1 Department of Pharmacokinetics & Pharmacodynamics, Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, 11 Bolshaya Pirogovskaya Street, Moscow, 119021; 2 Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow, Russia; 3 Department of Medicine, Mount Auburn Hospital, Harvard Medical School, Cambridge, MA, USA
Received 2 December 2003; returned 12 February 2004; revised 18 March 2004; accepted 19 March 2004
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Abstract |
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Methods: Three clinical isolates of S. aureusincluding two ciprofloxacin-susceptible S. aureus, 201 and 480and a ciprofloxacin-resistant S. aureus 866, were exposed to clinically achievable ratios of area under the curve (AUC) to MIC in a dynamic model that simulated human pharmacokinetics of ABT492 (400 mg) and levofloxacin (500 mg) as a single dose. In addition, S. aureus 201 was exposed to single and multiple doses of ABT492 and levofloxacin (both once daily for 3 days) over wide ranges of 24 h AUC/MIC (AUC24/MIC) including clinically achievable AUC24/MIC ratios.
Results: With each isolate, ABT492 at clinically achievable AUC/MICs produced greater anti-staphylococcal effects than levofloxacin. Areas between the control growth and the timekill curves (ABBC in single dose simulations and the sum of ABBCs determined after the first, second and third dosing in multiple dose simulationsABBC1+2+3) were higher with ABT492 than levofloxacin. Moreover, at comparable AUC/MICs and AUC24/MICs, the maximal reductions in the starting inoculum of ABT492-exposed S. aureus were more pronounced than with levofloxacin. Loss in susceptibility of S. aureus 201 exposed to ABT492 or levofloxacin depended on the simulated AUC24/MIC. Although the maximal increase in MIC (MICfinal) related to its initial value (MICinitial) was seen at a higher AUC24/MIC ratio of ABT492 (120 h) than levofloxacin (50 h), similar AUC24/MICs (240 and 200 h, respectively) were protective against the selection of resistant S. aureus. These threshold values are readily achievable with 400 mg ABT492 (AUC24/MIC 870 h) but not with 500 mg levofloxacin (AUC24/MIC 70 h).
Conclusion: Overall, these findings predict greater efficacy of clinically achievable AUC/MIC (or AUC24/MIC) of ABT492 both in terms of the anti-staphylococcal effect and prevention of the selection of resistant mutants.
Keywords: S. aureus , resistance , in vitro models
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Introduction |
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Materials and methods |
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ABT492 and levofloxacin (kindly provided by Abbott Laboratories, North Chicago, IL, USA and Ortho-McNeill Pharmaceuticals, Raritan, NJ, USA, respectively) were used in the study. Three differentially susceptible clinical isolates of S. aureus including two ciprofloxacin-susceptible strains, S. aureus 201 and 480 (MICs of ciprofloxacin 0.8 and 2 mg/L, respectively) and a ciprofloxacin-resistant strain 866 (MIC of ciprofloxacin 4 mg/L) were selected for the study. S. aureus 201 was kindly provided by Dr E. Gugutsidze from Moscow Clinical Hospital and S. aureus 480 and 866 were kindly provided by Dr M. Edelstein from the Institute of Antimicrobial Chemotherapy of Smolensk State Medical Academy (Russia).
Susceptibility testing was performed in triplicate using broth microdilution techniques3 at 24 h post-exposure, with the organism grown in Ca2+ (2025 mg/L)- and Mg2+ (1012.5 mg/L)-supplemented MuellerHinton broth (MHB, BBL, Becton Dickinson and Company, Sparks, MD, USA) at an inoculum size of 106 cfu/mL. To establish precise values, MICs were determined using doubling dilutions, with starting concentrations of 3, 4 and 5 mg/L, as described previously.4 The MICs of ABT492 for S. aureus 201, 480 and 866 were 0.02, 0.06 and 0.12 mg/L, respectively. The respective MICs of levofloxacin were 0.6, 1 and 3 mg/L.
