JAC Advance Access originally published online on July 21, 2008
Journal of Antimicrobial Chemotherapy 2008 62(5):1070-1077; doi:10.1093/jac/dkn294
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Original research |
Development of an integrated semi-automated system for in vitro pharmacodynamic modelling
1 Department of Infectious Disease Research, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, NJ 07065, USA 2 Department of Research Operations, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, NJ 07065, USA
* Corresponding author. Tel: +1-732-594-2145; Fax: +1-732-594-6708; E-mail: liangsu_wang{at}merck.com
Received 2 November 2007; returned 2 February 2008; revised 6 June 2008; accepted 20 June 2008
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Abstract |
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Objectives: The aim of this study was to develop an integrated system for in vitro pharmacodynamic modelling of antimicrobials with greater flexibility, easier control and better accuracy than existing in vitro models.
Methods: Custom-made bottle caps, fittings, valve controllers and a modified bench-top shaking incubator were used. A temperature-controlled automated sample collector was built. Computer software was developed to manage experiments and to control the entire system including solenoid pinch valves, peristaltic pumps and the sample collector. The system was validated by pharmacokinetic simulations of linezolid 600 mg infusion. The antibacterial effect of linezolid against multiple Staphylococcus aureus strains was also studied in this system.
Results: An integrated semi-automated bench-top system was built and validated. The temperature-controlled automated sample collector allowed unattended collection and temporary storage of samples. The system software reduced the labour necessary for many tasks and also improved the timing accuracy for performing simultaneous actions in multiple parallel experiments. The system was able to simulate human pharmacokinetics of linezolid 600 mg intravenous infusion accurately. A pharmacodynamic study of linezolid against multiple S. aureus strains with a range of MICs showed that the required 24 h free drug AUC/MIC ratio was 
30 in order to keep the organism counts at the same level as their initial inoculum and was about 
68 in order to achieve >2 log10 cfu/mL reduction in the in vitro model.
Conclusions: The integrated semi-automated bench-top system provided the ability to overcome many of the drawbacks of existing in vitro models. It can be used for various simple or complicated pharmacokinetic/pharmacodynamic studies efficiently and conveniently.
Keywords: in vitro models , linezolid , Staphylococcus aureus
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Introduction |
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Pharmacokinetic/pharmacodynamic (PK/PD) studies have been becoming increasingly important in bridging preclinical data and clinical trials. Numerous studies have been published on using in vitro or in vivo models to study antimicrobial effect, to determine the PK/PD parameters of antimicrobials for the purpose of optimizing dosing strategy, to study the emergence of resistance, to select breakpoints and to study combination therapies.1–7 The in vitro pharmacodynamic models (IVPMs), both the one-compartment models and the two-compartment models with a wide range of model designs, have been used successfully by several groups, as exemplified in the cited publications,7–17 to complement in vivo animal models for such studies.
Most IVPM systems use continuous culture systems with pumps, tubing, filters and flasks to reproduce drug pharmacokinetics. They have the advantages of being flexible, adaptable and relatively low cost.2 They are able to conveniently simulate the human/animal pharmacokinetics or permutations of pharmacokinetics of antibiotics and to closely monitor the time course of microbial killing and growth events. IVPM systems, however, have some drawbacks and are, therefore, often limited to their use for simple descriptive studies. Some of the drawbacks are as follows. First, as with animal models, experiments with IVPM are quite laborious involving pump calibrations, tubing preparations, drug dosing and sample collections as manifested in dose fractionation studies. Secondly, even with the availability of multi-channel pump heads from pump vendors, multiple pumps are often required for drug oral administration, for combination therapy and for parallel experiments. This makes IVPM systems bulky and difficult to control. Thirdly, many investigators place their IVPM systems in a warm room because such bulky systems usually do not fit in a regular incubator. This requires researchers to work inside the warm environment for the processes of model set-up and sample collections. Fourthly, with multiple lines of fluid flow going in and out of flasks over one or more days of the experiment running period, the chances of contamination are increased.
