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

Abstract

Objectives: Protein binding decreases antibacterial activities as the free fraction only crosses membranes thus reaching intracellular targets. However, serum components may also increase antibacterial activities. Therefore, the effect of serum proteins on activities of ß-lactams, macrolides, and fluoroquinolones was examined. Several preparation-and cultivation-conditions were examined as some preparative methods like freeze-thawing of sera may cause artifacts. Furthermore, previous studies have indicated that data may vary depending on the endpoints studied. Therefore, the aim of this study was to avoid an impact of methodological factors on the data generated and to analyse the activities of the study drugs by examining different static-or dynamic endpoints.Methods: Bacteria were grown in Brain Heart Infusion Broth (BHI), plus 50% of either fresh inactivated, pH 7.2, or fresh inactivated-, pH 7.2 or 8.2, frozen inactivated-serum, pH 8.2 as compared to BHI without any supplementations, pH 7.2 or 8.2, and BHI plus 45 g/L albumin. MICs of faropenem, amoxicillin, clarithromycin, azithromycin, moxifloxacin, and levofloxacin were determined and kill-kinetics were recorded following exposure to constant or fluctuating drug concentrations simulating serum pharmacokinetics of the agents. Kill-rates and areas under the bacterial kill curves were calculated.Results: Albumin and inactive serum increased MICs and reduced kill-rates of the agents studied in conformity with their protein binding, whereas active serum increased the activities of the agents. MICs and kill-rates did not change in parallel. The impact of protein binding in decreasing order was: MICs>kill-rates in time-kill experiments>kill-rates in kinetic-simulations. Macrolides and fluoroquinolones but not ß-lactams were more active at an alkaline-than neutral pH. Use of frozen sera caused alkalinization of media, thus generating artifacts. Conclusions:The impact of serum proteins on antibacterial activities is strongly dependent from three factors: the methods applied to prepare the serum pool, the incubation conditions, and the enpoints studied.

Seventy-Five Years of Research on Protein Binding

“…A variety of physicochemical factors and methodological variables may have affected serum-antibiotic interactions. Storage of serum in the refrigerator and freezethaw cycles rose the pH of the serum pool from 7.4 to Ն8.0 (6,(32)(33)(34). Heat treatment used for complement inactivation or acidification used for the preparation of commercially available products induced conformational changes and dimerization of serum proteins, affecting their interaction with bacterial membranes and antigens (34)(35)(36)(37)(38)(39)(40)(41), and it affected the stability of some serum proteins, including albumin (42)(43)(44).…”

“…Heat treatment used for complement inactivation or acidification used for the preparation of commercially available products induced conformational changes and dimerization of serum proteins, affecting their interaction with bacterial membranes and antigens (34)(35)(36)(37)(38)(39)(40)(41), and it affected the stability of some serum proteins, including albumin (42)(43)(44). Changes in the pHs of the test media increase significantly in vitro activities of macrolides, fluoroquinolones, and aminoglycosides but leave ␤-lactams unaffected (33,(45)(46)(47)(48)(49), so that serum macrolide, aminoglycoside, and fluoroquinolone synergistic effects were simply caused by pH shifts. Preparative techniques, methods for quantification of protein binding, models used in determining protein binding impact (50,51), and the endpoints to be analyzed (33,34) should be standardized; otherwise, data generated by the use of different serum preparations and/or different models may be contradictory or may even represent artifacts.…”

“…Changes in the pHs of the test media increase significantly in vitro activities of macrolides, fluoroquinolones, and aminoglycosides but leave ␤-lactams unaffected (33,(45)(46)(47)(48)(49), so that serum macrolide, aminoglycoside, and fluoroquinolone synergistic effects were simply caused by pH shifts. Preparative techniques, methods for quantification of protein binding, models used in determining protein binding impact (50,51), and the endpoints to be analyzed (33,34) should be standardized; otherwise, data generated by the use of different serum preparations and/or different models may be contradictory or may even represent artifacts.…”

The Impact of Protein Binding on Antibacterial Activities of Antibiotics is more than Predicted by Considering its Numerical Value Alone: Effects of Serum Proteins on Activities of Poorly Penetrating Agents and Inhibition of ß- Lactamase Activity-Part II

“…The methods used in this study are identical to those described in the companion manuscript [36]. Briefly, blood was sampled from twelve healthy volunteers immediately prior to commencement of each experimental series and pooled.…”

“…MICs were determined according to CLSI guidelines [37]; time-kill curves were generated by exposure to multiples (1-,4-,8-,16-, and 32-times) of the MICs recorded in the corresponding media to ensure that bioequivalent concentrations were used. Sampling, processing of samples, quatitation of viable counts as well as drug concentration-assays has been described in detail in the companion manuscript [36]. In general, growth controls in the corresponding drug free media, MIC determinations or time-kill assays were run in parallel under the four experimental conditions studied.…”