Bactericidal Activity and Resistance Development Profiling of Dalbavancin

Dalbavancin, a semisynthetic lipoglycopeptide being developedfor the treatment of skin and skin structure infections (SSSIs),has a half-life of 5 to 7 days in humans and offers promisefor a convenient weekly dosing regimen. We studied the in vitrobactericidal activity of dalbavancin against target organisms,using the concentrations that are maintained in human bloodwith the proposed dosage regimen. Dalbavancin minimal bactericidalconcentrations (MBCs) were $≤$ 0.5 µg/ml for eight staphylococcalisolates; and for six of these strains, including one vancomycin-intermediateStaphylococcus aureus (VISA) isolate, the MBCs were equal toor within 1 doubling dilution of the MIC. Dalbavancin MICs forall three Streptococcus pyogenes strains were 0.008 µg/ml,as were the MBCs for two of the isolates. In time-kill studiesconducted with a different set of seven strains (two methicillin-susceptibleS. aureus isolates, three methicillin-resistant S. aureus isolates,one VISA isolate, and one S. pyogenes isolate), all strainsexhibited a $≥$ 3-log₁₀ decrease in their viable counts when theywere exposed to $≥$ 1 µg/ml of dalbavancin for 24 h. Resistancedevelopment studies by both direct selection (resistance frequency,<10^–10) and serial passage failed to produce stablemutants with decreased susceptibility to dalbavancin. Theseobservations suggest that dalbavancin will be an effective choicefor the management of patients with SSSIs.

INTRODUCTION

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Introduction

Dalbavancin is a novel semisynthetic lipoglycopeptide that isadministered intravenously and that is currently under regulatoryreview for the treatment of complicated skin and skin structureinfections (SSSIs) caused by gram-positive bacteria. Takingadvantage of the dalbavancin half-life of 5 to 7 days, the proposeddosing regimen in humans, which consists of 1 g on day 1 and0.5 g on day 8, was designed to maintain bactericidal concentrationsof dalbavancin in blood over the entire 2-week treatment period.The maintenance of therapeutic levels throughout therapy shouldcontribute to improved outcomes and should also reduce the likelihoodof the emergence of resistance. The convenience of once-weeklyintravenous infusion was another consideration in developingthis regimen. While a number of studies have been conductedto establish dalbavancin's MIC profile, studies of its bactericidalactivity, particularly at pharmacokinetically relevant concentrations,have been limited (9, 10, 12, 13, 18, 19). Using informationon its pharmacokinetics and estimates of protein binding, westudied the bactericidal activity of dalbavancin against themain SSSI target organisms, Staphylococcus aureus and Streptococcuspyogenes (5, 7, 18), over a range of concentrations that includedapproximations of the levels of free drug present in human bloodafter infusion of a dose and 1 week later.

At the time that this study was initiated, the pharmacokineticprofile of dalbavancin in humans had been characterized. Theconcentrations of total drug of 20 to 40 µg/ml were present7 days after a therapeutic dose, and peak concentrations atthe end of infusion were >200 and >150 µg/ml forthe first and second doses, respectively. In addition, dalbavancinis approximately 93% protein bound (2, 16).

MATERIALS AND METHODS

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Materials and Methods

The staphylococcal and S. pyogenes isolates used in this studywere selected as clinical strains of known resistance phenotypes(e.g., methicillin-resistant S. aureus [MRSA] and vancomycin-intermediateS. aureus [VISA]) from various hospitals in the United States.MICs, minimal bactericidal concentrations (MBCs), and time-killprofiles were determined according to Clinical and LaboratoryStandards Institute (CLSI; formerly NCCLS) guidelines (14, 15).MICs and MBCs were determined with validated dry-form microtiterpanels provided by TREK Diagnostics (8). Time-kill studies wereperformed by using plastic tubes and cation-adjusted Mueller-Hintonbroth with 0.02% polysorbate-80 (P-80). Addition of P-80 preventsdalbavancin from sticking to plastic; and in broth microdilutionMIC determinations by the CLSI methodology, P-80 has been shownto provide the same results obtained with dry-form panels, whichdo not require P-80 (1, 8). Broth microdilution determinationof the MIC for dalbavancin by the CLSI methodology has now beenstandardized with a 0.002% final concentration of P-80 (4).For S. pyogenes, the medium was supplemented with 2 to 5% lysedhorse blood. The starting inoculum was approximately 10⁷ CFU/ml,and the concentrations of dalbavancin tested were chosen toreflect the calculated free levels of dalbavancin in plasmaover a 1-week dosing interval (1, 4, and 16 µg/ml) aswell as a lower concentration (0.25 µg/ml) which moreclosely reflected the MBCs for some strains. Estimates of freedalbavancin levels in blood were based on the extent of proteinbinding (93%) in human plasma and total human blood levels ( $≥$ 200and ca. 150 µg/ml after infusion of the first and seconddoses, respectively; >20 µg/ml at the end of each 1-weekdosing interval) (2).

