Abstract

Objective: Daptomycin exhibits bactericidal activity against clinically significant Gram-positive bacteria despite being highly bound to human proteins. Evaluations characterizing the effect of protein on daptomycin pharmacodynamics are warranted.

Methods: We utilized an in vitro pharmacodynamic model to simulate daptomycin regimens of 6 mg/kg/day under controlled conditions of pH, calcium and/or protein. Free concentrations were simulated in broth, whereas total concentrations were simulated in broth supplemented with human albumin. Bacterial density was profiled over 48 h for two methicillin-resistant Staphylococcus aureus (MRSA) and two vancomycin-resistant Enterococcus faecium (VREF) clinical isolates.

Results: Daptomycin exhibited bactericidal activity against both MRSA isolates, with time to 99.9% killing occurring at 0.5 h and 8 h in broth and in albumin-supplemented broth, respectively. Initial kill was observed against both VREF isolates followed by regrowth. There was no statistical difference (P>0.05) in extent of bacterial kill at 24 or 48 h between the different media.

Conclusions: Although delayed, the extent of kill for daptomycin was unaltered against all isolates in albumin-supplemented broth. Further antimicrobial studies that incorporate protein are warranted to assess the influence of protein in the pharmacodynamic evaluation of antimicrobials.

Introduction

Daptomycin is a cyclic lipopeptide antibiotic with potent Gram-positive activity against a wide variety of organisms, including methicillin-resistant staphylococci and vancomycin-resistant enterococci.1,2 Despite its high affinity for protein (>90% bound), daptomycin exhibits rapid concentration-dependent killing.35

The influence of protein binding remains uncertain given the observation of both clinical and microbiological success and failure with highly protein bound antimicrobials. Nevertheless, there have been several in vitro studies that have demonstrated that the presence of serum or albumin may enhance the activity of certain antibiotics.58 However, the creation of testing conditions that mimic the dynamic physiological interaction between binding of proteins to antimicrobials remains difficult, expensive and lacks standardization. We conducted this in vitro study to determine the influence of protein on daptomycin's activity, with antibiotic exposures reflecting one of the current suggested clinical doses, 6 mg/kg/24 h.

Materials and methods

Bacterial strains:

A total of four strains were evaluated: two clinical strains of methicillin-resistant Staphylococcus aureus (MRSA-67 and MRSA-494) and two clinical strains of vancomycin-resistant Enterococcus faecium (VREF-SF12047 and VREF-12366).

Susceptibility testing and media

Daptomycin minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were performed as previously described.9 Supplemented (calcium at 75 mg/L and magnesium at 12.5 mg/L) Mueller–Hinton broth (SMHB) with 4 g/dL of human albumin (Red Cross, Detroit, MI, USA) and SMHB with 50% human serum were used for susceptibility tests and for preliminary time–kill experiments.9 All broth media for all experiments were further titrated with sodium hydroxide and calcium chloride solution, as necessary, to achieve a pH of 7.4 and physiological calcium levels (free: 1.15–1.32 mmol/L and total: 2.1–2.6 mmol/L).9,10 Samples from broth were verified for albumin and free and total calcium content by Detroit Medical Center chemistry laboratories. All samples from time–kill and model experiments for bacterial enumeration were plated on Tryptic Soy Agar (TSA).

Preliminary time–kill experiments

To determine the potential impact of protein binding on daptomycin, time–kill experiments were performed with the four study isolates. Initial inocula of 1 × 106 log10 cfu/mL were targeted in 24-well tissue culture plates (final volume, 2.0 mL per well). Time–kill experiments were performed with the above media and each organism with daptomycin concentrations as follows: daptomycin at four-fold the MIC in broth and at total concentrations (derived from free concentrations at four-fold the MIC) in broth plus protein. Total concentrations tested were based on 100×(4 × MIC)/8 to account for the change in MIC due to protein binding and to provide equivalent exposures to free concentration tests.5 Samples from wells were extracted at 0, 8 and 24 h and diluted in cold normal saline at 10–10 000-fold dilutions to circumvent some antimicrobial carryover.

