Prediction of the antimicrobial effects of trovafloxacin and ciprofloxacin on staphylococci using an in-vitro dynamic model

Alexander A. Firsova,*, Raif G. Vasilova, Sergey N. Vostrova, Olga V. Kononenkoa, Irene Yu. Lubenkoa and Stephen H. Zinnerb

a Department of Pharmacokinetics, Centre of Science &Technology LekBioTech, 8 Nauchny proezd, Moscow 117246, Russia; b Division of Infectious Diseases, Roger Williams Medical Center, Rhode Island Hospital, Brown University, Providence, RI, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To compare the pharmacodynamics of trovafloxacin and ciprofloxacin, three clinical isolates of Staphylococcus aureus with different MICs (0.03, 0.15, 0.6 and 0.1, 0.25, 1.25 mg/L, respectively) were exposed to decreasing concentrations of the quinolones according to their half-lives of 9.25 and 4 h, respectively. With each organism, single doses of trovafloxacin and twice-daily doses of ciprofloxacin were designed to provide 8-fold ranges of the ratio of area under the concentration—time curve (AUC) to the MIC, 58—466 and 116—932 (mg·h/L)/(mg/L), respectively. The antimicrobial effect was expressed by its intensity: the area between the control growth in the absence of antibiotics and the antibiotic-induced time—kill/regrowth curves (I E). Linear relationships established between I E and log AUC/MIC were bacterial strain-independent but specific for the quinolones (r 2= 0.99 in both cases). At a given AUC/MIC ratio, the I Es of trovafloxacin were greater than those of ciprofloxacin, suggesting that the antimicrobial effect of trovafloxacin compared with ciprofloxacin against staphylococci may be even greater than might be expected from the difference in their MICs. These data were combined with previous results obtained with three Gram-negative bacteria. Again, I E correlated well with the log AUC/MIC of trovafloxacin and ciprofloxacin in a strain- and species-independent fashion (r 2= 0.94 and 0.96, respectively). On this basis, a value of the AUC/MIC of trovafloxacin which might be equivalent to Schentag's s AUC/MIC = 125 (mg·h/L)/(mg/L) reported as the breakpoint value for ciprofloxacin was estimated at 71 (mg·h/L)/(mg/L) with the respective MIC breakpoint of 0.27 mg/L. Based on the I E— log AUC/MIC relationships, the I Es were plotted against the logarithm of trovafloxacin and ciprofloxacin dose (D) for hypothetical representatives of S. aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa with MICs corresponding to the MIC 50s. These I E log D relationships allow prediction of the effect of a given quinolone on a representative strain of the bacterial species.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the presumed advantages of in-vitro dynamic models is the ability to compare pharmacokinetically different antimicrobials. However, most such comparisons have not been valid either because of inappropriate experimental design (one-dose levels, simulating noncomparable concentration:MIC ratios, insufficient observation periods, etc.) and/or incorrect quantification of the antimicrobial effect. We recently described a new approach to the in-vitro comparison of fluoroquinolones based on the analysis of relationships between intensity of the antimicrobial effect (I E, the area between control growth and bacterial killing/regrowth curves 1) and the ratio of the area under the concentration- time curve (AUC) to the MIC as established over a wide range of AUC/MICs. 2 Based on the relationships between I E and log AUC/MIC, which were bacterial species- independent but quinolone-specific, greater effects of trovafloxacin on Gram-negative bacteria than those of ciprofloxacin were demonstrated at each of the AUC/MIC ratios studied. The predicted single-dose of trovafloxacin which might be of similar efficiency to two 500 mg doses of ciprofloxacin averaged 209 mg and it appeared to be consistent with the clinically established daily dose of the new quinolone (200 mg). 3,4,5 This estimated dose of trovafloxacin was assumed to be at least as efficient as ciprofloxacin against Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa 2despite the findings that these organisms are less susceptible to trovafloxacin. 6,7,8,9,10,11,12

A similar approach was applied in the present study to compare the antimicrobial effects of trovafloxacin and ciprofloxacin on Staphylococcus aureus which are more susceptible to trovafloxacin than to ciprofloxacin. 6,7,8,9,11,13 For this reason this study was not designed to propose a specific lower dose of trovafloxacin targeted at S. aureus (which might be insufficient to treat Gram-negative infections). The intention was to establish the quinolone doses (D 223) that provide the same I E (average 223 (log cfu/mL) x h) that was considered acceptable at 209 mg of trovafloxacin or 2 x 500 mg of ciprofloxacin against Gram-negative bacteria. 2 Other objectives included an examination of whether the relationships between I E and log AUC/MIC are bacterial strain-independent (as was shown earlier with Gram-negative strains 14) and the prediction of the AUC/ MIC and MIC breakpoints of trovafloxacin. Since our previous study 14 did not confirm the hypothesis that relationships between the antimicrobial effect and AUC/ MIC were independent on the specific quinolone, 15 the present experiments were designed to provide comparable antimicrobial effects (IEs) rather than the same AUC/ MICs. Therefore, the range of the simulated AUC/MIC ratios for ciprofloxacin was shifted towards higher AUC/ MICs relative to the corresponding range for trovafloxacin.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial agents and bacterial strains