We developed an integrated semi-automated IVPM system to overcome the above physical limitations. This system uses a bench-top shaking incubator to hold flasks and hollow-fibre cartridges with custom ports on the sides to allow tubing to pass through. All bottles and flasks are equipped with multiple custom inlets and outlets to allow connections with tubing using standard Luer fittings. A custom temperature-controlled sample collector has been made for automated sample collections. The entire IVPM system is computer-controlled, from pump calibrations and drug dosing to sample collections. This integrated system has been validated by measuring the human pharmacokinetics of linezolid intravenous (iv) dosing. The pharmacodynamics of linezolid against multiple clinical isolates of Staphylococcus aureus were studied using this integrated IVPM system as a proof of concept.
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Materials and methods |
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Description of IVPM system
Overview of model configurations. One-compartment and two-compartment hollow-fibre models were adapted from previously published models as described by Grasso et al.8 and Blaser et al.9 Figure 1(a and b) shows the schematic diagrams of one-compartment and two-compartment models with iv infusion. Both models used a central compartment fabricated from a flask placed in a bench-top shaking incubator. Liquid was continuously introduced into this vessel via a peristaltic pump either from a diluent bottle or from a drug bottle at a constant rate calculated based on the drug's pharmacokinetic parameters.8,9 Liquid was also continuously removed from the central compartment by the same method at the same constant rate and directed either to a waste container or into a 96-well plate as a collected fraction.
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For the two-compartment model, a hollow-fibre cartridge was placed in the same incubator and connected to the central compartment through a circulation loop (Figure 1b). Microbial culture in the peripheral compartment of the hollow-fibre cartridge was circulated by a peristaltic pump to ensure uniform culture growth. Samples from this secondary loop were collected into a 96-well plate.
The entire system was controlled by custom computer software developed internally. A picture of the actual set-up is shown in Figure S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
System hardware. Peristaltic pumps (Model EW-07550-50, Cole-Parmer) were used for fluid transfer. These pumps were equipped with an RS-232 interface and controlled by the system computer. Each peristaltic pump was equipped with an eight-channel pump head (Model EW-07535-08, Cole Parmer). This pump head allowed the use of tubing with pre-installed stops (EW-06421-13 or EW-06421-16, Cole Parmer) to reduce tube-to-tube variations in flow rate. All flow rates were confirmed by gravimetric calibration prior to starting an experiment.
Where necessary, fluid flow switching was performed using electromagnetically actuated 3-way pinch valves (Model 100P3MP24-03S, BioChem Valve Inc., Boonton, NJ, USA) under computer control. A custom controller was fabricated, allowing for up to 32 valves to be connected to the system.
All bottles and flasks used were equipped with GL-45 thread tops. We used custom-fabricated bottle caps with integral 1.60 mm ID 3.18 mm OD stainless steel inlet and outlet tubes (TSS262, Valco Instruments Co. Inc.) on the interior of the vessel and stainless steel female Luer connections (31507-34, Cole Parmer) on the exterior of the vessel. Vessels were vented to atmosphere through 0.20 µm filters (Millipore).
Various components in the system were connected with L/S 13 silicone tubing (Cole Parmer). For the two-compartment model, L/S 16 silicone tubing (Cole Parmer) was used to connect the central compartment and the hollow-fibre cartridges (C3001, FiberCell Systems, Frederick, MD, USA), and was also used for circulation loop of each cartridge. Where necessary, various polypropylene barbed and barbed to Luer fittings were used (McMaster-Carr).
All central compartment vessels and hollow-fibre cartridges were incubated during the experiment in a bench-top shaking incubator (Innova 4000, New Brunswick Scientific; or DR1959, ECE Scientific Co.). The incubator was modified with a set of custom pass-through ports, allowing up to 16 pieces of tubing to enter or exit from either the left or right sides of the incubator without the need for fittings while maintaining the desired temperature. This design allows multiple experiments to be run in parallel. Near the back, inside the shaker, a rack with clamps was installed to hold hollow-fibre cartridges if two-compartment hollow-fibre models were run.
