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Antimicrobial Agents and Chemotherapy, June 2009, p. 2687-2689, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.00197-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Impact of Human Serum Albumin on Oritavancin In Vitro Activity against Enterococci

Geoffrey A. McKay, Sylvain Beaulieu, Ingrid Sarmiento, Francis F. Arhin, Thomas R. Parr Jr., and Gregory Moeck^*

Targanta Therapeutics Incorporated, 7170 Frederick Banting, St. Laurent, Quebec, Canada H4S 2A1

Received 13 February 2009/ Returned for modification 15 March 2009/ Accepted 29 March 2009

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Oritavancin is a lipoglycopeptide with activity against gram-positive pathogens including vancomycin-resistant enterococci. The impact of human serum albumin (HSA) on oritavancin activity against enterococci was compared to those of vancomycin, daptomycin, teicoplanin, and linezolid in vitro using MIC and time-kill methods. Oritavancin MICs increased between 0- and 8-fold in the presence of HSA. In time-kill assays with HSA, oritavancin retained activity, killing or inhibiting enterococci more rapidly than did comparators when peak concentrations were simulated.

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Oritavancin, a lipoglycopeptide with activity against drug-resistant gram-positive bacteria, is 85 to 90% protein bound (11, 15). Most of the binding of oritavancin in human serum is to albumin (15). The effect of protein binding on the bactericidal activity of antimicrobial agents has been a source of controversy—arguments for protein binding either diminishing or augmenting the activity of agents have been advanced by different researchers, although it has been established that the free, unbound fraction is responsible for antimicrobial activity (3, 12). Most in vitro antibiotic susceptibility tests and time-kill assays are performed with standard microbiological media, such as cation-adjusted Mueller-Hinton broth (CAMHB), without supplementation by serum or serum albumin. However, studies that take into account the influence of biological fluids, such as human serum or its purified constituents, on the activities of antibacterial agents in vitro may better predict in vivo drug efficacy. In this study, we have investigated the effect of human serum albumin (HSA) at physiological concentrations (14) on the in vitro activities of oritavancin and comparator antibiotics against reference strains and clinical isolates of enterococci. The tested strains represented vancomycin-susceptible and both VanA and VanB vancomycin-resistant isolates.

(Part of this work was presented at the joint meeting of the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy and the Infectious Diseases Society of America 46th annual meeting, held at Washington, DC, 25 to 28 October 2008 [9].)

Oritavancin concentrations (Table 1) were chosen to approximate the closest MIC doubling dilution to the total (protein bound plus unbound) peak (C_max) and total trough levels in plasma following administration of a 200-mg dose (6, 7, 13).

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TABLE 1. Concentrations of antimicrobial agents evaluated in this study

Comparator drug concentrations (Table 1) were likewise chosen to approximate (to the closest MIC doubling dilution) their respective total C_max and total trough levels in plasma when administered at FDA-approved dosages for complicated skin and skin structure infections (daptomycin, Cubicin insert; linezolid, Zyvox insert; vancomycin, Vancocin insert; teicoplanin, Targocid insert).

Predicted free drug concentrations in the presence of 4% (wt/vol) HSA (Table 1) were calculated as the product of the total peak or total trough concentration (Table 1) and the free fraction (Table 1), as determined from protein binding data from the literature (11) or package inserts.

MICs against oritavancin, vancomycin, and teicoplanin were determined by the broth microdilution assay according to the CLSI M7-A7 and M100-S18 guidelines (4, 5). Tests with oritavancin included polysorbate 80 (final test concentration of 0.002%) as described previously (1, 5): oritavancin was dissolved in 0.002% polysorbate 80 in water, and polysorbate 80 was maintained at this concentration in drug dilution steps and in the MIC assays. For assays with daptomycin, CAMHB was supplemented with 50 µg/ml CaCl₂ (final concentration) (4).