The mutant prevention concentrations (MPCs) of ABT492 and levofloxacin for S. aureus 201 were determined as described elsewhere.5 Briefly, the tested microorganisms were cultured in MHB and incubated for 24 h. Then, the suspension was centrifuged (4000g for 10 min) and re-suspended in MHB to yield a concentration of 1010 cfu/mL. A series of agar plates containing known fluoroquinolone concentrations was then inoculated with 1010 cfu of S. aureus 201. The inoculated plates were incubated for 48 h at 37°C and logarithms of bacterial numbers were plotted against fluoroquinolone concentrations. MPC was taken as the point where the plot intersected the x-axis, i.e. the lowest fluoroquinolone concentration that completely inhibited growth. The MPCs of ABT492 and levofloxacin were estimated at 0.07 and 1.75 mg/L, respectively.
Simulated pharmacokinetic profiles
Reported concentrationtime data obtained in humans after a single 400 mg dose of ABT492 and after daily dosing for 5 days6 were fitted by a three-exponential equation with an absorption half-life of 0.2 h, a distribution half-life of 1.7 h and an elimination half-life (terminal half-life) of 25 h (Figure 1, upper panel). Best fit estimates were obtained by a non-linear regression analysis using TOPFIT software (V. 1.1Gödeke, Schering, Thomae, 1991). Then, the same fitting procedure was applied to combined data obtained after ABT492 administration at doses 100, 200 and 400 mg.6
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Because of the relatively minor impact of the absorption phase on the observed pharmacokinetics [the partial area that reflects the contribution of the absorption phase was estimated at only 9% of the total area under the curve (AUC)], a bi-exponential concentration decay of ABT492 was simulated with t1/2 of 2 h and t1/2ß of 23 h. Two quasi-linear portions of each concentration-time curve (Figure 2, left panel) approximated this bi-exponential profile. With levofloxacin, a series of mono-exponential profiles were simulated with t1/2ß of 6.8 h (Figure 2, right panel) that represent weighted means of the values reported in humans: 6.07.4 h.710
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A previously described dynamic model11 was used in the study. Briefly, the model consists of two connected flasks, one containing fresh MHB and the other a magnetic stirrer; and a central unit, with the same broth containing either a bacterial culture alone (control growth experiments) or a bacterial culture plus antimicrobial (killing/regrowth experiments). Peristaltic pumps circulate fresh nutrient medium to the flasks: from the central 70 mL unit at a flow rate of 22.4 mL/h during first 16 h after dosing of ABT492 and then at a flow rate of 2.1 mL/h. With levofloxacin, the central unit volume was 60 mL and the flow rate 6.1 mL/h. The reliability of fluoroquinolone pharmacokinetic simulations and the high reproducibility of the timekill curves provided by the model have been reported elsewhere.11
The system was filled with sterile MHB and placed in an incubator at 37°C. The central unit was inoculated with an 18 h culture of S. aureus. After the bacteria had been incubated for 2 h, the resulting exponentially growing cultures reached 106 cfu/mL in single-dose simulations, or 108 cfu/mL (6 x 1097 x 109 per 6070 mL central compartment) in multiple dose simulations, at which time ABT492 or levofloxacin was injected into the central unit.
Quantification of the timekill curves and antimicrobial effect
In each experiment, multiple sampling of bacteria-containing media from the central compartment was performed throughout the observation period. One hundred microlitre samples were serially diluted as appropriate, and 100 µL was plated onto agar plates. The duration of the experiments was defined in each case as the timeafter a single or the final dosewhen antibiotic-exposed bacteria reached the maximum numbers observed in the absence of antibiotic (>108 cfu/mL). In multiple dose studies, the minimum duration was 72 h if no regrowth occurred. The lower limit of accurate detection was 2 x 102 cfu/mL. A level of 10 cfu/mL was considered to be the theoretical limit of detection.
Based on timekill data obtained in single-dose simulations, the area between the normal growth curve and the curve of bacteria exposed to antibiotic (ABBC)12 was calculated from time zero to 24 h. In multiple dose simulations, a cumulative antimicrobial effect was expressed as the sum of ABBC determined within the first, second and third dosing interval (ABBC1, ABBC2 and ABBC3, respectively): ABBC1 + ABBC2 + ABBC3 = ABBC1+2+3. The upper limit of bacterial numbers, i.e. the cutoff level on the regrowth and control growth curves used to determine ABBC and ABBC1+2+3 was 109 cfu/mL. The computation of ABBC, ABBC1, ABBC2 and ABBC3 is depicted graphically in Figure 4.