Trovafloxacin mesylate and ciprofloxacin lactate powders (kindly provided by Pfizer, Inc. (Groton, CT, USA)and by Bayer Pharmaceuticals (West Haven, CT, USA)) were used in the study. Clinical isolates of S. aureus, one methicillin-susceptible (MSSA, S. aureus 48) and two methicillin- resistant (MRSA, S. aureus 944 and 916) were used in the study. The MICs of trovafloxacin for these organisms at an inoculum size of 10 6 cfu/mL, i.e. 0.03, 0.15 and 0.60 mg/L, respectively, were approximately half the corresponding MICs of ciprofloxacin (0.10, 0.25 and 1.25 mg/L).

In-vitro dynamic model and simulated pharmacokinetic profiles

A previously described dynamic model 1 was used in the study. The operation procedure, reliability of simulations of the quinolone pharmacokinetic profiles and the high reproducibility of the time- kill curves provided by the model have been reported elsewhere. 14 A series of monoexponential profiles that mimic single-dose administration of trovafloxacin and twice-daily dosing of ciprofloxacin were simulated. The simulated half-lives (9.25 h for trovafloxacin and 4.0 h for ciprofloxacin) were consistent with values reported in human volunteers: 7.2—9.9 h 16,17 and 3.2—5.0 h, 18,19,20 respectively. The four simulated AUC/MIC ratios for trovafloxacin and ciprofloxacin were 58, 116, 233 and 466, and 116, 233, 466 and 932 (mg·h/L)/(mg/L), respectively. With ciprofloxacin, the designed AUC/MICs reflect the sum of two AUC/MICs provided by the two doses of the quinolone administered at 12 h intervals taking into account the residual concentrations at the end of the first interval. The respective range of the simulated peak concentration:MIC ratios for trovafloxacin was 4—32 and that for ciprofloxacin was 10—80.

Quantification of the antimicrobial effect and its AUC/MIC and dose relationships

The procedure for quantifying viable counts and the antimicrobial effect by the I E parameter 1 has been reported elsewhere. 2,14 The I E versus log AUC/MIC data sets obtained with each quinolone against S. aureus in this study and S. aureus, E. coli, K. pneumoniae and P. aeruginos{alpha} (combined data from this and a previous study 2) were described by the equation


   (1)

To express the antimicrobial effects as a function of quinolone dose (D), AUC in a linear relationship between I E and log AUC that corresponds to equation (1) written for a given quinolone- pathogen pair was substituted by D according to the polynomial equation


   (2)

where c, d, and e for trovafloxacin are equal to -0.01, 7.5 x 10 -2 and 9.6 x 10 -5, and those for ciprofloxacin are equal to 0.10, 1.4 x 10 -2 and 7.5 x 10 -6, respectively, as calcu-lated based on the quinolone's s pharmacokinetic data in humans. 16,18

To generalize the results of the comparison of the effects of trovafloxacin and ciprofloxacin in terms of the I E-log {Delta} relationships, such relationships were constructed not only for the strains studied but also for hypothetical strains of S. aureus, E. coli, K. pneumoniae, and P. aeruginos whose MICs corresponded to reported MIC 50s for these organisms. The MIC 50s of trovafloxacin and ciprofloxacin were calculated as weighted geometric means of the values reported elsewhere. 6,7,8,9,10,11,12,13 Since the MIC 50s for MRSA reported in one study 8 differed substantially from the estimates reported in five other studies, 6,7,9,11,13 only MIC 50s for MSSA 8 were considered. The weighted geometric means of the MIC 50s of trovafloxacin for S. aureus, E. coli, K. pneumoniae and P. aeruginos were 0.05, 0.03, 0.09 and 0.72 mg/L and those of ciprofloxacin were 0.52, 0.01, 0.03 and 0.31 mg/L, respectively.

