Radezolid Synthesis Essay

Cellular Pharmacodynamics of the Novel Biaryloxazolidinone Radezolid: Studies with Infected Phagocytic and Nonphagocytic cells, Using Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Legionella pneumophila

  1. Sandrine Lemaire1,
  2. Klaudia Kosowska-Shick2,
  3. Peter C. Appelbaum2,
  4. Gunther Verween3,
  5. Paul M. Tulkens1 and
  6. Françoise Van Bambeke1,*
  1. 1Unité de Pharmacologie cellulaire et moléculaire and Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium
  2. 2Hershey Medical Center, Hershey, Pennsylvania
  3. 3Keratinocyte Bank, Burn Wound Center, Queen Astrid Military Hospital, Brussels, Belgium

ABSTRACT

Radezolid is a novel biaryloxazolidinone in clinical development which shows improved activity, including against linezolid-resistant strains. In a companion paper (29), we showed that radezolid accumulates about 11-fold in phagocytic cells, with ∼60% of the drug localized in the cytosol and ∼40% in the lysosomes of the cells. The present study examines its activity against (i) bacteria infecting human THP-1 macrophages and located in different subcellular compartments (Listeria monocytogenes, cytosol; Legionella pneumophila, vacuoles; Staphylococcus aureus and Staphylococcus epidermidis, mainly phagolysosomal), (ii) strains of S. aureus with clinically relevant mechanisms of resistance, and (iii) isogenic linezolid-susceptible and -resistant S. aureus strains infecting a series of phagocytic and nonphagocytic cells. Radezolid accumulated to similar levels (∼10-fold) in all cell types (human keratinocytes, endothelial cells, bronchial epithelial cells, osteoblasts, macrophages, and rat embryo fibroblasts). At equivalent weight concentrations, radezolid proved consistently 10-fold more potent than linezolid in all these models, irrespective of the bacterial species and resistance phenotype or of the cell type infected. This results from its higher intrinsic activity and higher cellular accumulation. Time kill curves showed that radezolid's activity was more rapid than that of linezolid both in broth and in infected macrophages. These data suggest the potential interest of radezolid for recurrent or persistent infections where intracellular foci play a determinant role.

Intracellular infections are difficult to treat because bacteria are shielded from many of the humoral and cellular means of natural defenses while being also partially protected from the action of most antibiotics (7, 12, 47, 58). While intracellular survival is part of the pathogenic cycle of obligatory or facultative intracellular bacteria like Listeria monocytogenes or Legionella pneumophila (7, 38, 51), it contributes to the recurrent or persistent character of infections caused by opportunistic intracellular bacteria like staphylococci (16). The treatment of such intracellular infections, therefore, requires the use of antibiotics that can express their activity at the site of infection. This, however, cannot be predicted simply on the basis of the ability of drugs to accumulate in cells, as several other factors may play a critical role in enhancing or impeding their local antimicrobial properties (7, 58). For example, previous work in our laboratory using a model of Staphylococcus aureus-infected THP-1 cells showed that β-lactams, which do not accumulate in these cells, nevertheless display significant intracellular activity provided their extracellular concentration is brought to sufficiently high but still clinically meaningful levels (31). Conversely, azithromycin, which is known to accumulate in large amounts in cells (6, 18), proves only marginally active against S. aureus phagocytosed by macrophages (1, 32). This occurs despite the fact that bacteria persist and thrive for prolonged periods in phagolysosomes after their engulfment by these cells (5, 24, 35), which is also where the bulk of the drug accumulates (6). The difficulty of predicting intracellular activity on the simple basis of pharmacokinetics therefore warrants individual evaluation of new drugs in appropriate models. While animal models are being developed (49), models of cultured cells remain helpful because they offer the possibility of exploring in detail the pharmacological descriptors governing the intracellular activity of antibiotics in the absence of host factors.