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Quasi-linear portion of the AUC24/MIC plots of ABBC1+2+3 were fitted by a linear equation:
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To express the antimicrobial effect (ABBC1+2+3) as a function of quinolone dose (D), D was calculated for each simulated AUC24/MIC according to linear equation:
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Quantification of bacterial resistance
To reveal possible changes in the susceptibility of ABT492- and levofloxacin-exposed S. aureus 201, precise fluoroquinolone MICs of bacterial cultures sampled from the model were determined daily over the 3 day treatment. The stability of resistance was determined by consecutive passaging of ABT492- and levofloxacin-exposed S. aureus onto antibiotic-free agar plates for 5 days.
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Results |
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Time courses of killing and regrowth of S. aureus 201 exposed to bi-exponentially decreasing concentrations of ABT492 and mono-exponential concentration decay of levofloxacin at AUC/MIC of 60480 h are shown in the upper panel of Figure 5. The timekill curves observed with both quinolones yielded similar patterns: regrowth followed a prompt reduction in bacterial numbers. At a given AUC/MIC ratio, the maximal reductions in the starting inoculum of ABT492-exposed S. aureus were greater than those with levofloxacin, but it showed longer times to regrowth than ABT492. At AUC/MIC of 60, 120, 240 and 480 h, the minimal numbers (Nmins) of surviving S. aureus exposed to ABT492 were lower than with levofloxacin: 1.8 x 103 versus 1.5 x 104, 1.3 x 103 versus 3.5 x 103, 7 x 102 versus 1 x 103 and 1.5 x 102 versus 1 x 103 cfu/mL, respectively. The respective times to regrowth were 9 versus 16 h, 11 versus 22 h, 11 versus 26 h and 15 versus 30 h. However, at AUC/MIC ratios provided by the clinical dose of ABT492 (400 mg), regrowth of all three strains of S. aureusS. aureus 201, 480 and 866was observed much later than in simulations of the clinically achievable AUC/MIC of levofloxacin (500 mg) (Figure 5, bottom panels). Again, the maximal reductions in viable counts after ABT492 exposure were greater than with levofloxacin: Nmin of 1.5 x 102 versus 1.1 x 104 cfu/mL (S. aureus 201), 6.5 x 102 versus 9.3 x 103 cfu/mL (S. aureus 480) and 7 x 102 versus 1.5 x 104 cfu/mL (S. aureus 866), respectively.
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Timekill dynamics. Time courses of ABT492- and levofloxacin-exposed S. aureus 201 are shown in Figure 8. At most studied AUC24/MIC ratios, regrowth followed a prompt reduction in bacterial numbers after each dosing. However, at the clinically achievable AUC24/MIC ratio of ABT492 (870 h) but not levofloxacin (70 h), no bacterial regrowth occurred. AUC24/MIC-dependent relationships of ABBC1+2+3 (Figure 6, right panel) were similar to the AUC/MIC relationships of ABBC delineated in single-dose simulations. Because of fewer points with ABT492, these data could not be accurately approximated by equation 1. Therefore, quasi-linear portions of both ABT492 and levofloxacin plots were fitted by equation 2. Like single-dose simulations, with ABT492 the 870 h clinical value of the AUC24/MIC ratio produced a 1.7 times greater antimicrobial effect than a 70 h value for levofloxacin: ABBC1+2+3 of 505 versus 290 (log cfu/mL) x h.
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Significant increases in MIC of ABT492 were observed at AUC24/MIC of 60 and 120 h, and with levofloxacin at AUC24/MIC of 25100 h after 3 days exposure of S. aureus 201 (Figure 9). With both quinolones, these increases were most pronounced after the third quinolone dose. Serial passages of resistant isolates onto antibiotic-free plates revealed minimal or no changes in the elevated MICs, showing stable resistance after five passages (data not shown). At the higher AUC24/MIC (240 h with ABT492 and 200 h with levofloxacin), no loss in the susceptibility of S. aureus was documented. Therefore, these threshold values may be considered to be protective against the selection of resistant mutants.