Correlation and regression analyses of the relationships between I E and log AUC/MIC for each quinolone were performed at the level of significance of P = 0.05.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Quinolone pharmacodynamics with staphylococci

The time courses of killing and regrowth of the three strains of S. aureus exposed to trovafloxacin and ciprofloxacin yielded similar patterns (Figure 1). The regrowth followed a rapid and considerable reduction in bacterial numbers and its appearance was distinctly dependent on the simulated AUC/MIC: the higher the AUC/MIC, the later the regrowth. For all three bacterial strains exposed to trovafloxacin at a given simulated AUC/MIC ratio, bacterial regrowth was observed later than with ciprofloxacin, except at 116 (mg·h/L)/(mg/L).



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Figure 1. The kinetics of killing and regrowth of S. aureus 48 ({blacktriangleup}, {triangleup}), 944 ({blacksquare}, 2{square}) and 916 ({blacktriangledown}, {triangledown}) exposed to (a) trovafloxacin and (b) ciprofloxacin. The simulated AUC/MIC (in (mg·h/L)/(mg/L)) is indicated by the number above each curve.

 
The I E—log AUC/MIC plots were linear, virtually superimposed for all strains studied but specific for each quinolone (Figure 2). The I E—log AUC/MIC plots fitted by equation (1) differed in both position and slope: that with trovafloxacin was 1.9-fold higher than that with ciprofloxacin. At the same AUC/MIC ratio (116, 233 or 466 (mg·h/L)/(mg/L)), the antimicrobial effect of trovafloxacin was greater than that of ciprofloxacin. For example, at an AUC/MIC ratio of 125 (mg·h/L)/(mg/L), which has been considered to be a significant breakpoint for predicting acceptable clinical outcome, 21 the I E of trovafloxacin was 26% higher than that of ciprofloxacin. The proposed equivalent AUC/MIC breakpoint for trovafloxacin corresponding to the AUC/MIC breakpoint for ciprofloxacin was 79 (mg·h/L)/(mg/L).



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Figure 2. AUC/MIC-dependent antimicrobial effects of trovafloxacin (— , open symbols) and ciprofloxacin (- - - , filled symbols) on S. aureus 48 ({blacktriangleup}, {triangleup}), 944 ({blacksquare}, {square}) and 916 ({blacktriangledown}, {triangledown}) (left panel) and on S. aureus (symbols as for left panel), E. coli (, {diamond}), K. pneumoniae (•, {circ}) and P. aeruginosa(+,) (right panel). Predicted values of the AUC/MIC of trovafloxacin which are equivalent to the AUC/MIC breakpoint for ciprofloxacin at IE = 223 (left panel) and 198 log cfu/mL x h (right panel) are indicated by the bold numbers.

 
By combining equations (1) and (2) with a and b values specific for each quinolone, the relationships between I E and D were determined. As seen in Figure 3, the slopes and positions of the I E—log D plots are both quite different for the two quinolones. With the most susceptible, S. aureus 48, even a dose of 40 mg of trovafloxacin may produce an ` acceptable' I E (223 (log cfu/mL) x h), 2 which might be provided by two 435 mg doses of ciprofloxacin. With the intermediately susceptible S. aureus 944, such an effect might be produced by 170 mg of trovafloxacin and 2 x 950 mg doses of ciprofloxacin, whereas with the least susceptible, S. aureus 916, the required doses would be 490 and 2 x 2400 mg, respectively.



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Figure 3. Dose-dependent antimicrobial effects of trovafloxacin (— ) and ciprofloxacin (- - - ) on S. aureus. The doses providing IE = 223 (log cfu/mL) x h are indicated by the bold numerals.