Radezolid is a novel oxazolidinone currently in phase II of clinical development (see our companion paper for its structure [29]). In comparison to linezolid, it shows improved activity against a series of bacterial species capable of surviving intracellularly, such as Staphylococcus, Chlamydia, and Legionella species, and remains active against linezolid-resistant strains (25). In the companion paper, we showed that radezolid accumulates to about 12-fold-higher levels than linezolid in human THP-1 cells and localizes in lysosomes for about 40% of the total cell load, while the remainder is found in the cytosol (29). This triggered us to examine the intracellular activity of radezolid using models allowing a quantitative assessment of its pharmacodynamic properties. We selected different types of bacteria with distinct subcellular localizations. We used L. monocytogenes, which thrives in the cytosol (15, 21), L. pneumophila, which is found in specific replication vacuoles (20), and S. aureus and the coagulase-negative Staphylococcus epidermidis, which show a phagolysosomal localization in most cell types (1, 3, 13, 40) but may also partially escape in the cytosol of endothelial or epithelial cells (17, 37, 52). We also assessed the intracellular activity of radezolid against different strains of S. aureus with various resistance mechanisms, including to linezolid. Finally, we used different cell types as models of territories where S. aureus can survive intracellularly (endothelial cells, osteoblasts, respiratory epithelial cells, keratinocytes, and fibroblasts [11, 14, 22, 36, 41]), together with phagocytic cells (macrophages).

Although the maximal effects of both drugs are similar, we found that radezolid acts more rapidly and is consistently more potent than linezolid, mainly due to its higher intrinsic activity and larger cellular accumulation.

(Parts of this study were presented at the 19th European Congress of Clinical Microbiology and Infectious Diseases, Helsinki, Finland, May 2009, as oral presentations O29 and O30, at the 26th International Conference on Chemotherapy, Toronto, Ontario, Canada, June 2009, and at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, September 2009 [33, 34].)

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MATERIALS AND METHODS

Antibiotics and main reagents.Radezolid (RX-1741, supplied as microbiological standard powder with a potency of 93%) and [14C]RX-1741 (4 μCi/ml, 25 mCi/mmol, labeled on the C of the methylacetamide replacing the oxazolidinone ring) were obtained from Rib-X Pharmaceuticals (New Haven, CT). [14C]RX-1741 was diluted with cold drug to obtain a stock solution at 1 mg/liter (4 μCi/ml). Linezolid was obtained as the corresponding branded product (Zyvoxid) distributed in Belgium for human use by Pfizer SA/NV (Brussels, Belgium). Unless stated otherwise, cell culture media and sera were from Invitrogen Corp. (Carlsbad, CA) and other reagents from Sigma-Aldrich or Merck KGaA (Darmstadt, Germany).

Bacterial strains, susceptibility testing, and extracellular activity.The bacterial strains used in the present study are listed in Table 1. MIC determinations were performed in Mueller-Hinton broth (S. aureus or S. epidermidis, 24 h, pH 7.3 to 7.4, unless stated otherwise), tryptic soy broth (L. monocytogenes, 24 h, pH 7.4), or α-ketoglutarate-buffered yeast extract broth (L. pneumophila, 48 h, pH 6.9). For S. aureus, time kill curves or concentration response experiments in acellular medium were performed in Mueller-Hinton broth as described previously (1).

TABLE 1.

Strains and their susceptibilities to linezolid and radezolid

Cell lines.Most of the experiments were performed with human THP-1 cells (ATCC TIB-202 [American Tissue Culture Collection, Manassas, VA]), a myelomonocytic cell line displaying macrophage-like activity (55). These cells were maintained in our laboratory as previously described (9). Experiments were also conducted with primary cultures of (i) embryonic rat fibroblasts (isolated as described earlier [56]), (ii) Clonetics normal human osteoblasts (NHOst, cultivated in osteoblast growth medium according to the manufacturer's instructions [Lonza, Inc., Walkersville, MD]), (iii) Clonetics human umbilical vein endothelial cells (HUVEC, cultivated in endothelial cell growth medium and gelatin-treated flasks according to the manufacturer's instructions [Lonza, Inc.]), (iv) immortalized cultures of human bronchial epithelial cell line (Calu-3 [ATCC HBT-55]) maintained in our laboratory as previously described (19) except for the use of uncoated culture flasks, and (v) primary cultures of human epidermidal keratinocytes from neonatal foreskin obtained as described previously (46). These were frozen at passage 7 in Synth-a-Freeze (Cascade Biologics, Portland, OR) and then thawed and seeded on multiwell plates coated with 1 μg/ml collagen type 1 (1 ml/well, coating for 4 h at 37°C) and cultured in EpiLife medium supplemented with supplement S7 (defined growth supplement; Cascade Biologics).