Temperature-controlled automated sample collector. A custom sample collector was fabricated, allowing for unattended sample collection and storage of samples in cold plates from up to eight fluid collection channels (Figure 2). The sample fluid from each channel was collected into a 96-well microplate (Costar 3956, Corning) sealed with a silicone septum mat (03-396-49, Fisher Scientific). The eight microplates were placed on an aluminium deck at 4°C. A circulating water bath (RTE111, NesLab) filled with a 50% water 50% ethylene glycol solution was connected to a liquid heat exchanger attached to the deck of the sample collector to maintain the temperature. Between each microplate and the deck was an aluminium heat sink designed to surround each well in the plate. This heat sink was intended to increase the rate of cooling for the collected samples and to improve the uniformity of the incubation temperature once the sample had been collected. A computer-controlled XYZ positioning stage was suspended over the microplate deck. The stage used computer-controlled servomotors for motion control (SM2315D and SM2337D SmartMotors, Animatics Corp., Santa Clara, CA, USA). This XYZ stage was equipped with a 22 gauge sample introduction needle and a 22 gauge vent needle for each microplate. The vent needle was vented to atmosphere through a 0.20 µm filter (Whatman 6780-1302). A waste trough was included to collect liquid from needle purging and washing prior to sample collection. The stage would purge the sample needle, locate the appropriate wells, plunge to pierce the septum with both needles, fill the well with samples and retract the following sample collection. A solenoid pinch valve (Model 100P3MP24-03S, BioChem Valve Inc.) located on the XYZ stage was used to select between fluid flow to waste and fluid flow to the sample needle for each channel. In the case of the two-compartment model, the solenoid valve was located close to the peripheral compartment to reduce the tubing length of the circulation loop. The sample was introduced into the tubing and bracketed by air gaps to reduce the volume of fluid required for a sample. The lines were then flushed with alcohol and media. A 6-way rotary valve (V-1341-DC, Upchurch) was used to select the flushing solvent. This modification allowed a sample of small volume to be collected from the secondary circulation loop without removing more culture fluid than necessary.
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System control software. A custom control application was written in Microsoft VB.NET to reduce the labour necessary for many tasks involved in running this system and to improve the timing accuracy when it was necessary to perform drug dosing and sample collection simultaneously for multiple parallel experiments.
The software allowed the user to perform gravimetric flow rate calibrations for the peristaltic pumps, which improved the matching of the inlet and outlet flow rates for the central compartment. Experiment definitions such as the type of models to use and drug administration schedules were defined using a method set-up editor and mapped into the system set-up. Sample collection was then scheduled for sampling time and sample volumes. Once all of the set-up had been completed, progress could be monitored during the experiment. The software also provided a utility screen for maintenance, cleaning and troubleshooting. Some screen shots of IVPM system control software are shown in Figure S2, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
Temperature profile test of automated sample collector. Prior to fabricating the sample collector, we performed tests to determine whether the rate of cooling in a 96-well microplate would be sufficient to stop culture growth in a manner similar to manually collected samples. We determined that the best method for temperature control would be to use an aluminium microplate with glass vials (Symyx), but that liquid handling and cleaning issues would be difficult for this type of plate. Therefore, we fabricated a custom heat sink that allowed us to use a disposable polypropylene microplate. Comparison of the temperature profiles from different collection methods after sample introduction, measured by IOtech DaqBook 200 (IOtech, Cleveland, OH, USA), indicated that the rate of cooling from the polypropylene plate (Costar 3956, Corning) on an aluminium block with a heat sink was sufficiently fast, comparable to that from the aluminium microplate with glass vials, and even faster than using a 5 mL Falcon 2063 tube (Becton–Dickinson) on ice from manual sampling (Figure 3). Costar 3956 polypropylene 96-well plates on an aluminium block with heat sink was thus chosen for automated sample collector.
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Antibiotics, bacterial strains and antimicrobial susceptibility testing
Linezolid was obtained as a marketed injectable solution (Zyvox I.V. bag 600 mg/300 mL) purchased from Belle Medical Supply, Marlboro, NJ, USA.
