Evaluation of once-daily vancomycin against methicillin-resistant ...

AAC Accepts, published online ahead of print on 14 November 2011
Antimicrob. Agents Chemother. doi:10.1128/AAC.05664-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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Version 2: AAC05664-11
Evaluation of once-daily vancomycin against methicillin-resistant
Staphylococcus aureus in a hollow fiber infection model
Anthony M. Nicasio 1* , Jürgen B. Bulitta 2,a , Thomas P. Lodise 1 , Rebecca E. D’Hondt 2 ,
Robert Kulawy 2 , Arnold Louie 2 and George L. Drusano 2
1. Albany College of Pharmacy & Health Sciences, 2. Ordway Research Institute, Albany,
NY
Running Title: ONCE-DAILY VANCOMYCIN HOLLOW FIBER MODEL
Current word count (218 words abstract)
Major Article limits: 250 words abstract
*Corresponding Author:
Anthony M. Nicasio, Pharm.D.
Assistant Professor
Department of Pharmacy Practice-Infectious Diseases
Albany College of Pharmacy and Health Sciences
106 New Scotland Avenue, Albany, NY 12208
(T):518-694-7324, (F): 518-694-7062 (E):Anthony.nicasio@acphs.edu
a : Present address: Centre for Medicine Use and Safety, Faculty of Pharmacy and
Pharmaceutical Sciences, Monash University, Melbourne, Victoria 3052, Australia
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Abstract
For methicillin-resistant Staphylococcus aureus (MRSA) infections, data suggest that
clinical response is significantly better if the total vancomycin AUC/MIC ratio is ≥ 400.
While AUC/MIC ratio is the accepted pharmacokinetic/pharmacodynamic index for
vancomycin, this target has been achieved using multiple daily doses. We are unaware
of a systematically designed dose-fractionation study to compare the bactericidal
activity of once-daily administration versus traditional twice daily administration.
A dose-fractionation study was performed with vancomycin in the in vitro hollow-fiber
infection model against a MRSA USA300 strain (MIC of 0.75 µg/mL) using an inoculum
of ~10 6 CFU/mL. The three vancomycin regimens evaluated for 168 h were 2g every
24h (Q24h) as 1h infusion, 1g Q12h as 1h infusion, and 2g Q24h as a continuous
infusion. Free steady-state concentrations (assuming 45% binding) for a total daily
AUC/MIC ratio of ≥400 were simulated for all regimens. A validated liquid
chromatography-tandem mass spectrometry method was used to determine
vancomycin concentrations. Although once-daily and twice-daily dosage regimens
exhibited total trough concentrations

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Keywords: vancomycin, once-daily administration, hollow-fiber infection model, MRSA,
methicillin-resistant Staphylococcus aureus, pharmacokinetics, pharmacodynamics, in
vitro
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Introduction
Given the dual threat of diminishing vancomycin efficacy against MRSA and
recent reports of rising nephrotoxicity (10, 20) (albeit potentially due to concomitant
dosing increases), new vancomycin dosing strategies are urgently needed. From a
pharmacodynamic viewpoint, there is an opportunity to alter standard dosage regimen
of vancomycin in clinical practice to optimize outcomes and minimize toxicity. Data
suggest that killing by vancomycin is concentration-dependent and a near maximal
bactericidal effect is achieved against MRSA when the ratio of the area under the total
vancomycin concentration-time curve and the MIC (AUC:MIC) exceeds 400 (20). While
an AUC:MIC of 400 is a well-recognized pharmacokinetic/pharmacodynamic (PK/PD)
target for vancomycin, this target has been determined using multiple daily doses which
has resulted in the default stance of using multiple daily dosing regimens in clinical
practice (4,5,7,9,12,13,18,19). The possibility of once-daily administration is appealing
from a PK/PD perspective as it affords the ability to achieve more robust AUCs in a
defined interval (i.e. during the first 6-12 h) while minimizing trough concentrations,
which predicts nephrotoxicity (10). However, we are unaware of a systematically
designed dose-fractionation study that compared the bactericidal activity of once-daily
administration versus twice-daily administration of vancomycin.
Our objective was to compare the rate and extent killing of MRSA USA300
between vancomycin regimens at 2g/day given as 1h infusion every 12h or every 24h,
or as continuous infusion in the hollow fiber infection model (HFIM). To our knowledge,
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this is the first dose-fractionation study that compared the bactericidal activity of once-
daily versus twice-daily administration of vancomycin.