Accumulation and assay of cell-associated radezolid.Antibiotic accumulation was determined following the general procedure used in our previous studies (45, 59), and the cellular content of [14C]radezolid was assayed in cell lysates by liquid scintillation counting (lowest limit of detection, 0.003 mg/liter; linear response between 0.01 and 0.78 mg/liter; R2 = 0.999; see the companion paper for further details [29]). All cell drug contents were expressed by reference to the total cell protein content (determined using Lowry's method) and converted into apparent total cell concentrations using a conversion factor of 5 μl per mg of cell protein (45, 57).

Cell infection and assessment of antibiotic intracellular activities.Infection of THP-1 cells and assessment of the intracellular activity of antibiotics were performed exactly as described earlier for L. monocytogenes (8), for S. aureus (1) (the same protocol was used here for S. epidermidis), and for L. pneumophila (32). For adherent cell lines, we used the general protocol developed previously for J774 macrophages infected by S. aureus (50), except that we used an initial inoculum of 5 × 107 to 1 × 108 (2 × 106 for HUVEC) bacteria/ml and a phagocytosis time of 2 h (1 h for HUVEC), as described earlier for infected keratinocytes or Calu-3 cells (28) and for HUVEC (39). This allowed us to reach, for all cell types, a postphagocytosis inoculum of 1.0 × 106 to 4.0 × 106 CFU per mg of cell protein, a value close to that used for THP-1 macrophages.

Statistical analyses.Curve fitting statistical analyses were made with GraphPad Prism, version 4.03, and GraphPad Instat, version 3.06 (GraphPad Software, San Diego, CA).

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RESULTS

Antibiotic susceptibilities of S. aureus, S. epidermidis, L. monocytogenes, and L. pneumophila.Table 1 shows the susceptibility to linezolid and radezolid of the S. aureus strains (with the corresponding relevant resistance mechanisms) and of the other bacterial strains used in the present study. For S. aureus and S. epidermidis, the linezolid MICs were 1 to 2 mg/liter for all strains, excluding two isolates for which the MICs were 16 mg/liter. These strains will be referred to as linezolid resistant hereinafter. In contrast, the radezolid MICs ranged between 0.25 and 2 mg/liter for all strains. A strain-by-strain comparison shows that radezolid MICs were systematically equal to or lower (up to 3 log2 dilutions) than those of linezolid for all linezolid-susceptible strains, with an 8-fold difference for the linezolid-resistant strains. For L. monocytogenes and L. pneumophila, the radezolid MICs were also systematically lower than those of linezolid (3 to 6 log2 dilutions). When compared on a molar basis, the MICs of radezolid are systematically 1- to 10-fold lower than those of linezolid against S. aureus and 5- to 43-fold lower for the other bacteria.

Effect of concentration on oxazolidinone activity against intracellular bacteria in THP-1 cells.In a first set of experiments, we compared the intracellular activity of radezolid to that of linezolid against bacteria showing different subcellular localizations and intracellular growth rates (estimated by the increase in CFU as extrapolated for an infinitely low drug concentration [Emin]) in the THP-1 macrophage model. Dose response experiments (at fixed time points [24 h for S. aureus, S. epidermidis, and L. monocytogenes and 48 h for L. pneumophila]) were performed to obtain the pertinent pharmacological descriptors of oxazolidinone activity (relative maximal efficacy [Emax], relative potency [50% effective concentration {EC50}], and apparent static concentration [Cs]; see reference 1 for a complete description of the models and of these parameters). A graphical representation of the data is presented in Fig. 1, with the numerical values for each pharmacological descriptor shown in Table 2. The activity of both oxazolidinones was concentration dependent within a range extending from roughly 0.1- to 10-fold the MIC, with a plateau reached when exceeding the latter concentration, as indicated by the sigmoidal shape of the curves. Two main observations can be made. First, radezolid shows a greater potency (about 5- to 10-fold lower Cs and EC50 values) than linezolid, independent of the bacteria tested, when concentrations are expressed on a weight (mg/liter) basis. When data are expressed as multiples of MICs (extracellular equipotent concentrations), however, the differences between the two molecules are minimized, highlighting the importance of the higher intrinsic activity of radezolid in this context. Second, the maximal relative efficacies (Emax) of linezolid and radezolid were similar when comparing the same bacterium. The Emax values for both drugs were lower against bacteria that exhibit robust intracellular growth (L. monocytogenes and S. aureus) than for those showing minimal growth (L. pneumophila and S. epidermidis). However, the amplitude of the antibacterial response (i.e., by considering the EminEmax difference) was larger for L. monocytogenes and S. aureus (3 to 2.5 log10 CFU difference) than for L. pneumophila or S. epidermidis (2 to 1 log10 CFU difference).