In vitro pharmacodynamic infection models

A previously described in vitro pharmacodynamic model was utilized with an initial inoculum of 1 × 106 log10 cfu/mL.10 Daptomycin was administered as follows: for media containing protein, a 6 mg/kg/24 h simulating total concentrations (peak concentration = 80 mg/L); media without protein, a 6 mg/kg/24 h simulating free concentrations (peak concentration = 6 mg/L).9,10 A peristaltic pump was used to simulate the half-life of 8 h for daptomycin. pH was monitored throughout all experiments. All model experiments were performed in triplicate to ensure reproducibility.

Pharmacodynamic analysis

Duplicate samples were removed at 0, 0.5, 1, 2, 4, 6, 8, 24, 28, 32 and 48 h and diluted in cold saline prior to plating onto TSA plates. Plates were incubated at 35°C for 24 h prior to colony enumeration. Samples were diluted 10–10 000-fold in cold normal saline and vacuum-filtered to reduce antibiotic carryover when plating. The limit of detection for this method of colony count determination is 2.0 log10 cfu/mL. Pharmacodynamic profiles were constructed by plotting bacterial density (log10 cfu/mL) over time. Bactericidal activity was defined as a bacterial density reduction of 99.9% from the initial inoculum.

Pharmacokinetic analysis

Samples were obtained at 0.5, 1, 2, 4, 8, 24, 32 and 48 h for determination of antibiotic concentrations and were stored at −70°C until ready for analysis. Concentrations of daptomycin were determined by a previously described microbioassay.1,9 The half-lives, peak concentrations and area under the concentration-time curves (AUCs) were determined by the trapezoidal rule with PK Analyst software (Micromath Research, St Louis, MO, USA).

Resistance

Samples (100 μL) from 24 and 48 h were plated on TSA plates containing four- and eight-fold the MIC of daptomycin to assess the development of resistance. Plates were then examined for growth after 48 h of incubation at 35°C.

Statistical analysis

Bacterial densities at sampled time points were compared by two-way analysis of variance with Tukey's post-hoc test. A P value of ≤0.05 was considered to be significant. All statistical analyses were performed using SPSS Statistical Software (Release 10.07; SPSS, Inc., Chicago, IL, USA).

Results

Susceptibility testing and media parameters

Daptomycin susceptibility results for MRSA-67, MRSA-494, VREF-SF12047 and VREF-12366 are presented in Table 1. Overall, MIC values increased two to eight-fold in the presence of albumin and decreased two-fold in the presence of serum. Calcium, albumin and pH was confirmed with each batch of broth (mean±standard deviation): total calcium=2.41±0.28 mM, ionized calcium=1.20±0.09 mM, albumin=4.0±0.1, pH = 7.4±0.1.

Pharmacodynamics

Preliminary time–kill experiments revealed no statistical differences in bacterial densities at 24 h between broth and broth plus serum for both MRSA and VREF isolates. Significantly (P>0.05) higher densities were noted between 4–8 h with broth plus albumin than broth plus serum for all tested isolates (results not shown). Therefore, broth plus albumin was selected for all in vitro model simulations as a conservative measure to prevent overestimation of pharmacodynamic effects. Model simulation studies for daptomycin simulated as free and as total plus albumin are depicted in Figure 1 for MRSA (a) and for VREF (b). All growth control profiles reached ∼9.0 log10 cfu/mL by 8 h and were maintained for the duration of the study. Rapid and pronounced bactericidal activity to near undetectable levels was noted to occur for each free concentration model simulation against both MRSA isolates by 0.5 h. Bactericidal activity was also noted when total daptomycin concentrations were simulated in broth plus albumin when tested against MRSA-67 and MRSA-494, and occurred by 8 and 24 h, respectively. Daptomycin achieved bactericidal activity against VREF isolates by 1 and 8 h for free concentration simulations and total concentrations plus protein, respectively. Regrowth was noted for all simulations of daptomycin against VREF, independent of the presence of protein. In all pharmacodynamic profiles, there were no statistical differences (P >0.05) in the extent of bacterial kill at 24 or 48 h between the different media. Plating of detectable 48 h samples revealed no resistant colonies.

Pharmacokinetic exposures and parameters were within 10% of targeted values. Peak concentrations achieved for total drug simulations and for free drug simulations were 77±7.1 and 6.0±0.1 mg/L, respectively. Half-lives were 8.0±0.3 h for all simulations. Mean AUC values for simulations of broth with and without protein were 515 and 53 mg·h/L, respectively.

