Protein Binding of Antimicrobials: Methods for Quantification and for Investigation of its Impact on Bacterial Killing

Abstract

Abstract. Plasma protein binding of antimicrobial agents is considered to be a key characteristic of antibiotics as it affects both their pharmacokinetics and pharmacodynamics. However, up to the present, no standard methods for measuring protein binding or for quantification of the influence of protein binding on antimicrobial activity exist. This short-coming has previously led to conflicting results on antibacterial activity of highly protein-bound antibiotics. The present review, therefore, set out to summarize (1) methods for quantification of protein binding, (2) microbiological growth media used for determination of the impact of protein binding on antimicrobial activity of antibiotics, and (3) different pharmacodynamic in vitro studies that are used in this context. The advantages and disadvantages of a wide range of different approaches are discussed and compared. The urgent call for international standardization by microbiological societies and laboratories may be considered as a logical consequence of the presented data.

Importance of Relating Efficacy Measures to Unbound Drug Concentrations for Anti-Infective Agents

“…Available techniques for measurement of protein binding include equilibrium dialysis, ultrafiltration, ultracentrifugation, microdialysis, chromatographic methods, capillary electrophoresis, fluorescence spectroscopy, and ultrafast immunoextraction. These methods have been compared extensively in published review articles, and only an overview is provided here (16,48,49). With the exception of microdialysis, which may be applied in vivo to measure unbound drug concentrations, these methods are used in vitro for protein binding determination.…”

“…The impact of protein binding on drug efficacy will depend on the extent of the binding, PK properties, and intrinsic activity of the drug (11)(12)(13)(14)(15). In vitro, the impact of protein binding on antimicrobial activity is often investigated through determination of the MIC, time-kill curves, and cell culture assays (13,16). Numerous in vitro studies have been published, which evaluated the impact of protein binding on antimicrobials (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), antivirals (29,30), and antifungals (31,32) by using protein supplements and/or serum to mimic in vivo conditions.…”

“…In the majority of studies, free drug concentrations are not measured directly in the experimental setting, and the extent of protein binding is accounted for by the using binding values reported in the literature. This approach can be misleading if the protein binding reported in the literature differs from the actual protein binding in the experimental setting (16,33).…”

The Impact of Protein Binding on Antibacterial Activities of Antibiotics is more than Predicted by Considering its Numerical Value Alone: Impact of Preparative and Incubation Methods on Different Pharmacodynamic Endpoints of ß-Lactams, Macrolides, or Fluoroquinolones Against Gram-positive and Gram-negative Bacteria-Part I

“…Such differences really matter, as the free fraction used as basis for PK/PD calculations could be assumed to be as low as 30% and 48% or as high as 60% and 75%, respectively. These differences are very likely due to methodological differences as reviewed previously [3,59], but the question is which of the published data should be selected for PK/PD calculations and based on which criteria?…”

“…It would therefore have been desirable to quantitate protein binding as well as free drug concentrations not only in the different serum preparations studied but also in the course of PK-simulations because of continuously fluctuating drug concentrations. However, quantitation of free drug concentrations is laborious and time consuming depending on the methods applied [3,59], so that total drug concentrations in the 7,224 samples withdrawn in this study were assayed instead and unspecific adsorption was avoided by siliconization of the glass-and plastic ware used. Although total drug concentrations varied by <5% from the theoretical concentrations suggesting that methodological errors can be excluded, this finding does not indicate that free drug levels were identical under the various conditions studied.…”