FIG. 1.

Dose response curves of linezolid and radezolid toward the intracellular forms of L. monocytogenes (strain EGD), S. aureus (strain ATCC 25923), L. pneumophila (strain ATCC 33153), or S. epidermidis (strain CN362) after phagocytosis by human THP-1 cells. Cells were incubated with the antibiotic for 24 h (for S. aureus, S. epidermidis or L. monocytogenes) or 48 h (for L. pneumophila) at the concentrations (total drug) indicated on the abscissa, with values expressed in mg/liter or in multiples (X) of the MIC. The ordinate shows the change in the number of CFU per mg of cell protein compared to the postphagocytosis inoculum. All values are means ± standard deviations (n = 3; when not visible, the standard deviation bars are smaller than the size of the symbols). The horizontal line corresponds to an apparent static effect. L, liter.

TABLE 2.

Pertinent pharmacological descriptors of antibiotic activity and statistical analysis of the dose response curves illustrated in Fig. 1a

Effect of concentration on oxazolidinone activity against intracellular forms of linezolid-susceptible and linezolid-resistant S. aureus within THP-1 cells.The activity of radezolid and linezolid was then compared against a series of S. aureus of the MSSA (methicillin-susceptible S. aureus), MRSA (methicillin-resistant S. aureus), or VISA (vancomycin-intermediate S. aureus) phenotype and against 2 linezolid-resistant strains selected in vitro by exposure to linezolid (SA238L and SA040L, isogenic to SA238 and SA040) (23) (Fig. 2 and Table 3 show regression parameters). For all strains, radezolid shows an improved potency compared to that of linezolid when concentrations are expressed on a weight (mg/liter) basis (Table 3). The data were plotted as multiples of the MIC to allow comparison of the activities against the different strains at equipotent concentrations. Interestingly, a single sigmoidal function could be fitted to the whole set of data against linezolid-susceptible strains for both drugs (Fig. 2, upper panel). The Cs values (static concentrations) were close to the respective MIC of each strain (and therefore lower for radezolid than for linezolid). Against linezolid-resistant strains (Fig. 2, lower panel), radezolid's activity was indistinguishable from that observed for linezolid-susceptible strains (similar Emin, Emax, and EC50 values and Cs values slightly lower than the MIC [0.8 mg/liter]). Linezolid showed a Cs value close to its MIC (16 mg/liter) for strain SA040L but was poorly effective against strain SA238L, for which a static effect was never reached.

FIG. 2.

Dose response curves of linezolid and radezolid toward different strains of S. aureus phagocytosed by THP-1 cells (upper panel, linezolid-susceptible strains; lower panel, linezolid-resistant strains). Cells were incubated with the antibiotic for 24 h at the concentrations (total drug) indicated on the abscissa and expressed in multiples (X) of the MIC. The ordinate shows the change in the number of CFU per mg of cell protein compared to the postphagocytosis inoculum. All values are means ± standard deviations (n = 3; when not visible, the standard deviation bars are smaller than the size of the symbols). The horizontal line corresponds to an apparent static effect. The vertical dotted line corresponds to the MIC, the value (or range of values) of which is indicated below each panel. L, liter.

TABLE 3.

Pertinent pharmacological descriptors of antibiotic activity and statistical analysis of the dose-response curves for the individual strains used in Fig. 2a

Concentration-dependent activity of radezolid versus linezolid against intracellular forms of linezolid-susceptible and linezolid-resistant S. aureus within nonphagocytic cells.Given that intracellular forms of S. aureus can be found in many other cell types than macrophages, we next examined the intracellular activity of radezolid against S. aureus strains internalized by human (HUVEC, Calu-3, keratinocytes, and osteoblasts) or animal (fibroblasts) nonphagocytic cells in comparison with its activity against S. aureus internalized by THP-1 macrophages (Fig. 3). Radezolid showed concentration-dependent activity that was indistinguishable against both strains and in all cells tested, with Cs values ranging from 0.6 to 3.3 mg/liter (1.2× to 1.5× MIC) and the Emax corresponding to CFU reductions of 0.6 to 1.5 log10 compared to the original inoculum. As shown in Fig. 1, larger decreases in CFU (corresponding to more-negative Emax values) were observed in cells where bacterial growth was slower, so that the amplitudes of the effects of the antibiotics (difference between Emin and Emax values) were similar (about 3.5 log10 CFU) in all cases. Linezolid was less potent than radezolid in all models, with Cs values ranging from 2.6 to 9.5 mg/liter (1.3× to 4.8× MIC) for the linezolid-susceptible strain and from 15 (0.9× MIC) to >100 mg/liter for the linezolid-resistant strain. Linezolid's overall activity was also markedly reduced against the linezolid-resistant strain but to various levels in the different cell types.