Methods
Bacterial isolates, susceptibility testing, and drug preparation:
Vancomycin analytical grade powder was purchased from Fisher Scientific
(Fairlawn, NJ). A sterile stock solution of 10,000 µg/mL was prepared in sterile water,
diluted and aliquoted to the appropriate concentration, and stored at -80°C. MRSA
pulse-field gel electrophoresis type USA300 (obtained as a gift from Brian Tsuji, SUNY
Buffalo) was used for the susceptibility testing, time-kill studies, and HFIM. A quality
control strain (methicillin-susceptible S. aureus, ATCC 25925) was only used for all
susceptibility testing. Both of the strains were stored at -80°C in 20% glycerol and
cation-adjusted Mueller-Hinton II broth (MHB) (BBL, Sparks, MD). Prior to
experimentation, fresh isolates were grown on trypticase soy agar plates (TSA) with 5%
sheep blood and incubated at 35°C for 24h.
Susceptibility Testing:
The susceptibility testing was performed in triplicate using the broth microdilution
and agar dilution methods in MHB and MH agar following the CLSI guidelines (2).
Dilutions were performed by arithmetic increments of 0.25μg/mL for the minimum
inhibitory concentration (MIC) range of 0.25-2 μg/mL and increments of 1μg/mL for the
MIC ≥2 μg/mL. The minimum bactericidal concentration (MBC) was determined by sub-
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culturing of broth from the microdilution wells that were previously incubated for 24h and
plated onto TSA plates.
Time-kill assay:
A 48 hour time-kill experiment was conducted to evaluate the bactericidal activity
of various concentrations of vancomycin against MRSA USA 300. The vancomycin
concentrations used in the time-kill study (0, 0.75, 2.25, 8, 16, 25, and 50 µg/mL)
reflected the free vancomycin peak and trough concentrations for the dosing regimens
evaluated in the HFIM. A starting inoculum was used of the same density as that used
in the HFIM (10 5.8 CFU/mL) and the varying concentrations of vancomycin were added
to conical tubes. The time-kill experiment was performed in a water-shaker bath at
35°C and 100 rpm. Both MHB and drug were replaced after 24h by spinning the
bacterial suspension, removal of the supernatant, and re-suspension in fresh (drug-
containing) MHB in order to precisely maintain vancomycin concentrations and to
maintain fresh MHB. Quantitative cultures were assessed at 0 (pre-dose), 3, 6, 24, 27,
and 48h. Prior to culturing the bacterial samples on TSA agar, each sample was
centrifuged and re-suspended twice with normal saline to remove vancomycin.
Studies on the vancomycin less-susceptible subpopulations:
The mutation frequencies of less susceptible subpopulations at different multiples
of the MIC in agar were calculated as the ratio of colonies that grew onto drug
containing agar divided by the colonies that grew onto drug-free agar as previously
described by Louie et al. (11). Briefly, aliquots of the inoculum (1 - 5 ml) were plated
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onto agar containing 3X, 5X, 6X, 10X the baseline vancomycin agar MIC of the
organism and incubated at 35°C. After as long as 72h of incubation, the colonies on the
drug-containing media plates were enumerated and compared to the colony counts of
the total bacterial population previously assessed at 24h. Due to the limited bacterial
growth on plates with 5x, 6x, and 10x MIC (data not shown), 3X MIC plates were used
to determine the mutational frequency and less susceptible subpopulations in the HFIM.
Mutation frequencies at 3X MIC were performed on at least four separate occasions.
Hollow fiber infection model (HFIM):
A description of the hollow-fiber bioreactor (Cellulosic cartridge, FiberCell
System, Inc., Fredrick, MD) has been detailed in previous publications (24). Along with
vancomycin being injected into the central compartment, a continuous flow of fresh
MHB was also infused in order to represent the human simulated terminal half-life of
vancomycin. In order to maintain an isovolumetric environment, MHB was removed
from the central reservoir into a waste reservoir via a peristaltic pump (Masterflex; Cole-
Palmer Instrument Co., Chicago, IL). In order to inoculate the HFIM, MRSA was
injected and confined to the hollow-fiber cartridge (i.e. the extracapillary space of the
hollow fiber system). Broth medium including nutrients and vancomycin was exchanged
by a rapid intercompartmental clearance between the central reservoir and the hollow
fiber cartridge.
