Cellular Pharmacokinetics and Pharmacodynamics of the Glycopeptide Antibiotic Oritavancin (LY333328) in a Model of J774 Mouse Macrophages

Abstract

The intracellular pharmacokinetics and pharmacodynamics of oritavancin (LY333328) were studied in cultured cells. Oritavancin was avidly accumulated by J774 and THP-1 macrophages and rat fibroblasts and to a lesser extent by LLC-PK1 and Caco-2 cells. In J774 macrophages, the level of accumulation reached a plateau (at 370-fold the extracellular concentration) within 24 h and was partly defeated by a rise in serum protein levels. Efflux was incomplete (with a plateau at two-thirds of the original level at 6 h). In short-term kinetic studies, oritavancin uptake was linear for up to 4 h (as was the case for horseradish peroxidase and small latex beads, used as markers of the fluid phase and adsorptive endocytosis, respectively), which was in contrast to azithromycin and chloroquine uptake (which accumulate in cells by diffusion and segregation). The rates of clearance of oritavancin and latex beads were comparable (150 and 120 l ؋ mg of protein ؊1 ؋ h ؊1 , respectively) and were approximately 200 times higher than that of horseradish peroxidase. Oritavancin accumulation was partially reduced by monensin but was unaffected by acidic pH (these conditions abolished chloroquine accumulation). Cell-associated oritavancin was found in lysosomal fractions after homogenization of J774 macrophages and fractionation by isopycnic centrifugation. Oritavancin was bactericidal against intracellular Staphylococcus aureus (phagolysosomal infection) but was unable to control the intracellular growth of Listeria monocytogenes (cytosolic infection), even though its cellular concentration largely exceeded the MIC (0.02 mg/liter) and minimal bactericidal concentration (2 mg/liter). We conclude that oritavancin enters cells by adsorptive endocytosis (favored by its lipophilic side chain and/or the presence of three protonatable amines), which drives it to lysosomes, where it exerts antibiotic activity.

Pharmacodynamic Evaluation of the Intracellular Activities of Antibiotics against Staphylococcus aureus in a Model of THP-1 Macrophages

“…For each of these antibiotics, the assay method was checked for linearity (telithromycin, 0.03 to 4 mg/liter; azithromycin, 0.05 to 4 mg/liter; ampicillin, 0.21 to 10 mg/liter, nafcillin, 1.1 to 120 mg/liter; oxacillin, 1.6 to 30 mg/liter; penicillin V, 0.04 to 10 mg/liter; rifampin, 0.2 to 15 mg/liter; linezolid, 12 to 190 mg/liter, vancomycin, 2.7 to 150 mg/liter; teicoplanin, 2.9 to 250 mg/liter; gentamicin, 0.6 to 240 mg/liter) and for reproducibility (coefficient of variation, Ͻ10%). Ciprofloxacin, moxifloxacin, and levofloxacin concentrations were measured by fluorimetry (12,56), and garenoxacin and oritavancin concentrations were measured by radiometry by using 14 C-labeled drugs (41,64). In pilot studies we checked that these assays detected genuine, bioactive drug.…”

Lipoglycopeptide Antibacterial Agents in Gram-Positive Infections: A Comparative Review

“…It also proved synergistic with other bactericidal antibacterials such as fluoroquinolones or rifampicin against small colony variants [74]. This remarkable intracellular activity was attributed to the capacity of oritavancin to accumulate within the lysosomes of cells to exceptionally high levels by a process of adsorptive endocytosis [75]. Telavancin was also more effective than vancomycin in THP-1 cells infected by MSSA, MRSA, or VISA, reaching a bactericidal effect after 3 h; however, its activity was much slower against VRSA, with a bactericidal effect being reached only after 24 h of incubation with concentrations higher than ten times the MIC [35].…”

Activity of sitafloxacin against extracellular and intracellular Staphylococcus aureus in vitro and in vivo: comparison with levofloxacin and moxifloxacin

“…9,10 However, the intracellular location of the bacteria might explain the slow response to antibiotic as they might be protected from the effects of antibiotics. 11 In general, intracellular antimicrobial activity is markedly impaired compared with the activity seen in broth or the extracellular milieu, [12][13][14] although we know of situations in which the opposite is true. Antibiotic treatments should therefore be optimized not only toward the extracellular forms of S. aureus but also toward the intracellular forms.…”

“…16 For these studies, the direct assessment of antibiotic activity in the pertinent models is warranted, and several in vitro models with either human or animal cells have been developed to study the extracellular and intracellular activities of antibiotics. 12,14 Therefore, we selected and used the cell line of RAW264.7 macrophages, because they present many of the characteristics of human macrophages while forming a homogeneous and reproducible population. 17 We used this cell line to analyze the potential relationship between the accumulation of antibiotics in cells and intracellular activity.…”