FIG. 3.

Dose response curves of linezolid (LZD) and radezolid (RZD) toward two isogenic strains of S. aureus that are linezolid susceptible (SA238) or linezolid resistant (SA238L), phagocytosed by different cell types. Cells were incubated with the antibiotic for 24 h at the concentrations (total drug) indicated on the abscissa and expressed in mg/liter. The ordinate shows the change in the number of CFU per mg of cell protein compared to the postphagocytosis inoculum. All values are means ± standard deviations (n = 3; when not visible, the standard deviation bars are smaller than the size of the symbols). The horizontal line corresponds to an apparent static effect. L, liter.

Cellular accumulation of radezolid in nonphagocytic cells and in infected cells.We showed in the companion paper that radezolid accumulates about 11-fold more in phagocytic cells than extracellularly (29). Therefore, we measured in this work its accumulation in nonphagocytic cells exposed to the drug during 2 or 24 h in comparison to its accumulation in THP-1 cells. At both time points, radezolid reached a cellular concentration of the same order of magnitude in all cell types (Table 4). We also determined the cellular accumulation of radezolid in THP-1 cells infected by S. aureus and did not find any difference from what was observed for noninfected cells (data not shown).

TABLE 4.

Comparative cellular accumulation of radezolid in phagocytic and nonphagocytic cells

Role of intracellular concentration of radezolid in activity.We showed that radezolid accumulates in cells and partially localizes in lysosomes (29). To assess whether this could account for its increased potency in comparison with that of linezolid, we replotted the data for strain SA238 (Lzds) as a function of the extracellular concentration expressed (i) as weight values (mg/liter), (ii) as multiples of the MIC at neutral pH or acidic pH (to mimic the conditions prevailing in the extracellular milieu and the phagolysosomes, respectively), and (iii) as a function of the cellular concentration, expressed also as multiples of the MIC at acidic pH (Fig. 4 and Table 5). As a first approximation, and since no data were available regarding the subcellular distribution of linezolid, we used total cellular concentrations for both drugs. Extracellular activity was also determined in parallel. Radezolid proved about 23-fold more potent (lower Cs and EC50 values) intracellularly than extracellularly based on these criteria. Interestingly, linezolid showed a similar effect, with a 5-fold-lower Cs value in THP-1 cells than in broth.

FIG. 4.

Dose response curves of linezolid and radezolid toward S. aureus SA238 in broth or phagocytosed by THP-1 cells. Activity was determined after 24 h of incubation with an antibiotic at the concentrations (total drug) indicated on the abscissa and expressed as (i) weight concentrations (mg/liter); (ii) multiples of the MIC as determined in broth adjusted to pH 7.4 (linezolid MIC, 2 mg/liter; radezolid MIC, 0.5 mg/liter); (iii) multiples of the MIC as determined in broth adjusted to pH 5.5 (linezolid MIC, 4 mg/liter; radezolid MIC, 8 mg/liter); (iv) multiple of the cellular concentration expressed in multiples of the MIC at pH 5.5, using accumulation factors of 1.7-fold (linezolid) and 9.8-fold (radezolid), respectively. The ordinate shows the change in the number of CFU per mg of cell protein compared to the initial inoculum. All values are means ± standard deviations (n = 3; when not visible, the standard deviation bars are smaller than the size of the symbols). The horizontal line corresponds to an apparent static effect. L, liter; X MIC, multiple of the MIC.

While the intracellular relative potency of radezolid was clearly higher than that of linezolid expressed on a weight basis (mg/liter), the two drugs behaved alike when compared on the basis of multiples of their MICs at neutral pH. Interestingly enough, the relative potency of radezolid, which was 10-fold higher (10-fold-lower Cs value) than that of linezolid when expressed in multiples of the MIC at acidic pH, returned to its original value when taking into account its cellular accumulation level. In all cases, the maximal relative efficacies of both oxazolidinones were measurably lower (less-negative Emax values) against intracellular bacteria than against bacteria grown in broth.