Schedule-response experiment:
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A dose-fractionation study was performed in duplicate with the same USA300
MRSA isolate using the HFIM in a humidified incubator at 35°C. The isolate was taken
from the -80° freezer and sub-cultured twice on TSA plates prior to preparation of the
bacterial suspension. The inoculum was prepared by taking 3–5 freshly grown mid-size
colonies and placing them into fresh MHB at 35°C. The inoculum was grown to
logarithmic state and diluted with MHB to approximately 10 5.8 CFU/mL; this was
measured with a spectrophotometer to an optical density of 630nm.
Twelve milliliters of the inoculum were injected into the peripheral port of each of
the four hollow-fiber cartridges. The four cartridges (for each of the two experiments)
consisted of a growth control (drug-free) and three vancomycin dosing arms that were
designed to achieve a total AUC/MIC ratio of ≥400 over 168 h. The three vancomycin
regimens simulated the steady-state human concentrations of unbound vancomycin at a
percent protein binding of 45% (1, 26). The mean pharmacokinetic parameters and
CLCR values for these vancomycin regimens were derived from a vancomycin
population pharmacokinetic model (6, 21, 22). A glomerular filtration rate of 88 mL/min
to reflect elderly patients with normal renal function yielded a total vancomycin
clearance of 5.0 L/h. For a daily dose of 2 g, this yielded a total drug AUC of 400
µg•h/mL and an unbound AUC of 220 µg•h/mL. The half-life simulated in the HFIM was
approximately 4.8 h. The vancomycin dosing arms of 1g every 12h, 2g every 24h
(intermittent 1h infusion), and 2g every 24h (continuous-infusion) were achieved by
simulating a peak non-protein bound (free) drug concentration of 30.5, 18.2, 9.2 µg/mL
(16). To achieve the targeted half-life for vancomycin, the central compartment volume
was 485 mL and the medium within the compartment was isovolumetrically replaced
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with fresh MHB at a flow rate of 1.17 ml/min. Vancomycin concentration samples for
each of the drug-containing arms were acquired at ≥12 different timepoints, mainly
within the first 48h, from the drug sampling port of the central reservoir. These
concentrations were later confirmed with a validated liquid chromatography-tandem
mass spectrometry (LC/MS/MS) method performed at Ordway Research Institute
(Albany, NY).
Viable counts of the total population were determined at 0 (pre-dose), 6, 24, 30,
72, 96, 120, 144, and 168h. Viable counts of less-susceptible population(s) on 3x MIC
plates were determined every 24 h. Samples of the inoculum were washed twice with
normal saline to minimize drug carry-over. The samples were serially diluted 10-fold in
normal saline and cultured onto TSA for each of the timepoints. Agar plates were
incubated for 24h (TSA plates) or 72h (vancomycin agar plates) at 35°C. A volume of
200 µL was plated for all samples for which the lower limit of detection was 6 colonies
(1.5 log10 CFU/mL).
To determine the susceptibility, and therefore to confirm the development of
resistance, MICs were performed in duplicate on colonies that were obtained at each
timepoint from each experimental arm, excluding the control arm.
LC/MS/MS assay:
The samples (vancomycin in MHB) were diluted with high pressure liquid
chromatography water (0.050mL sample into 1.00mL 50:50 methanol:water), and were
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analyzed by LC/MS/MS. The LC/MS/MS system was comprised of a Shimadzu
Prominence UFLC system and a Thermo Scientific TSQ Vantage LC/MS/MS.
Chromatographic separation was performed using a Thermo Scientific Hypersil
C8 column, 5 µm, 50 x 3.0 mm column and a mobile phase consisting of 85% 5 mM
ammonium acetate pH 3.5 and 15% methanol, at a flow rate of 0.6 ml/min. Vancomycin
concentrations were obtained using LC/MS/MS monitoring the MS/MS transition m/z
725 (doubly charged ion) → m/z 144. Analysis run time was 5.0 minutes. The assay
was linear over a range of 0.05 – 50.0 µg/ml (r 2 > 0.995). The inter-day precision (%CV)
for the quality control samples (which were analyzed in triplicate at three concentrations
[1.0, 5.0, and 10.0 µg/mL]) ranged from 2.68 to 3.40%; the accuracy (%recovery)
ranged from 97.5 to 101%.
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Results
Susceptibility and mutation frequency: The agar dilution and broth microdilution
modal MICs for the USA300 MRSA strain were both 0.75 µg/mL; the MBC was 1
µg/mL. The log10 mutation frequency ranged from -5.8 to -6.9 for agar plates with 3x the
baseline MIC and from -7.1 to -7.7 for 5x MIC based on mutation frequencies
determined on three different days. The less-susceptible sub-populations that grew in
the presence of 3X MIC exhibited a higher MIC (2-3 µg/mL).