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Discussion |
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It is fair to say that t1/2ß of 23 h simulated in this study differs from values reported in human studies with ABT492: 4.28.514 and 8.39 h.15 This difference is due to the fact that the reported t1/2ßs ignore the terminal phase of the concentration-time curves (from 1248 h) that reflects enterohepatic recycling of ABT492. In this light, t1/2ß simulated in the present study may be considered as an apparent half-life that describes ABT492 pharmacokinetics as observed in humans, regardless of the underlying mechanisms.
Despite the relatively smaller anti-staphylococcal effects of ABT492 compared with levofloxacin at a given AUC/MIC or AUC24/MIC ratio, the clinically achievable AUC/MIC or AUC24/MIC ratios, i.e. those provided by a 400 mg dose of ABT492, produced much greater ABBCs with S. aureus 201 (Figure 6), 480 and 866 (Figure 7) than did clinically achievable AUC/MICs or AUC24/MICs of 500 mg levofloxacin. Moreover, similar 1.7-fold differences in the antimicrobial effect in terms of ABBC or ABBC1+2+3 were observed in single- and multiple-dose simulations. Concordant estimates of relative anti-staphylococcal efficacy have been reported with moxifloxacin and levofloxacin in previous single-dose and 3 day dosing in vitro studies.16
Like our study with four fluoroquinolones,2 loss in the susceptibility of ABT492- and levofloxacin-exposed S. aureus 201 (MICfinal/MICinitial) depended on the simulated AUC24/MIC (Figure 10, upper panel). However, with ABT492, the maximal increase in MICfinal was seen at a higher AUC24/MIC ratio (120 h) than with levofloxacin (50 h). Resistance of quinolone-exposed S. aureus 201 was also related to T>MPC: the longer the T>MPCs, the less pronounced the loss in susceptibility. Like AUC24/MIC relationships, a specific T>MPC relationship with MICfinal/MICinitial was inherent in each drug (Figure 10, bottom panel). To protect against the selection of resistant S. aureus, shorter T>MPCs were needed with ABT492 (<10 h) than levofloxacin (18 h). In contrast to data reported with more slowly eliminated quinolones,2 increases in ABT492 MICs did not correlate with the time when ABT492 concentrations were within the mutant selection window (MSW), i.e. between MPC and MIC (TMSW). With four of five simulated regimens (including AUC24/MIC of 60 and 120 h) where loss in susceptibility was observed and AUC24/MIC of 240 and 480 h without such a loss, TMSWs were similar (17% of the dosing interval). At the same time, no increases in MIC were associated with AUC24/MIC of 870 h when TMSW was 38% of the dosing interval.
Both AUC24/MIC and T>MPC relationships of the MICfinal/MICinitial ratio allow prediction of threshold values that prevent the selection of resistant S. aureus. In terms of the AUC24/MIC ratio, these threshold values are similar for ABT492 and levofloxacin (240 and 200 h, respectively), but in terms of T>MPC they are different: 10 versus 18 h. The clinically achievable AUC24/MIC ratio of ABT492 (870 h) is far in excess of the 240 h protective value. Unlike ABT492, the clinically achievable AUC24/MIC ratio of levofloxacin (70 h) is less than its protective value (200 h).
Based on linear relationships of AUC to the dose for ABT492 and levofloxacin13 (equation 3), the AUC24/MIC relationships of ABBC1+2+3 and MICfinal/MICinitial can be presented as respective dose relationships. As seen in the left upper panel of Figure 11, a 400 mg dose of ABT492 provides a 60% greater effect (ABBC1+2+3) on S. aureus 201 than a 500 mg dose of levofloxacin. Also, the clinical dose of ABT492 but not levofloxacin may be able to prevent the loss in susceptibility (Figure 11, left bottom panel).
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Overall, these findings predict greater efficacy of clinically achievable AUC/MIC and AUC24/MIC of ABT492 both in terms of the anti-staphylococcal effect and prevention of the selection of resistant mutants.
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Acknowledgements |
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Footnotes |
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References |
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2
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