TABLE 5.

Pertinent pharmacological descriptors of antibiotic activity and statistical analysis of the dose response curves toward S. aureus SA238 illustrated in Fig. 4a

Time effect on oxazolidinone extracellular and intracellular activities against S. aureus (strain ATCC 25923).To further characterize the pharmacodynamic profiles of the oxazolidinones, their activities against S. aureus growing in broth or phagocytosed by THP-1 cells were then examined over shorter incubation periods (Fig. 5). Intracellular growth in the absence of antibiotic was minimal over the 5 hours of the experiment. Radezolid exerted a time-dependent effect in both environments, causing a 2 log10 CFU decrease extracellularly and a 0.5 to 1 log10 CFU decrease intracellularly for concentrations as low as 1 mg/liter. The extracellular effect of linezolid was never greater than about 1.3 log10 CFU extracellularly. Linezolid remained static intracellularly at the highest concentration tested.

FIG. 5.

Influence of time on the extracellular (broth) and intracellular (THP-1 cells) activities of linezolid and radezolid against S. aureus ATCC 25923. The ordinate shows the change in CFU compared to the initial inoculum. All values are means ± standard deviations (n = 3; when not visible, the standard deviation bars are smaller than the size of the symbols). The horizontal line corresponds to an apparent static effect. L, liter.

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DISCUSSION

Using a previously established general experimental design (1), the present study describes the intracellular activity of radezolid in comparison with that of linezolid against 4 bacterial species and in 6 different cell types. This allowed us to show that radezolid is consistently more potent than linezolid in relation to its ability to accumulate to high levels inside those cells.

The models used are representative of true pathological situations seen in the clinics, as they use cell types as models of territories where S. aureus can persist in the body, including inside the cells (see references 4 and 10 for examples). At the same time, they permit observation of the characteristics associated with different pathogenic mechanisms at the cellular level, as illustrated from the respective rates of intracellular growth of the different bacteria used in our model (see Table 2). Thus, L. monocytogenes, a true facultative intracellular parasite, was found to multiply as efficiently inside cells as in broth (see reference 8 for additional data). This is probably due to its capacity to reach the cytosol (escaping much of the host defense mechanisms) and to modify its metabolism to become fully adapted to this new intracellular environment (21, 42, 60). L. pneumophila, which is known more as an intracellular organism in humans, actually shows a slower growth rate than Listeria in THP-1 cells, probably because its growth capabilities markedly depend on the culture conditions and environment (48). For staphylococci, which are considered opportunistic pathogens, we observed a fairly robust intracellular growth for S. aureus but only after a lag period of about 8 to 10 h, and no apparent intracellular growth of the coagulase-negative S. epidermidis (compared to 3 log10 within 24 h in broth [1, 43]). This suggests a reduced capacity of S. epidermidis to resist the weak but nevertheless active cell defense mechanisms of THP-1 cells. These defense mechanisms may be somewhat defeated for S. aureus by the expression of virulence factors, such as staphyloxanthin, which is under the control of RsbU (39). Within the context of the evaluation of antibiotics, the concomitant use of these models offers us an opportunity to compare antibiotic activities against intracellular bacteria that differ not only by their subcellular localization but also by their multiplication rate.

A critical observation made during this study is that radezolid proves approximately 10-fold more potent than linezolid in all intracellular models when compared at equivalent weight concentrations (this difference being larger for linezolid-resistant strains). This occurs irrespective of the subcellular localization of the bacteria, their intracellular growth rate, the type of cell infected, or the resistance phenotype of the strain. This favorable activity profile of radezolid may result from its higher intrinsic activity (with MICs typically 3 to 6 dilutions lower than those of linezolid) and/or from its higher cellular accumulation. Recent studies with torezolid, another oxazolidinone in development, have suggested that the MIC is the main driver for intracellular potency, as improvement in potency over that of linezolid is normalized when concentrations are expressed in multiples of the MICs (30). The importance of the MIC is also highlighted here, as we see that a single sigmoidal regression can be fitted to the data obtained for all S. aureus strains once the linezolid-resistant strains have been excluded. This is also what we observed for ceftobiprole (26), suggesting that this concept can perhaps be generalized. The situation with radezolid, however, is probably more complex. The potencies of radezolid and linezolid are indeed similar when recalculated as a function of cellular concentration expressed in multiples of the MIC at acidic pH. As the activity of radezolid, but not that of linezolid, is reduced at low pH, this recalculation of the data suggests that cellular accumulation is a key property of radezolid's activity, at least against those organisms (staphylococci and Legionella spp.) that thrive in acidic compartments. A similar effect has been reported previously for aminoglycosides (2). However, we cannot ascertain that pH exerts similar effects toward bacteria grown in broth and those thriving in cells (where other environmental factors may also influence their susceptibility to antibiotics). Yet, the fact that radezolid is more potent intracellularly than extracellularly against S. aureus also lends strong support for a potential role of accumulation. Of interest also is that intracellular activity is observed regardless of the intracellular location of bacteria. This is consistent with radezolid's dual localization in the cytosol and acidic vacuoles (29). In this context, our companion paper (29) showed no association of radezolid with mitochondria, as assessed by cell fractionation studies.