Time-kill studies: The rate of bacterial killing was 1.5 to 2 log10 killing at 6h for all
tested vancomycin concentrations. Between 6 and 24h, extensive regrowth was
observed for 0.75 µg/mL. Bactericidal activity (i.e., ≥3 log-kill) was observed for all
concentrations within 2.25 to 50 µg/mL and the overall extent of killing and lack of
regrowth up to 48 h for these concentrations was comparable (Figure 1).
Hollow fiber infection model: The observed vancomycin concentration time
profiles for the three vancomycin dosage regimens are provided in Figure 2. The free-
drug AUCs (determined by the linear trapezoidal rule) were well comparable between
regimens with 262.2 ± 29.5 for q24h dosing, 242.1 ± 31 for q12h dosing, and 244.2 ±
1.3 for continuous infusion.
All three vancomycin dosing regimens with a total AUC/MIC ratio of ≥400 had a
comparable bactericidal activity against MRSA. As shown in Figure 3, vancomycin
experienced a slow rate of kill within the first 6h (-0.91 to -1.24 log10 CFU/mL) and
attained bactericidal activity (-3.27 to -3.57 log10 CFU/mL) at 24h which was maintained
up to 168h. Although bactericidal activity was achieved with all of the vancomycin
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dosing regimens in the HFIM, it is important to note that sterility was not attained. Of
the colonies that did exist near or above the limit of detection for any of the dosing
regimens, the MICs of 0.75-1 µg/mL did not change from baseline. Based on the
results from the drug-containing agar with vancomycin at 3X MIC, all dosing regimens
prevented breakthrough of non-susceptible sub-populations for each of the
experimental treatment arms during the seven day HFIM experimental period. No
resistant colonies were found on 3x MIC plates for the drug treatment arms, whereas
the growth control showed a number of colonies in agreement with the mutation
frequency on 3x MIC plates.
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Discussion
After half a century of clinical use, the optimal mode of administration has not yet
been elucidated for vancomycin. Although vancomycin is classified as an AUC/MIC
ratio-driven antimicrobial, the data that support this were derived from multiple daily
dosing regimens (4, 5, 7, 9, 12, 13, 18, 19). To our knowledge, this is the first
contemporary dose-fractionation study designed to evaluate whether killing by
vancomycin is driven by the AUC/MIC ratio. Overall, when dosed to simulate a total
AUC/MIC ratio of ≥400 in the HFIM, we were able to demonstrate comparable bacterial
efficacy between once-daily vancomycin administration, continuous infusion, and twice-
daily administration. Within each of the dosing regimens, vancomycin exhibited
bactericidal activity after 24h and maintained this activity for seven consecutive days. In
addition to the sustained bactericidal effects, all three regimens suppressed the
development of non-susceptible subpopulations and were able to maintain MIC values
≤1 µg/mL throughout the seven days.
The exploration of dosing strategies to enhance the utility of an antibiotic agent is
not a new approach (23, 25). This approach was utilized with daptomycin and was
pivotal in the discovery that skeletal muscle toxicity issues were associated with more
frequent dosing rather than a maximum concentration (15). Similar findings were
exhibited with aminoglycosides where it was determined that more frequent dosing
intervals led to higher incidences of nephrotoxicity (17). To date, we are only aware of
one clinical and one in vitro study to compare vancomycin once-daily versus twice-daily
dosing regimens. In the clinical study, which focused on efficacy and safety, the
outcomes were comparable between once-daily and twice-daily groups; however, it
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should be noted that the patients studied were not severely ill (3). In the in vitro study,
Houlihan et al. utilized a simulated endocarditis vegetation model at a high initial
inoculum (10 9 CFU/mL) in order to investigate the use of 2g-per-day regimen,
administered as once-daily, twice-daily, or continuous-infusion, for a total of 72h (8).
Similar to our study, the authors observed that trough levels did not dictate bacterial
efficacy despite a bacterial inoculum as high as 10 9 CFU/mL when the total AUC/MIC
ratios associated with the dosing regimens were all >400.