Time-kill assays in the presence of 4% HSA were performed according to CLSI M26-A guidelines (10) with the following alteration: polymethylpentene Erlenmeyer flasks were used in an effort to minimize binding of oritavancin to vessel surfaces (2). Exponential-phase cells were diluted to approximately 5 x 10⁵ to 1 x 10⁶ CFU/ml and exposed to oritavancin or comparator antibiotics over a 24-h period in a total volume of 12.5 ml of CAMHB plus 4% HSA. For assays with oritavancin, 0.002% polysorbate 80 (1) was included to minimize the loss of oritavancin to surfaces. For assays with daptomycin, CAMHB was supplemented with 50 µg/ml CaCl₂ (final concentration) (4). Viable-cell counts were determined by serial dilution plating. Culture aliquots were mixed with a suspension of activated charcoal (25 mg/ml) to minimize antibiotic carryover. Bactericidal activity was defined as a 3-log decrease in cell counts compared to the inoculum (10). Assays were performed at least twice independently, with similar rates and extents of kill between experiments seen for oritavancin and for comparators; results presented are from a single experiment. All data points are an average of duplicate determinations within an experiment.

Table 2 shows MIC results for oritavancin and comparators in the presence and absence of 4% HSA. Oritavancin MICs increased two- to eightfold in the presence of 4% HSA against five of the six isolates of enterococci; oritavancin MICs remained unchanged for Enterococcus faecium strain 1119175 (VanB vancomycin-resistant Enterococcus [VRE]) in the presence and absence of 4% HSA. Vancomycin and linezolid MICs against enterococci determined in the presence of 4% HSA remained unchanged, while teicoplanin and daptomycin MICs increased 2- to 8-fold and 8- to 16-fold, respectively, in the presence of 4% HSA. The extents of these HSA-induced MIC shifts for comparator agents against enterococci are similar to those reported for vancomycin, teicoplanin, and daptomycin using a similar approach for Staphylococcus aureus (12).

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TABLE 2. Broth microdilution MICs for oritavancin and comparator drugs against enterococci in the presence or absence of 4% HSAa

Time-kill curves of oritavancin at its total peak or total trough concentration in the presence of 4% HSA were similar to previously reported time-kill data with predicted free peak or free trough concentrations of drug in CAMHB in the absence of albumin (8). This suggests that the oritavancin serum protein binding value of 88% (11) that was used in calculations to represent free C_max and free trough concentrations in vitro is useful for predicting actual free drug concentrations. The same was found to hold true for the comparator agents.

Oritavancin demonstrated concentration-dependent activities against the enterococci used in this study in the presence of 4% HSA (Table 3 and data not shown). This was expected, since oritavancin activity in vitro and in vivo against enterococci, streptococci, and staphylococci is best described as concentration dependent (8). When tested in the presence of 4% HSA at drug concentrations approximating its total peak and total trough levels, oritavancin was the only compound that exerted bactericidal activity against vancomycin-susceptible Enterococcus (Table 3). Furthermore, oritavancin was the only compound to retain bacteriostatic or bactericidal activity against all tested strains at the total peak concentration (Table 3). In the presence of 4% HSA, only oritavancin retained activity against the VanA (n = 3) and VanB (n = 2) VRE strains tested in this study (Table 3). Linezolid demonstrated bacteriostatic activity at both peak and trough levels for all the strains tested.

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TABLE 3. Comparative bactericidal activities of oritavancin against enterococcal isolates, determined by time-kill assay in the presence of 4% HSAa

The studies presented herein demonstrate that oritavancin retains in vitro activity against difficult-to-treat VRE in the presence of physiologically relevant levels of HSA and is a potential candidate in the treatment of infections caused by enterococci regardless of their glycopeptide susceptibility phenotype.

 
We thank the scientific staff at Targanta Therapeutics for their contributions to this research.

 
* Corresponding author. Mailing address: Targanta Therapeutics Incorporated, 7170 Frederick Banting, St. Laurent, Quebec, Canada H4S 2A1. Phone: (514) 332-1008, ext. 232. Fax: (514) 332-6033. E-mail: gmoeck{at}targanta.com

Published ahead of print on 6 April 2009.

