JAC Advance Access originally published online on August 5, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):802-805; doi:10.1093/jac/dkl311
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Pharmacodynamics of dalbavancin studied in an in vitro pharmacokinetic system
Bristol Centre for Antimicrobial Research & Evaluation, North Bristol NHS Trust and University of Bristol Bristol, UK
*Correspondence address. Bristol Centre for Antimicrobial Research & Evaluation, North Bristol NHS Trust, Department of Medical Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, UK. Tel: +44-117-959-5651/2; Fax: +44-117-959-3154; E-mail: alasdair.macgowan{at}nbt.nhs.uk
Received 24 April 2006; returned 18 May 2006; revised 26 June 2006; accepted 5 July 2006
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Abstract |
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Objectives: The antibacterial effect of dalbavancin was studied against Staphylococcus aureus using stepwise declining concentrations designed to model a range of free drug concentrations observed in human serum.
Methods: Initial concentrations ranged from 0.6 to 21 mg/L and experiments were conducted over 240 h. Three vancomycin-susceptible and one vancomycin-intermediate strain of S. aureus were used.
Results and conclusions: Dalbavancin showed non-concentration-dependent killing against the three vancomycin-susceptible strains in the range 3–21 mg/L and the vancomycin-intermediate strain at 15 and 21 mg/L. AUC/MIC could be related to antibacterial effect. The AUC/MIC for a bacteriostatic effect was 36 at 24 h, 55 at 120 h and 100 at 240 h. A larger AUC/MIC was required to produce a 2 log reduction in counts, being 214 at 24 h, 195 at 120 h and 331 at 240 h.
Keywords: AUC/MIC , S. aureus , VISA
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Introduction |
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In vitro pharmacokinetic systems are widely used to study the pharmacodynamic properties of developmental or marketed antibacterial agents.1 They provide highly flexible tools with which to describe the antibacterial effects of drugs at concentrations designed to reproduce human serum concentrations and also study selection or emergence of antibiotic resistance. The present pharmacodynamic paradigm would indicate that only free antibiotic is microbiologically active hence it is now usual to only simulate free drug concentrations in pharmacokinetic systems.2
Dalbavancin (BI 397) is a semi-synthetic glycopeptide derived from a teicoplanin-like molecule A40926.3 Its in vitro potency is greater than that of vancomycin; the dalbavancin MIC90 for Staphylococcus aureus and coagulase-negative staphylococci being 0.06 mg/L (irrespective of oxacillin susceptibility), dalbavancin MIC90s for vancomycin-susceptible Enterococcus sp. being 0.06–0.12 mg/L and dalbavancin MIC90 for Streptococcus pneumoniae or ß-haemolytic streptococci being 
0.03 mg/L.4,5 The pharmacokinetics of dalbavancin in man is characterized by high protein binding. Use of isothermal titration micro colorimetry suggested that protein binding was greater than 98% to albumin in solution.3 However, more recent data indicate 93% binding, the free fraction therefore is 
7%. The protein binding means that the steady-state volume of distribution is 10–12 L, roughly equating to extracellular water and the serum half-life is prolonged at around 240 h. The initial total drug plasma concentration is 300 mg/L after a 1 g dose, a free drug concentration of 21 mg/L.6,7
The objective of the present study was to investigate the pharmacodynamics of dalbavancin in an in vitro pharmacokinetic system. Free drug concentrations associated with an initial concentration of 21 mg/L and half-life of 240 h were simulated over a period of 10 days and the antibacterial effect against S. aureus was measured. In addition, initial concentrations of 15, 3 and 0.6 mg/L were also simulated, as part of a dose ranging study design.