Our results carry important implications for clinical practice. First, they challenge
the need to obtain frequent trough concentrations: bactericidal efficacy was observed
despite a trough concentration that was well below the lowest recommended total
trough concentration of 10µg/mL for the total daily vancomycin dose of 2g given as a
once-daily dosing regimen. Second, they call into question the need for trough
concentrations of 15-20 µg/mL for all patients. Our results are similar to that of another
study by our group (16) which found that regimens producing trough values ≥ 15µg/mL
were not always necessary to provide a total AUC/MIC ratio of ≥ 400, especially if the
vancomycin MIC of the MRSA is ≤ 1 µg/mL.
A second clinical implication of our data is the convenience associated with once-
daily administration of vancomycin. Multiple daily dosing of a drug is inconvenient due
to the increased use of nursing time and the increased risk of medication errors
associated with the frequency of administration and the time intervals between doses
(14). The administration of vancomycin as a once-daily drug would alleviate these
inconveniences, in addition to facilitating the transition to an outpatient treatment
protocol.
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Finally, by minimizing the trough concentration needed to achieve the desired
AUC/MIC ratio, one may be able to reduce the risk of nephrotoxicity associated with
vancomycin. Since the daily AUC value is independent of dosing frequency (16), and
nephrotoxicity appears to be linked to the trough concentration (10), it may be
advantageous to move from multiple daily dosing to once-daily vancomycin dosing in
clinical practice. This approach needs explicit testing in randomized trials.
Overall, at a moderate inoculum, we demonstrated with a HFIM that vancomycin
given as a once-daily dosing regimen exhibited similar bactericidal activity as conferred
by continuous infusion and twice-daily dosing regimens against MRSA USA 300 (MIC:
0.75 µg/mL) while maintaining the same AUC/MIC ratio of ≥400. Vancomycin was able
to maintain efficacy and suppress the development of non-susceptible subpopulations
during a clinically relevant treatment duration of seven days. Therefore, these results
question the need for aggressive vancomycin trough concentrations of 15-20 µg/mL for
all infections and suggests that the efficacy of vancomycin is not predicated on the
trough concentration but rather the AUC when the MIC of the pathogen is

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Acknowledgments:
We would like to thank Brian van Scoy and David Brown for their technical assistance,
and Allison Krug MPH for her editorial support.
Potential Conflicts of Interest:
A.M.N., R.E.D., R.K., and A.L. have no conflicts of interest. JBB has received grant
support from Pfizer and Trius Therapeutics. T.P.L. is a consultant and speaker for
Pfizer, Cubist Pharmaceuticals, Astellas, and Forest. T.P.L. has received grant support
from Astellas, Cubist, and Pfizer. G.L.D. is a consultant for Forest, Cerexa
Pharmaceuticals and AstraZeneca. G.L.D. has received grant support from Forest.
Funding:
This study was non-funded.
Presented in part:
Interscience Conference on Antimicrobial Agents and Chemotherapy, 51 st Annual
Meeting, Chicago, IL, September, 2011.
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Fig 1: Activity of vancomycin tested at concentrations ranging from 0.75 to
Bacterial Density (log 10 CFU/mL)
10
8
6
4
2
50µg/mL in a static time-kill assay against USA300 MRSA strain at an
inoculum of 10 5.8 CFU/mL.
0 12 24 36 48
Time (h)
22
growth control
0.75μg/mL
2.25μg/mL
8μg/mL
16μg/mL
25μg/mL
50μg/mL

399
400
401
402
403
404
405
406
407
408
409
410
Fig 2. Observed vancomycin concentration time profiles for (A) 2g every 24h, (B)
(A)
Vancomycin Concentration (μg/mL
(B)
Vancomycin Concentration (μg/mL)
25
20
15
10
5
40
35
30
25
20
15
10
5
1g every 12h, (C) 2g continuous infusion in the HFIM.
0
0 20 40 100 120 140 160 180 200
Time (hr)
Time (hr)
0
0 20 40 100 120 140 160 180 200
23

411
412
C)
Vancomycin Concentration (μg/mL)
40
30
20
10
0
0 20 40 100 120 140 160 180 200
Time (hr)
24

413
414
415
416
417
418
419
420
421
422
423
Fig 3. An in vitro HFIM was used to compare three different vancomycin regimens
Bacterial D ensity (log 10 CFU/mL)
14
12
10
8
6
4
2
(and a growth control): 2g every 24h, 1g every 12h, and 2g continuous-
infusion. The figure show the average ± standard deviation of the log 10
CFU/mL from 0 to 168h based on two replicated for all cohorts except for the
2g continuous infusion for that one replicate was available.
0
0 20 40 60 80 100 120 140 160 180
Time (hr)
25
Control
2g every 24h
1g every 12h
2g continous infusion