Direct selection and serial passage studies for the detectionof resistance development were performed with one methicillin-susceptibleS. aureus (MSSA) isolate, three MRSA isolates, one VISA isolate,and one methicillin-resistant Staphylococcus epidermidis isolate.The procedures described by Silverman et al. (17) were usedas a guideline for these studies. To detect spontaneous mutants,an inoculum of approximately 1 x 10⁹ to 1 x 10¹⁰ CFU was platedonto the surfaces of Mueller-Hinton agar plates containing dalbavancinat concentrations of 0.5x, 1x, 2x, 4x, and 8x the initial brothmicrodilution MIC. The dalbavancin MICs of any potential mutantsgrowing on these selective plates were determined by broth microdilution.

The same staphylococcal strains were subjected to serial passagein the presence of sub-MICs of dalbavancin over 20 consecutivedays by a broth microdilution method with dry-form panels. Foreach passage day the entire content of the dalbavancin microtiterwell (100 µl) with the highest concentration of drug thatallowed growth was used to generate the inoculum for a freshmicrotiter plate. After 20 passages, the dalbavancin MICs ofeach strain were determined and compared with those obtainedprior to the initiation of serial passage.

RESULTS AND DISCUSSION

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Results and Discussion

The MICs and MBCs of dalbavancin, vancomycin, and teicoplaninfor five S. aureus isolates, five coagulase-negative staphylococci,three S. pyogenes isolates, and two isolates each of Enterococcusfaecalis and Enterococcus faecium are presented in Table 1.In every case, dalbavancin had lower MICs and MBCs than bothvancomycin and teicoplanin, including those for a VISA strainand coagulase-negative staphylococcal isolates with reducedsusceptibilities to vancomycin and/or teicoplanin. The dalbavancinMICs ranged from 0.03 to 0.12 µg/ml for 9 of 10 staphylococcalstrains, and the dalbavancin MIC was 1 µg/ml for the VISAstrain. Dalbavancin MBCs were $≤$ 0.5 µg/ml for eight of thestaphylococcal isolates; and for six strains, including theVISA strain, the dalbavancin MBCs were equal to or within 1doubling dilution of the MIC. The dalbavancin MICs for all threeS. pyogenes strains were 0.008 µg/ml, as were the MBCsfor two of the isolates. The one S. pyogenes isolate that hadthe high dalbavancin MBC/MIC ratio also had high ratios withteicoplanin and vancomycin, which indicates that this particularstrain may have some underlying physiology that allows it tobe relatively refractory to this class of agents. Nonetheless,the dalbavancin MBC for this strain was well below the therapeuticlevels of this drug. The MICs and MBCs of dalbavancin for thisspecies were at least an order of magnitude lower than thoseof vancomycin. Against both the E. faecalis and the E. faeciumstrains, the MBC-to-MIC ratios for dalbavancin, teicoplanin,and vancomycin were all high. Although this group of agentsis not considered effectively bactericidal against enterococci,the MBCs obtained with dalbavancin were lower than those obtainedwith either vancomycin or teicoplanin.

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TABLE 1. Comparison of minimal bactericidal concentrations of dalbavancin, vancomycin, and teicoplanin

Time-kill studies were conducted with a different set of sixstrains of S. aureus (two MSSA strains, three MRSA strains,and one VISA strain) and one S. pyogenes strain. Regardlessof phenotype, all strains exhibited a $≥$ 3-log₁₀ decrease in viablecounts and, in most cases, exhibited about a 5-log₁₀ decreasewhen they were exposed to $≥$ 1 µg/ml of dalbavancin for 24h. Two strains (one MSSA strain and one MRSA strain) were alsokilled to that extent by the lowest dalbavancin concentrationtested, 0.25 µg/ml. The data for three of the S. aureusisolates (one isolate each of MSSA, MRSA, and VISA) are shownin Fig. 1A to C, respectively. No further killing was obtainedby increasing the dalbavancin concentration to 4 µg/ml(Fig. 1) or even to 16 µg/ml (data not shown). At higherconcentrations (8 and 32 µg/ml), a $≥$ 3-log₁₀ killing ofall of the S. aureus isolates was also seen with vancomycinand teicoplanin, but in some cases the extent of killing wasless than that achieved with 0.25 or 1 µg/ml of dalbavancin(data not shown). Dalbavancin also produced >3-log₁₀ killingof S. pyogenes at 0.25 µg/ml and about a 5-log₁₀ reductionat $≥$ 1 µg/ml (Fig. 1D). Vancomycin and teicoplanin had notablypoorer bactericidal activities against this organism, achieving<2-log₁₀ killing. Thus, even when it was tested at very lowconcentrations, dalbavancin had more potent bactericidal activitythan vancomycin and teicoplanin. These observations correlatewith the findings of studies that demonstrated that lower andless frequent doses of dalbavancin were effective in animalinfection models (3, 6). The strong bactericidal activity ofdalbavancin at concentrations of free drug that are sustainedin humans throughout the therapeutic interval (approximately15 to 20 µg/ml after infusion and 1.5 to 3 µg/mlafter 7 days) suggests a low potential for the selection ofresistance in patients.