Discussion

Significant antibiotic binding to proteins has been a topic of controversy for years. Historically, the degree of protein binding has been more of an issue for antibiotics with targeted activity against Gram-positive organisms than for Gram-negative organisms. Studies completed by Brier et al.3 and our laboratory have demonstrated that daptomycin is ∼91%–95% bound to serum proteins. Both teicoplanin activity and daptomycin activity are somewhat diminished by the presence of protein; however, the extent of suppression may be less with daptomycin.6 This may be due, in part, to the affinity rates of the antibiotic to the target protein as well as the length of time the antibiotic is bound.

The observed rapid and sustained bactericidal activity by daptomycin in this study is congruent with previous studies.1,4,9,10 Also consistent with previous findings was the observation of slight regrowth when daptomycin was studied against VREF at dose simulations of less than or equal to 6 mg/kg given every 24 h. Additional pharmacodynamic studies looking into dose–effect relationships revealed that larger doses of 7 mg/kg achieved and maintained bactericidal activity.10 Regrowth may be due to the clearance of daptomycin at the end of the dosing interval in conjunction with changes in a heterogeneous bacterial population since no susceptibility changes were noted. These findings may also be limited by the small number of isolates studied and quantification errors. There is also no clear reason for the reduced or lack of effect of subsequent doses; this requires further investigation.10

The main discovery was that the time to achieve bactericidal activity was prolonged in the presence of human albumin, whereas the extent of overall kill was generally unchanged. These observed differences are probably due to susceptibility alterations when protein was supplemented to broth media. Similar to previous investigations evaluating free AUC/MIC ratios that correlate bactericidal activity for daptomycin against S. aureus, we observed ratios >175 for both MRSA strains.4,9,10 However, despite the reduction in AUC/MIC based on the increases in MICs in the presence of albumin (MICs increased 2–8 times when tested in protein-supplemented broth for study isolates), bactericidal activity to our limits of detection was achieved by 24 h and was maintained for the duration of the MRSA experiments. Since no data are available for the differential clearance of free and unbound daptomycin, we were unable to simulate them separately. Human albumin was selected in this study as the protein supplement in broth as a conservative measure for daptomycin evaluations. Currently, there are no standard methods for incorporation of protein to study the pharmacodynamics of antimicrobials. Therefore, further studies are needed to investigate different methodologies incorporating protein. Such studies should help differentiate between free and total concentration effects in both susceptibility and pharmacodynamic evaluations that incorporate analysis on the affinities between protein and antimicrobials.

Figure 1.

Pharmacodynamic profiles of daptomycin simulated as free and as total with protein (human albumin) against both MRSA (a) isolates and against both VREF (b) isolates in in vitro pharmacodynamic models. Lower limit of detection is 2.0 log10 cfu/mL.

Figure 1.

Pharmacodynamic profiles of daptomycin simulated as free and as total with protein (human albumin) against both MRSA (a) isolates and against both VREF (b) isolates in in vitro pharmacodynamic models. Lower limit of detection is 2.0 log10 cfu/mL.

Table 1.

Susceptibility results of tested isolates

 MIC/MBC (mg/L) for strains:
 
   
 MRSA-67 MRSA-494 VREF-SF12047 VREF-12366 
Daptomycin 0.125/0.5 0.25/0.25 4.0/4.0 2.0/4.0 
Daptomycin     with albumin 1.0/2.0 1.0/4.0 8.0/32 8.0/16 
Daptomycin     with human     serum 0.063/0.125 0.125/0.25 2.0/4.0 2.0/4.0 
 MIC/MBC (mg/L) for strains:
 
   
 MRSA-67 MRSA-494 VREF-SF12047 VREF-12366 
Daptomycin 0.125/0.5 0.25/0.25 4.0/4.0 2.0/4.0 
Daptomycin     with albumin 1.0/2.0 1.0/4.0 8.0/32 8.0/16 
Daptomycin     with human     serum 0.063/0.125 0.125/0.25 2.0/4.0 2.0/4.0 

MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration.

We would like to thank J. Alder and T. Chen of Cubist Pharmaceuticals for their financial support and advice.

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Author notes

1Anti-Infective Research Laboratory, Department of Pharmacy Practice, Eugene Applebaum College of Pharmacy and Health Sciences; 2School of Medicine, Wayne State University, 259 Mack Ave., Detroit, MI 48201, USA