 
Quinolone pharmacodynamics with different bacterial species

The combined data sets for S. aureus (this study) and the three Gram-negative organisms 2 were properly fitted by equation (1) for each quinolone (Figure 2). As seen in the figure, I E correlated well with log AUC/MIC of trovafloxacin and ciprofloxacin: in both cases r2 exceed 0.9. By comparing the I E—log AUC/MIC plots, an equivalent AUC/MIC value of trovafloxacin which corresponds to an AUC/MIC of 125 (mg·h/L)/(mg/L) and gives an I E of 198 (log cfu/mL) x h, was estimated at 71 (mg·h/L)/(mg/L). This estimated value might be proposed as an equivalent AUC/MIC breakpoint that in turn might be used to predict the MIC breakpoint of trovafloxacin. From equation (2), a clinically accepted dose of trovafloxacin, namely 200 mg, would give an AUC of 18.9 mg·h/L. The MIC breakpoint is thus equal to 18.9/71 = 0.27 mg/L. The corresponding value for a ciprofloxacin dose of 2 x 500 mg, estimated using equation (2), is lower: 22/125 = 0.18 mg/L.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This comparative study demonstrates that trovafloxacin has greater antimicrobial effects on S. aureus than ciprofloxacin over a wide range of in-vitro simulated AUC/MIC ratios. At a given AUC/MIC, the regrowth of S. aureus exposed to trovafloxacin was observed later than with ciprofloxacin, despite similar rates of initial killing and minimal numbers of surviving bacteria. These results are consistent with the recently reported important role of bacterial regrowth in discriminating the effects produced by different AUC/MICs and quinolones on E. coli, K. pneumoniae and P. aeruginos. 14 As in our previous study, linear relationships between I E and log AUC/MIC were shown to be independent of the bacterial strain but specific for each quinolone. As with the Gram-negative organisms, 2 the I E- log AUC/MIC plot showing the effects of trovafloxacin on staphylococci was positioned higher and was steeper than that for ciprofloxacin. Moreover, the combined data sets on the three Gram-positive (this study) and three Gram-negative organisms 2 were properly fitted by equation (1) for each quinolone. Hence, at a given AUC/MIC ratio trovafloxacin might be used more effectively against Gram-positive bacteria than ciprofloxacin at the same AUC/MIC. Furthermore, the critical value of the AUC/ MIC for trovafloxacin was substantially lower than the equivalent significant breakpoint for ciprofloxacin, 21 i.e. 71 vs 125 (mg·h/L)/(mg/L).

The findings obtained in this and the previous study 2 with specific bacterial strains may also be used to predict the antimicrobial effects of trovafloxacin and ciprofloxacin on hypothetical strains of S. aureus E. coli, K. pneumoniae and P. aeruginos with MICs equal to the respective reported MIC 50s. Due to the bacterial species- and strain-independent relationships between I E and AUC/MIC, equation (1) may be applied to any strain, including those with MICs equal to MIC 50, MIC 90 or the geometric mean of MICs. For example, when the MIC is equal to MIC 50, equation (1) may be rearranged as follows:


   (3)

where á = a-b log MIC 50. By using equation (3) and the estimates of a and b presented in Figure 2, the I E-log AUC plots were reconstructed for trovafloxacin and for ciprofloxacin to account for the MIC 50s specific for each of the four bacterial species. In this case, I E = 198 (log cfu/mL) x h at AUC/MIC = 71 (trovafloxacin) or 125 (ciprofloxacin) (mg·h/L)/(mg/L) was used as a reference level of an ` acceptable' antimicrobial effect. The MIC 50-adjusted plots of dose-dependent I Es for the two quinolones and four bacterial species are shown in Figure 4.



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Figure 4. Dose-dependent antimicrobial effects of trovafloxacin (— ) and ciprofloxacin (- - - ) on hypothetical strains of S. aureus, E. coli, K. pneumoniae and P. aeruginosa with MICs which are equal to the respective MIC 50s. The doses giving an I E of 198 (log cfu/mL) x h are indicated by the bold numerals.

 
As seen in the figure, the I E-log D plots for trovafloxacin were positioned to the left of those for ciprofloxacin, showing that the same antimicrobial effect (I E = 198 (log cfu/mL) x h) against each organism might be provided by much lower absolute doses of the new quinolone. The I E-log D plot that reflects the antimicrobial effect of trovafloxacin on a hypothetical strain of S. aureus (MIC 50 = 0.05 mg/L) is shifted from the corresponding plot for ciprofloxacin (MIC 50 = 0.52 mg/L) much more than any plot shown in Figure 3. This results in striking contrasts between doses of trovafloxacin and ciprofloxacin that would be necessary to produce the same antimicrobial effect: the predicted D 198s of equal efficiency are almost 50-fold different: 45 vs 2 x 1070 mg, respectively. On the other hand, a 200 mg dose of trovafloxacin is able to provide much greater effect (390 (log cfu/mL) x h) than the effect that might be produced by the highest clinically relevant dose of ciprofloxacin (1500 = 2 x 750 mg): 163 (log cfu/mL) x h. Thus, while the clinically accepted 200 mg dose of trovafloxacin might be far in excess of what is needed to treat susceptible staphylococcal infections, 2 x 500 mg and even 2 x 750 mg doses of ciprofloxacin might not be sufficiently efficient. Less pronounced differences between the I E-log D plots for the two quinolones were inherent in E. coli, K. pneumoniae and P. aeruginosa. The quinolone doses which are necessary to efficiently suppress the growth of E. coli (27 and 74 mg, respectively) are much lower than a 200 mg dose of trovafloxacin or 2 x 500 mg dose of ciprofloxacin. The same clinically accepted doses of both quinolones also might be highly effective against K. pneumoniae but they are not sufficiently effective against P. aeruginosa, although in the latter case the highest dose of ciprofloxacin (2 x 750 mg) does produce an ` acceptable' I E = 198 (log cfu/mL) x h.