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FIG. 1. Time kill curves of dalbavancin versus S. aureus and S. pyogenes isolates. (A) Methicillin-susceptible S. aureus (strain FB1109397; dalbavancin MIC, 0.06 µg/ml; teicoplanin MIC, 1 µg/ml; vancomycin MIC, 1 µg/ml); (B) methicillin-resistant S. aureus (strain FB1109400; dalbavancin MIC, 0.06 µg/ml; teicoplanin MIC, 1 µg/ml; vancomycin MIC, 1 µg/ml); (C) vancomycin-intermediate S. aureus (strain Mu50 Japan; dalbavancin MIC, 0.5 µg/ml; teicoplanin MIC, 4 µg/ml; vancomycin MIC, 4 µg/ml); (D) S. pyogenes (strain FB1110016; dalbavancin MIC, 0.008 µg/ml; teicoplanin MIC, 0.03 µg/ml; vancomycin MIC, 0.5 µg/ml).

The potential for staphylococci to develop resistance to dalbavancinwas studied by means of direct selection and by serial passageat subtherapeutic concentrations. In direct selection experimentswith the six strains tested (one MSSA strain, three MRSA strains,one VISA strain, and one methicillin-resistant Staphylococcusepidermidis strain), no colonies with increased MICs were obtainedon dalbavancin-containing plates (frequency, <10^–10).The same six strains were passaged in the presence of subinhibitoryconcentrations of dalbavancin. After 20 passages, four strains(including the VISA strain) had a dalbavancin MIC that was within1 doubling dilution of the MIC obtained prior to the 20 passages.For two MRSA strains, increases of 2 or 3 dilutions were seen(to 0.25 and 0.5 µg/ml). When these isolates were grownon drug-free medium for three consecutive days and retested,the MICs were equivalent or within 1 doubling dilution of thestarting MICs.

This study was conducted to evaluate the bactericidal activityof dalbavancin over a range of concentrations of free drug thatare present in human blood, as well as a lower concentration,and to assess the propensities of target organisms to developresistance on exposure to this agent. Due to the bactericidallevels present throughout the dosing interval, the selectionof secondary resistance in patients undergoing therapy withdalbavancin would seem unlikely. Also, it is thought that VISAstrains may be selected as a result of the twice-daily occurrenceof subtherapeutic trough levels of vancomycin in some patients;the effects of low but prolonged posttherapy levels could affectthe susceptibilities of colonizing strains. In the present study,the results of both direct selection and serial passage experimentsin vitro suggest a low potential for the selection of dalbavancin-resistantvariants. Notably, increased MICs were not seen with a VISAstrain. Similar results were previously reported from a morelimited study with single strains of S. aureus and Staphylococcushaemolyticus (11, 13).

In summary, dalbavancin has potent in vitro bactericidal activityagainst staphylococci and S. pyogenes at concentrations thatwill be maintained in human blood over the entire dosing interval.Concentrations of free dalbavancin considerably higher thanthe MBCs are present throughout most of the dosing interval.The potent bactericidal activity of dalbavancin for target organismsand the low propensity for the selection of resistant variantsin vitro, even with subtherapeutic concentrations, are desirableattributes for a new glycopeptide therapeutic agent.

ACKNOWLEDGMENTS

This study was funded by Pfizer Inc, Pfizer Global Pharmaceuticals,New York, NY.

FOOTNOTES

* Corresponding author. Mailing address: Focus Bio-Inova, Inc., 13665 Dulles Technology Drive, Suite 200, Herndon, VA 20171-4603. Phone: (703) 480-2536. Fax: (703) 480-2654. E-mail: dsahm{at}focusbioinova.com

Published ahead of print on 12 January 2007.

REFERENCES

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