Four S. aureus strains from the Merck Clinical Culture Collection were selected for the validation of the semi-automated IVPM system. These strains were CL5706 (methicillin-resistant, vancomycin-intermediate and constitutive macrolide-resistant), CL9161 (methicillin-sensitive and macrolide-sensitive), CL8072 (methicillin-resistant and constitutive macrolide-resistant) and CL5814 (methicillin-resistant, linezolid-resistant and macrolide-sensitive). These strains were chosen to include different resistance types and a range of linezolid MICs. Strains were grown and subcultured on trypticase soy agar (TSA) plates with 5% sheep blood and in cation-adjusted Mueller–Hinton broth (CAMHB). The MICs of linezolid for the S. aureus strains were determined by following the CLSI broth microdilution method. The MIC values of the four strains were 1, 2, 4 and 128 mg/L for CL5706, CL9161, CL8072 and CL5814, respectively.
IVPM operating procedure for validation
The integrated IVPM system with automated sample collector (Figure 1a) described earlier was used for the validation. For the pharmacokinetic study, free drug concentrations of linezolid 600 mg iv dose with 30 min infusion (half-life: 4.4 h) were simulated.18 The volume of the central compartment was 200 mL. The flow rate was set to be 0.53 mL/min to achieve the desired pharmacokinetic profile. Samples of 0.5 mL volume were collected by the automated sample collector at 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 h.
For the pharmacodynamic evaluations, 0.5 McFarland Standard from overnight culture on TSA plates with 5% sheep blood for each of the four S. aureus strains was prepared by suspending several colonies in saline. The initial inoculum was made by diluting 40 µL of the suspension into a flask with 200 mL of fresh CAMHB. The resulting suspension was incubated at 35°C with shaking (225 rpm). After the bacteria had been incubated for 4 h, the resulting exponentially growing cultures reached 106 cfu/mL, at which time the cultures were introduced to the IVPM. Linezolid was infused into the central compartment at 0 and 12 h with an infusion duration of 0.5 h each to simulate human 600 mg twice-daily dose regimens using free drug concentrations. For each S. aureus strain, a no-drug growth control was also set up. The central compartment was incubated at 35°C with shaking at 125 rpm to ensure proper mixing of the drug and even sampling. About 0.5 mL samples were collected at 0, 2, 4, 6, 8, 12, 14, 16, 18, 20 and 24 h, respectively, by the automated sample collector.
Dose–response studies were also carried out for strains CL5701 and CL9161 with linezolid 50, 100, 200, 400, 600, 800 and 1200 mg twice-daily dosing. The procedures for set-up and sample collections were the same as described earlier.
Measurement of drug concentrations
HPLC analysis of linezolid pharmacokinetic samples from IVPM was performed on a system consisting of a Waters multi-solvent delivery system interfaced with a Wisp autosampler. At harvest, linezolid was extracted from the sample with a 1:3 dilution with methanol. Extracted samples were vortexed for 30 s followed by 10 min at room temperature. The samples were then centrifuged (Costar-micro-centrifuge) for 10 min at 10 000 rpm. Samples for analysis were loaded onto a Waters symmetry B, C18 column 3.5 µ (4.6 mm x 100 mm) via the autosampler. The column temperature was maintained at 30°C with a column heater. The flow rate was 1.0 mL/min. The isocratic solvent system was 60% HPLC grade methanol and 40% HPLC grade water with 0.1% trifluoroacetic acid (TFA), and the UV absorption of the effluent was monitored at 254 nm. Under these conditions, the retention time of linezolid was –1.8 min.
Quantification of the antimicrobial effect
Bacterial viable counts were determined by serially diluting samples 10-fold in cold sterile saline. Aliquots of 100 µL of diluted samples (10–110–7) were plated onto TSA plates with 5% sheep blood with subsequent incubation at 35°C for 18–24 h. Drug carryover was negligible after serial dilutions. After incubation, the resulting colonies were counted and the numbers of cfu/mL were calculated.
