Cellular Pharmacokinetics of the Novel Biaryloxazolidinone Radezolid in Phagocytic Cells: Studies with Macrophages and Polymorphonuclear Neutrophils

Abstract

Radezolid (RX-1741) is the first biaryloxazolidinone in clinical development. It shows improved activity, including against linezolid-resistant strains. Radezolid differs from linezolid by the presence of a biaryl spacer and of a heteroaryl side chain, which increases the ionization and hydrophilicity of the molecule at physiological pH and confers to it a dibasic character. The aim of this study was to determine the accumulation and subcellular distribution of radezolid in phagocytic cells and to decipher the underlying mechanisms. In THP-1 human macrophages, J774 mouse macrophages, and human polymorphonuclear neutrophils, radezolid accumulated rapidly and reversibly (half-lives of approximately 6 min and 9 min for uptake and efflux, respectively) to reach, at equilibrium, a cellular concentration 11-fold higher than the extracellular one. This process was concentration and energy independent but pH dependent (accumulation was reduced to 20 to 30% of control values for cells in medium at a pH of <6 or in the presence of monensin, which collapses pH gradients between the extracellular and intracellular compartments). The accumulation at equilibrium was not affected by efflux pump inhibitors (verapamil and gemfibrozil) and was markedly reduced at 4°C but was further increased in medium with low serum content. Subcellular fractionation studies demonstrated a dual subcellular distribution for radezolid, with ϳ60% of the drug colocalizing to the cytosol and ϳ40% to the lysosomes, with no specific association with mitochondria. These observations are compatible with a mechanism of transmembrane diffusion of the free fraction and partial segregation of radezolid in lysosomes by proton trapping, as previously described for macrolides.

Contrasting Effects of Acidic pH on the Extracellular and Intracellular Activities of the Anti-Gram-Positive Fluoroquinolones Moxifloxacin and Delafloxacin againstStaphylococcus aureus

“…It is also interesting to note that both moxifloxacin and delafloxacin are found in the soluble fraction of the cells, with only a minimal amount detected in the organelles, as already observed for other fluoroquinolones (12,41). This is in opposition to what is seen with weak bases like macrolides or the biaryloxazolidinone radezolid, which are associated in large proportion with acidic vacuoles such as lysosomes (13,25). While such molecules show a marked decrease of accumulation when the pH gradient between subcellular compartments and the extracellular milieu is collapsed with monensin (25), the weak acid character of delafloxacin results in an opposite behavior.…”

“…This is in opposition to what is seen with weak bases like macrolides or the biaryloxazolidinone radezolid, which are associated in large proportion with acidic vacuoles such as lysosomes (13,25). While such molecules show a marked decrease of accumulation when the pH gradient between subcellular compartments and the extracellular milieu is collapsed with monensin (25), the weak acid character of delafloxacin results in an opposite behavior. Moxifloxacin behaves similarly to the weak bases in this respect, probably due to the presence of a protonatable amino group.…”

Investigational Antimicrobial Agents of 2013

“…It displayed MIC 90 values against cfr-containing linezolid-resistant S. aureus isolates that were 4-to 8-fold lower than those of linezolid (103). Another attractive property of this compound is higher intrinsic activity than linezolid in infected cells, with phagocytic cell internal concentrations increased 11-fold over extracellular concentrations (104,105). The compound has recently successfully completed two phase 2 clinical trials for community-acquired pneumonia (CAP) and for uncomplicated skin and skin structure infections (uSSSI) (http://www.clinicaltrials.gov).…”

Nonclinical and Pharmacokinetic Assessments To Evaluate the Potential of Tedizolid and Linezolid To Affect Mitochondrial Function

“…Cell fractionation and subcellular localization studies were therefore conducted to determine whether intracellular tedizolid would be associated with mitochondria, which might represent a possible toxicity hazard. Murine J774 macrophages were selected because these cells can easily be fractionated and their mitochondria adequately separated from other cell organelles by centrifugation (39). The methods were previously described in detail and validated to determine the subcellular localization of various antibiotics, such as aminoglycosides, fluoroquinolones, and macrolides (40) as well as oxazolidinones (39).…”

“…Murine J774 macrophages were selected because these cells can easily be fractionated and their mitochondria adequately separated from other cell organelles by centrifugation (39). The methods were previously described in detail and validated to determine the subcellular localization of various antibiotics, such as aminoglycosides, fluoroquinolones, and macrolides (40) as well as oxazolidinones (39). Cells were incubated for 2 h with 20 mg/liter tedizolid before collection.…”