Such an analysis exposes quinolones to the most rigorous conditions since many clinical isolates may in fact be more susceptible than those with MICs equal to MIC 50s. For example, a ciprofloxacin dose <500 mg (2 x 435 mg) was able to produce an ` acceptable' antimicrobial effect on S. aureus 48 (MIC = 0.1 mg/L), and a 2 x 950 mg dose (which is slightly higher than the maximal clinically used oral dose) was sufficient to provide the effect on S. aureus 944 (MIC = 0.25 mg/L) (Figure 3). Similarly, a 200 mg dose of trovafloxacin simulated in our previous study 2 was not less efficient than a 2 x 500 mg dose of ciprofloxacin against P. aeruginosa with MICs of 0.30 and 0.15 mg/L, respectively, barely two-fold lower than the MIC 50s.

Based on the predicted breakpoint value of AUC/MIC, the respective MIC breakpoint of trovafloxacin was predicted to be 0.27 mg/L. As shown in Figure 5, for trovafloxacin and ciprofloxacin against E. coli and K. pneumoniae, the MIC ranges limited from above by the respective MIC 50s do not cross the predicted breakpoint lines. The MIC ranges for ciprofloxacin against S. aureus and for both trovafloxacin and ciprofloxacin against P. aeruginosa do cross the predicted breakpoint line. This diagram demonstrates possible limitations of the two quinolones when administered at their clinically accepted doses.



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Figure 5. The MIC breakpoints of (a) trovafloxacin and (b) ciprofloxacin (indicated by the bold numbers) as compared with their MIC 50s for S. aureus, E. coli, K. pneumoniae and P. aeruginosa.

 
To predict quantitatively the doses which might be necessary for pathogens with lower or higher MICs than the predicted MIC breakpoints, quinolone doses that produce the same antimicrobial effect, i.e. I E = 198 (log cfu/mL) x h, as at AUC/MIC = 71 (trovafloxacin) or 125 (ciprofloxacin) (mg.{alpha}h/L)/(mg/L), D 198s, may be related to the MICs for the organisms studied (Figure 6). Despite a flatter D 198—MIC curve, a doubling in the MIC of trovafloxacin over the MIC breakpoint would require approximately the same 1.7- to 1.8-fold higher dose as a doubling in the MIC of ciprofloxacin over its MIC breakpoint. On the other hand, as follows from the D 198—MIC curve for ciprofloxacin, a 2 x 750 mg dose might increase the MIC breakpoint to a higher level (0.29 mg/L) which is close to the value reported elsewhere for ciprofloxacin (0.25 mg/L) 21 and to that for trovafloxacin (0.27 mg/L) as established in the present study.



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Figure 6. MIC-dependent doses (D 198s) of trovafloxacin (— ) and ciprofloxacin (- - - ) providing the same antimicrobial effect, I E = 198 (log cfu/mL) x h, on Gram-positive and -negative bacteria. The MIC breakpoints are indicated by the bold numbers.

 
In conclusion, unlike an earlier study with ciprofloxacin and two other quinolones against S. aureus, 22 this and our previous study 2 showed different AUC/MIC- and dose-dependent effects of the pharmacokinetically different quinolones. The lack of correspondence between visually compared viable count- time curves and repeated doses of each of three quinolones including ciprofloxacin reported in another study 22 might be attributed to insufficiently long observation periods after the last dose administration. As seen in Figure 1, in most cases the effects produced by different doses of the quinolones could be distinguished only with longer observations.

Overall, the data presented support the application of the relationships between the antimicrobial effect expressed by I E and the AUC/MIC to compare antimicrobials. 2 Based on these bacterial species-independent relationships, the findings obtained with specific strains might be generalized to other representatives of the same species to predict the antimicrobial effects against organisms of typical susceptibilities as expressed by MIC 50, MIC 90, etc. The suggested approaches to the prediction of quinolone antimicrobial effects might be applicable to other antibiotic classes.


    Acknowledgments
 
This study was supported by Pfizer, Inc. We are grateful to Yury A. Portnoy for assistance in computer presentation of the data.


    Notes
 
* Tel: +7-095-332-3435; Fax: +7-095-331-4116; E-mail:firsov{at}dol.ru Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 29 April 1998; returned 17 August 1998; revised 26 October 1998; accepted 7 December 1998