A one-compartment pharmacokinetic model was fitted to the drug concentrations in the IVPM central compartment (WinNonLin, Pharsight Corp.). The pharmacodynamic results were analysed by using the sigmoid Emax model to evaluate the relationship between linezolid exposure and its antimicrobial effect.
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Results |
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Pharmacokinetics simulation
The time course of linezolid free drug concentrations in the IVPM central compartment as determined by HPLC matched the target profile very closely (Figure 4). The derived pharmacokinetic parameters through one-compartment analysis of the observed data from the simulation of linezolid free drug concentrations of human 600 mg iv single dose had Cmax of 9.05 ± 1.20 mg/L, AUC of 63.26 ± 14.70 mg·h/L and half-life of 4.67 ± 0.48 h (Table 1), which were in good agreement with linezolid data observed in humans (Zyvox label18). The results indicated accurate set-up of the IVPM system and computer controlling software.
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Pharmacodynamics against S. aureus strains
In a pilot experiment, the growth of S. aureus strains was monitored with or without linezolid by sampling manually and by using the automated sample collector and leaving the samples in the cold microplates until the end of the run. Viable counts from manual sample collections and from automated collections were very similar (data not shown), indicating the suitability of the temperature-controlled automated sample collector for the IVPM system.
The antibacterial effect of linezolid against four clinical isolates of S. aureus was evaluated in the integrated semi-automated IVPM system with pharmacokinetics simulating free drug concentrations of linezolid human 600 mg twice-daily dosing regimens. The time–kill data over a 24 h period of time are shown in Figure 5. As expected, among these four strains, the one with the lowest MIC (CL5706, MIC = 1 mg/L) exhibited the most log reduction (–2.2 log10 cfu/mL relative to initial inocula). The growth of linezolid-resistant strain CL5814 with an MIC of 128 mg/L was not impacted by the drug. The log10 cfu/mL of strain CL8072 (MIC = 4 mg/L) showed little change over time compared with its initial inoculum (–0.66 log10 cfu/mL relative to initial inoculum). The change of log10 cfu/mL of strain CL9161 (MIC = 2 mg/L) was –1.39 log10 cfu/mL relative to initial inoculum.
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Besides examining the pharmacodynamics of linezolid against strains with different MICs, strains CL9161 and CL5706 were also exposed to different drug levels of linezolid to explore exposure–response relationship in IVPM. The results from both strains were then combined and analysed for the 0–24 h free drug AUC/MIC versus bacterial killing. As shown in Figure 6, an AUC/MIC ratio of
30 was required to keep the organism counts at the same level as their initial inocula. To achieve a >2 log10 cfu/mL reduction, the required free AUC/MIC ratio is 68 in IVPM. Since the free drug AUC0–24 of linezolid 600 mg twice-daily dosing is 110 mg·h/L based on the pharmacokinetic parameters described earlier, it is expected that this dosage regimen would result in >2 log10 cfu/mL reduction against S. aureus strains with an MIC of 1 mg/L but <2 log10 cfu/mL reduction against S. aureus strains with an MIC of 2 mg/L. This is consistent with linezolid antibacterial effect described in the last paragraph.
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Discussion |
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IVPMs were developed decades ago8,9 and have been used more and more frequently as a complementary tool to animal models for the study of antibiotic activities, resistance emergence and PK/PD parameter determinations.2,7 The improved IVPM system described in this article overcomes many drawbacks of existing in vitro models. In this integrated IVPM system, the custom bench-top shaking incubator allows researchers to work at room temperature. The custom caps for bottles and flasks with multiple inlets and outlets minimize contamination by limiting exposure of sterile medium or microbe-containing cultures to the environment, and they are convenient for combination therapy. The automated sample collector not only saves time and effort in sample collections but also avoids contamination associated with manual sampling. Computerization of the entire experiment running procedure ranging from calibration, drug dosing to sample collections reduces the amount of work and time associated with IVPM studies and increases the timing accuracy for many parallel processes. Many preclinical PK/PD studies, either in vivo or in vitro, require researchers to take early morning or late night shifts for drug administrations and/or sample collection. With the computer-controlled peristaltic pumps, pinch valves and temperature-controlled sample collector, drugs can be administered and samples can be collected and stored in cold plates without manual interventions. As a matter of fact, the sampling frequency can be scheduled to one's needs for more accurate pharmacokinetic and pharmacodynamic curves.