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Materials and methods |
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The following four strains of S. aureus were used: an oxacillin-susceptible strain (OSSA) SMH 30901, vancomycin MIC = 1.5 mg/L; two oxacillin-resistant strains (ORSA) SMH 30902, vancomycin MIC = 1.0 mg/L and gentamicin MIC = 0.5 mg/L, and SMH 7023, vancomycin MIC = 1.0 mg/L and gentamicin MIC = 128 mg/L; and a strain with intermediate vancomycin resistance (VISA) SMH 19898 (the Michigan strain), vancomycin MIC = 8 mg/L. Dalbavancin was provided by Vicuron Inc., King of Prussia, PA, USA, and MICs were determined by CLSI-based methods except that 0.2 mg/L steps were used.8 Mueller–Hinton broth (Oxoid, Basingstoke, UK) was used in all experiments. Nutrient agar plates (Oxoid) were used for the determination of viable counts. The test strains were exposed to a decreasing concentration of dalbavancin simulating a 240 h serum half-life in 10 mL polypropylene test tubes. The antibiotic serum profile was modelled using the stepwise process described by Bauernfeind.9 Dalbavancin peak (Cmax) concentrations simulated at time zero were 0.6, 3, 15 and 21 mg/L. A drug-free growth control for each strain was included. At 12 h and 24 h, and then every 24 h until 240 h the bacteria were spun at 3500 rpm for 10 min, the supernatant discarded and the pellet re-suspended in the next (lower) concentration of dalbavancin. At each time interval viable counts were performed using a spiral plater (Don Whitley Spiral Systems, Shipley, UK). The limit of detection is 2 x 102 cfu/mL and experiments were performed in triplicate. In the first set of experiments with each strain the viable counts were measured before and after centrifugation to assess any likely loss of bacteria. The initial inoculum in all the experiments was 106 cfu/mL.
In addition, aliquots were stored at –20°C for the determination of dalbavancin concentrations. Samples were assayed by bioassay using Bacillus subtilis NCTC 10400 as indicator.10 The limit of detection was 1 mg/L. The initial antibacterial effect was measured by the log change in viable count (log cfu/mL) compared with the initial count at 24 h (d24), 72 h (d72), 120 h (d120) 168 h (d168) and 240 h (d240) and the time to clear 99.9% or 99% of the initial inoculum (T99.9 or T99). The relationship between AUC24/MIC and the log change in viable count at 24, 120 and 240 h was explored using a sigmoid Emax model.
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The dalbavancin MICs were OSSA (SMH 30901), 0.14 mg/L; ORSA (SMH 7023), 0.12 mg/L; ORSA (SMH 30902), 0.08 mg/L; and VISA (SMH 19898), 0.8 mg/L. The actual and measured dalbavancin concentrations were in good agreement and the bacterial counts before and after centrifugation and suspension highly correlated (r2 = 0.98; CI = 0.92–1.04) (data not shown).
The antibacterial effect of dalbavancin is illustrated in Figure 1 (a–d) and the log change in viable count at 24, 72, 120, 168 and 240 h and time to 99% and 99.9% reduction in viable counts are shown in Table 1. All four strains grew rapidly in the absence of dalbavancin and viable counts remained 108 cfu/mL throughout the 240 h of the simulations. The pattern of antibacterial effect was similar with the OSSA strain and the two ORSA strains. Dalbavancin at 0.6 mg/L produced an initial 1–2 log reduction in viable counts by 48 h with regrowth occurring later in the simulation. Dalbavancin at 3 mg/L produced clearance from the system between 72 and 168 h and regrowth only occurred with one strain—SMH 30902. Dalbavancin at initial concentrations of 15 and 21 mg/L produced more rapid clearance from the model with counts being undetectable by 48–96 h. In contrast to these three strains the VISA strain displayed a different pattern of antibacterial effect. Dalbavancin at 0.6 mg/L had a minor antibacterial effect while 3 mg/L produced an initial 1–2 log drop by 24 h followed by regrowth. Dalbavancin at 15 and 21 mg/L produced clearance by 144 and 96 h, respectively.
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The log change in viable counts up to 240 h and the time to clear 99% or 99.9% of the initial inoculum was calculated to assess the antibacterial effect of dalbavancin. As the dalbavancin concentration increased from 3 to 21 mg/L there was no concentration-related effect for the vancomycin-susceptible strains. For the VISA strain, 15 and 21 mg/L had similar antibacterial effects (Table 1).