Top ABSTRACT INTRODUCTION REFERENCES

 

1
1. Arhin, F. F., I. Sarmiento, A. Belley, G. A. McKay, D. C. Draghi, P. Grover, D. Sahm, T. R. Parr, Jr., and G. Moeck. 2008. Effect of polysorbate 80 on oritavancin binding to plastic surfaces—implications for susceptibility testing. Antimicrob. Agents Chemother. 52:1597-1603.[Abstract/Free Full Text]
2
2. Arhin, F. F., I. Sarmiento, T. R. Parr, Jr., and G. Moeck. 2008. Binding of oritavancin to surfaces impacts choice of vessels for oritavancin in vitro assays, abstr. B2. 58th Annu. Meet. Can. Soc. Microbiol. Canadian Society of Microbiologists, Calgary, Canada.
3
3. Bailey, E. M., M. J. Rybak, and G. W. Kaatz. 1991. Comparative effect of protein binding on the killing activities of teicoplanin and vancomycin. Antimicrob. Agents Chemother. 35:1089-1092.[Abstract/Free Full Text]
4
4. Clinical and Laboratory Standards Institute. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, approved standard, 7th ed. M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA.
5
5. Clinical and Laboratory Standards Institute. 2008. Performance standards for antimicrobial susceptibility testing, 18th information supplement. M100-S18. Clinical and Laboratory Standards Institute, Wayne, PA.
6
6. Fetterly, G. J., C. M. Ong, S. M. Bhasvnani, J. S. Loutit, S. B. Porter, L. G. Morello, P. G. Ambrose, and D. P. Nicolau. 2005. Pharmacokinetics of oritavancin in plasma and skin blister fluid following administration of a 200-milligram dose for 3 days or a single 800-milligram dose. Antimicrob. Agents Chemother. 49:148-152.[Abstract/Free Full Text]
7
7. Hartman, C. S., M. M. Wasilewski, and B. M. Bates. 2008. Oritavancin in the treatment of complicated skin and skin structure infections (cSSSI): combined results of two phase 3 multinational trials, abstr. L-1414. Prog. Abstr. 48th Intersci. Conf. Antimicrob. Agents Chemother./Infect. Dis. Soc. Am. 46th Annu. Meet. American Society for Microbiology, Washington, DC.
8
8. McKay, G. A., S. Beaulieu, A. Belley, F. F. Arhin, T. R. Parr, Jr., and G. Moeck. 2007. In vitro time-kill experiments of oritavancin against drug-resistant isolates of Staphylococcus aureus and enterococci, abstr. E-1614. Prog. Abstr. 47th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.
9
9. McKay, G. A., S. Beaulieu, I. Sarmiento, F. F. Arhin, D. Moraitis, T. R. Parr, Jr., and G. Moeck. 2008. Impact of human serum albumin (HSA) on oritavancin (ORI) in vitro activity against enterococci, abstr. C1-3835. Prog. Abstr. 48th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.
10
10. National Committee of Clinical Laboratory Standards. 1999. Methods for determining bactericidal activity of antimicrobial agents, approved guideline. NCCLS document M26-A. National Committee of Clinical Laboratory Standards, Wayne, PA.
11
11. Rowe, P. A., and T. J. Brown. 2001. In vitro protein binding of [¹⁴C]oritavancin in human plasma at 1, 10 and 91 µg/ml employing a dextran coated charcoal adsorption method, abstr. 2193. Prog. Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.
12
12. Tsuji, B. T., S. N. Leonard, P. R. Rhomberg, R. N. Jones, and M. J. Rybak. 2008. Evaluation of daptomycin, telavancin, teicoplanin, and vancomycin activity in the presence of albumin or serum. Diagn. Microbiol. Infect. Dis. 60:441-444.[CrossRef][Medline]
13
13. Van Bambeke, F., Y. Van Laethem, P. Courvalin, and P. M. Tulkens. 2004. Glycopeptide antibiotics: from conventional molecules to new derivatives. Drugs 64:913-936.[CrossRef][Medline]
14
14. Zeitlinger, M. A., R. Sauermann, F. Traunmuller, A. Georgopoulos, M. Muller, and C. Joukhadar. 2004. Impact of plasma protein binding on antimicrobial activity using time-killing curves. J. Antimicrob. Chemother. 54:876-880.[Abstract/Free Full Text]
15
15. Zhanel, G. G., I. D. C. Kirkpatrick, D. J. Hoban, A. M. Kabani, and J. A. Karlowsky. 1998. Influence of human serum on pharmacodynamic properties of an investigational glycopeptide, LY333328, and comparator agents against Staphylococcus aureus. Antimicrob. Agents Chemother. 42:2427-2430.[Abstract/Free Full Text]

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Antimicrobial Agents and Chemotherapy, June 2009, p. 2687-2689, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.00197-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by McKay, G. A. Articles by Moeck, G. Search for Related Content PubMed PubMed Citation Articles by McKay, G. A. Articles by Moeck, G.