In the validation study, when linezolid human pharmacokinetics of a 600 mg single dose iv infusion for 30 min was simulated in this system, the measured pharmacokinetic profile closely mimicked the actual human data,18 indicating proper set-up of the system including the software and the automated sample collector. From the analysis of linezolid antibacterial effect against multiple S. aureus strains with different MICs when the pharmacokinetics of linezolid free drug was simulated, the required 24 h free drug AUC/MIC ratio was 30 in order to keep the organism counts at the same level as their initial inocula and was 68 in order to achieve >2 log10 cfu/mL reduction in IVPM. This is within the range of the in vivo results from the mouse thigh infection models published earlier.19 Based on these pharmacokinetic goals from our IVPM study, a dosage regimen of linezolid 600 mg twice daily should achieve success against S. aureus strains with MICs up to 2 mg/L, but would result in no significant change of bacterial burdens when up against S. aureus strains with an MIC of 4 mg/L.
Please note that, in this study, most of the samples were left in the cold microplates in the sample collector until the run was complete. However, the experiment control software has been implemented with multi-threading so that one can load/unload microplates from the aluminium deck of the sample collector between sampling times during the run without interrupting the rest of the processes. This is a useful feature for researchers to process the samples at a convenient time. If a drug is not very stable, sample plates can be taken out during the run and replaced with new ones.
In this validation study, the drug–pathogen pair was relatively easy to handle because linezolid is bacteriostatic against S. aureus. Despite the use of an automated rapid-cooling sample collector, precautions should be taken when working with rapid cidal agents or fastidious pathogens or extended-spectrum β-lactamase (ESBL) producers that rapidly degrade drugs. Pilot experiments such as growth/killing characterization of the pathogens in this system and test of drug stability in the cold microplates of the sample collector should be performed before PK/PD studies. In our laboratory, additional studies that were successfully run in this semi-automated IVPM system, either one-compartment models or two-compartment hollow-fibre models, include PK/PD studies of oxazolidinone compounds against Streptococcus pneumoniae, oxazolidinones against Haemophilus influenzae, piperacillin alone or piperacillin–tazobactam combinations against β-lactamase-producing Pseudomonas aeruginosa, echinocandins against Candida albicans, amphotericin B against C. albicans and a few other drug–pathogen pairs (authors’ unpublished results). Although samples were left in the cold microplates until the end of the run in some cases, we did take advantage of the convenient feature of microplate loading/unloading during the run (as described in the last paragraph) to have samples processed earlier whenever necessary or convenient to our needs.
Although the example used in this paper is a study from a one-compartment iv infusion model, the integrated semi-automated IVPM system is also suitable for other models including iv bolus, oral, continuous infusion and combination therapy with either one-compartment models or two-compartment hollow-fibre models (authors’ unpublished data). To handle these different models, the mechanism of sample collections remains the same as described. The system hardware has already been adapted for all scenarios, and the software has been implemented flexibly. The only thing one would need to change is the way that the tubing, flasks, pumps and valves are connected during model set-up. With this computerized integrated IVPM system, many of the complicated PK/PD studies can be carried out.
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Funding |
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The work was supported by internal funding from Merck Research Laboratories. No financial support was received from third parties.
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All authors are employees and stock option owners of Merck Research Laboratories.
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Supplementary data |
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Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
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Acknowledgements |
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We thank Dr Mary R. Motyl for her valuable input during the preparation of this manuscript. We also thank anonymous referees for their insightful feedback.
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References |
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Andes D, van Ogtrop ML, Peng J, et al. In vivo pharmacodynamics of a new oxazolidinone (linezolid). Antimicrob Agents Chemother (2002) 46:3484–9.
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