The relationship between AUC24/MIC and antibacterial effect, defined by change in viable count at 24, 120 and 240 h, is shown in Table 2. There is a good relationship between AUC24/MIC and antibacterial effect. The AUC24/MIC for static effect was 36–100 depending on the time of the end-point used and 214–331 for a –2 log drop.
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Discussion |
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The AUC/MIC and Cmax/MIC of dalbavancin have been related best to antibacterial effect in the neutropenic thigh and lung infection model using S. pneumoniae and S. aureus.11 We were also able to relate AUC24/MIC to antibacterial effect for S. aureus.
Recent data using static drug concentration time–kill curves have shown that dalbavancin is bactericidal against S. aureus producing at least a 3 log drop in cfu over 24 h at low multiples of the MIC. Higher dalbavancin concentrations were not more bactericidal than lower ones over 24 h.4,12 Our data are somewhat different as they are generated using likely pharmacological concentrations and viable counts were followed over up to 240 h. Analysis of the killing kinetics indicates a non-concentration-related effect, which is in contrast to observations of S. aureus infection in a rat granuloma pouch infection model where the degree of bacterial killing up to 50 h was dose-dependent.13
Dalbavancin does not easily fit into the existing pharmacodynamic paradigm for predicting clinical effect in man using non-human models. This is related to the drug's long half-life, which means that in some circumstances a single 1 g intravenous dose will be appropriate to treat clinical infection over a period of 7 days. Hence the relevance of using animal or in vitro models dosed over 24 h to predict the dominant pharmacodynamic index and its optimal size may not be of great relevance. We used an in vitro pharmacokinetic system to model free dalbavancin concentrations over 240 h and showed clearance of S. aureus strains with initial free dalbavancin concentrations of 3 mg/L or more. The appropriate size of the free drug AUC/MIC to best predict effect in man is unclear due to the unusual pharmacodynamics of dalbavancin and the lack of detailed knowledge of the glycopeptide drug class in general. However a free drug target based on the AUC24/MIC to produce a static effect may be best for mild to moderate infection. This produces AUC24/MIC values in the range 36–100. Greater drug exposures to produce –2 log clearance may be more appropriate in patients with moderate to severe infections giving AUC24/MIC values of 200–300. This would seem most appropriate for an intravenous agent such as dalbavancin.
In conclusion, dalbavancin at concentrations associated with a free fraction of 0.07 produced marked and non-concentration-dependent killing of S. aureus in this in vitro pharmacokinetic system. Free drug AUC24/MIC can be related to antibacterial effect at 24, 120 and 240 h and a range of potential free drug AUC/MIC targets generated depending on the degree of killing required and at what time.
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None to declare.
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Acknowledgements |
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We would like to thank Drs B. Goldstein, T. Henkel and J. Dowell of Vicuron Inc, King of Prussia, PA, USA, for their advice, and Vicuron for financial support.
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Candiani G, Abbondi M, Borgonvi M, et al. (1999) In vitro and in vivo antibacterial activity of BI 397, a new semi-synthetic glycopeptide antibiotic. J Antimicrob Chemother 44:179–92.
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Lin G, Credito K, Ednie LM, et al. (2005) Antistaphylococcal activity of dalbavancin, an experimental glycopeptide. Antimicrob Agents Chemother 49:770–2.
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10 Andrews JM. (1999) Microbiological assays. In Reeves DS, Wise R, Andrews JM. (Eds.), et al. Clinical Antimicrobial Assays (Oxford University Press, Oxford) pp. 35–44.
11 Andes DR and Craig WA. (2004) In vivo pharmacodynamic characterization of dalbavancin in the murine thigh infection model. Programs and Abstracts of the Forty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy2004Washington (American Society for Microbiology, Washington, DC, USA) pp. pp. 39 Abstract A-1872.
12 Flamm RK, Draghi DC, Goldstein BP, et al. (2005) Time kill kinetics of dalbavancin against clinical isolates of staphylococci and streptococci. One hundred and fifth General Meeting of the American Society for Microbiology2005Atlanta (American Society for Microbiology, Washington, DC, USA) pp. pp. 52.
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