mouse serum, and were linear in the range 0.1–2.0 mg/L for trovafloxacin. Standard curves for lung concentration determinations were prepared using infected lung homogenate; these were linear for trovafloxacin from 0.2 to 2.0 mg/L. Intra- and interassay coefficients of variance for trovafloxacin were 4.7% and 4.2%, respectively.
Pharmacokinetic and pharmacodynamic analyses
Pharmacokinetic analyses were performed using the computer program TopFit V2 (Gustav Fischer Verlag, Stuttgart, Germany). Serum and lung homogenate concentration- time curves were fitted to a noncompartmental model. The formula 1/y2 was used for weighting. Cmax was defined as the maximum concentration measured. Area under the concentration–time curve (AUC0-24) was calculated using the logarithmic trapezoidal rule.
The relation between pharmacodynamic indices (AUC/ MIC, Cmax/MIC
and T > MIC) in serum and lung and bacteria killing in lungs over 24 h was fitted to
a sigmoid Emax model using the computer program WinNonlin version
1.5. This program uses the formula E = (Emax x C
)/(C + EC
50), where E is the estimated bacteria killing in lungs over 24 h,
Emax is the maximum bacteria killing in lungs in 24 h, C is the
mean serum or lung AUC/MIC or Cmax/MIC ratio, EC50
is the C producing half-maximal bacteria killing in lungs over 24 h and is the
Hill coefficient indicating the slope of the sigmoid curve.
Statistical methods
Continuous variables were expressed as means ±S.D., and differences between treatment groups after 24 h of therapy were evaluated by ANOVA.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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MIC and MBC
The MICs and MBCs of the study antibiotics for S. pneumoniae were: trovafloxacin 0.06 mg/L and 0.125 mg/L; ceftriaxone 2 mg/L and 2 mg/L, respectively.
Serum and lung pharmacokinetics
In the trovafloxacin bioassay standard zone sizes obtained with similar concentrations in lung homogenate were markedly smaller than those obtained in serum. Time-concentration curves of single and multiple dosing regimens for trovafloxacin in serum and lungs are shown in Figure 1. All the dosing regimens of trovafloxacin achieved serum and lung concentrations above the MIC for the entire dosing interval. Pharmacokinetic indices are shown in the Table. Lung concentrations of trovafloxacin were two to three times greater than serum values (Table). The 10 mg/kg x 1 versus 3.3 mg/kg x 3 dosing regimens demonstrated similar AUCs for 24 h, with a lower Cmax in the latter regimen. Similar results were noted in the 30 mg/kg x 3 versus 10 mg/kg x 3 dosing regimens.
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); 10 mg/kg x
3
(•); 10 mg/kg x 1 (
); 3.3 mg/kg x 3 (
). The MIC of the S.
pneumoniae isolate was 0.06 mg/L.Bacterial clearance from lungs
The change in bacterial counts in the lungs over 24 h is illustrated in Figure 2. The mean bacterial count before the initiation of antibiotic therapy was 8.4 ± 0.3 log10 cfu/mL and in untreated control animals this increased to 9.6 x 0.8 log10 cfu/mL by 24 h. The decrease in bacterial counts in the lungs over 24 h were similar for the 3.3 mg/kg x 3 versus 10 mg/kg x 1 dosing regimens and the 10 mg/kg x 3 versus 30 mg/kg x 1 dosing regimens. However, the lower dosage regimens were less effective (P < 0.001, ANOVA). Bacterial killing with ceftriaxone therapy was similar to that with trovafloxacin 10 mg/kg x 3 or 30 mg/kg x 1 (P = 0.3, ANOVA).
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Pharmacodynamic analysis
The only pharmacokinetic index that could be fitted to the sigmoid Emax model was serum AUC/MIC ratio, r2 = 0.98 (Figure 3).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Trovafloxacin therapy was rapidly bactericidal in an immunocompetent murine pneumococcal pneumonia model infected with a cephalosporin-resistant S. pneumoniae. Trovafloxacin reduced bacterial counts in the lungs by approximately 104 cfu/mL (99.99% kill) in the 24 h study period. Lung cultures were not sterile at 24 h probably because of the very high counts present at initiation of therapy. A high inoculum was necessary to induce infection consistently.
We found that single and divided doses of trovafloxacin were similarly effective, suggesting that the efficacy of trovafloxacin is better predicted by AUC/MIC ratios rather than C max/MIC ratios. Moreover, only serum AUC/MIC could be fitted to the sigmoid Emax model. We found that trovafloxacin effectiveness was related to AUC/MIC ratios in the presence of high serum Cmax/MIC ratios (22/1 to 119/1). The strong relationship of AUC/MIC ratios with the effectiveness of quinolone therapy has been found by some investigators but not others.14,15,16,17,18 When treating infections where there is a significant risk of the emergence of resistant subpopulations, both in-vitro and in-vivo studies have shown that it is necessary to achieve a Cmax/MIC ratio of 8–10.14,19,20 In our study all four trovafloxacin regimens achieved serum and lung concentrations above the MIC for the whole study period, demonstrating that T > MIC was not a good predictor of efficacy. Trovafloxacin T1/2 in both the serum and lungs appeared to lengthen with the larger single dose of 30 mg/kg versus 10 mg/kg. This has been observed also in human studies.13
Trovafloxacin binds avidly to serum proteins; binding is concentration independent and averages 92, 75, 66 and 70% for rats, dogs, monkeys and humans, respectively, 13,21 but values for the mouse are not available. Despite the high protein binding in serum, concentrations of trovafloxacin in lung tissue were much higher than those in serum probably because trovafloxacin is concentrated within cells such as macrophages and leucocytes. 7,22 Bedos et al.7 found that immunocompetent mice had significantly higher PK/PD values than did immunosuppressed mice, mainly in the lungs. High intracellular accumulation was indicated in our study by small zone sizes obtained when trovafloxacin standards were prepared in lung homogenate. Thus, the amount of biologically active drug against extracellular organisms may actually be less than that measured in tissue homogenates because lung tissue homogenate contains about 80% intracellular fluid and 20% extracellular fluid derived from interstitial and intravascular fluid. In pneumococcal pneumonia, where the organisms are found extracellularly, interstitial fluid rather than tissue homogenate drug concentrations should be the best predictor of efficacy.
In conclusion, trovafloxacin was effective in the treatment of pneumonia caused by a cephalosporin-resistant S. pneumoniae isolate. The pharmacokinetic measure that correlated best with lung bacterial killing was the serum AUC/MIC ratio. Single daily doses were as effective as divided doses. Trovafloxacin could be considered as a useful alternative to ceftriaxone for treatment of cephalosporin-resistant S. pneumoniae pneumonia.
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Notes |
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* Corresponding address. Department of Paediatrics, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore 229899. Tel: +65-293-7933; Fax: +65-394-1043
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Received 4 September 1998; returned 27 December 1998; revised 5 February 1999; accepted 5 March 1999
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