Another important observation is that the activity of radezolid develops rapidly both intra- and extracellularly, as the maximal effect is already reached after 3 to 5 h of incubation even at 1 mg/liter. In contrast, linezolid remains static intracellularly for at least 5 h and shows only a modest drop in CFU in broth at 20 mg/liter. These observations are consistent with the improved interaction of radezolid with its ribosomal target (25, 53, 61). In spite of this, however, the maximal effects reached at 24 h are similar for radezolid and linezolid at the highest concentrations tested (if the comparison is limited to linezolid-susceptible strains). This suggests that in contrast to relative potency, which is markedly influenced by the intrinsic activity of each drug, the maximal relative efficacy should be related to the mode of action and pharmacodynamic profile of the drugs. In a broader context, we noted that bacteriostatic drugs, such as macrolides, also cause only a small reduction in the extracellular and intracellular bacterial counts (<1 log10 CFU). For bactericidal drugs like fluoroquinolones, synergistins, or lipoglycopeptides, the decrease in inoculum reaches the limit of detection extracellularly and 2 to 3 log10 CFU intracellularly (1, 27, 28, 32). It must, however, be pointed out that we deal here with an in vitro model where host defenses are minimal and contribute only poorly to the overall antibiotic response. Of interest also, the maximal relative efficacies (Emax values) depend on the target bacterial species but not on the strain (if compared in the same cell line) or on the type of cell infected (when comparing different bacterial species). This clearly shows that the maximal relative efficacy of radezolid is driven by species-specific differences that are more probably related to variations in permeability/efflux than in drug-target interactions. Radezolid indeed shows differences in maximal killing rates in broth when different bacterial species are examined (25) but has a very similar capacity to interact with prokaryotic ribosomes (54). On the other hand, differences among the intracellular models for a specified bacterial species may arise from their various rates of multiplication within the cells and/or from cell-related factors like their capacity for defense against bacteria (12, 44).

A third observation is that radezolid fully maintains its intracellular potency against linezolid-resistant strains in all models. This is an important result, as it supports the potential use of radezolid to fight infections with these strains. Of interest, the intracellular activity of linezolid against the two linezolid-resistant strains is not the same, and for SA238L, also varies depending on the cell type infected. The reasons for these differences need to be further investigated but may be underlying resistance mechanisms that are still largely undefined (23).

Altogether, and even with the limitations inherent to our model as discussed in our previous papers (use of static concentrations and fixed serum concentration [1, 27, 30]), the data presented here point to an improvement in intracellular activity for the new oxazolidinone radezolid, probably as a result of the combination of higher intrinsic activity, a higher level of accumulation within eukaryotic cells, and conserved activity against linezolid-resistant strains. The potential clinical impact of these findings will therefore also need to be reexamined in the light of the pharmacokinetic and pharmacodynamic properties of this molecule when administered to humans in order to define what advantage can be expected within the range of clinically meaningful concentrations.

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ACKNOWLEDGMENTS

M. C. Cambier and C. Misson provided dedicated technical assistance throughout this work.

S.L. is a Postdoctoral Researcher and F.V.B. a Senior Research Associate of the Belgian Fonds de la Recherche Scientifique (F.R.S.-F.N.R.S.). This work was supported by the Fonds de la Recherche Scientifique Médicale (grant nos. 3.4.597.06 and 3.8345.08) and by a grant-in-aid from Rib-X pharmaceuticals.

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FOOTNOTES

    • Received 8 December 2009.
    • Returned for modification 3 April 2010.
    • Accepted 6 April 2010.
  • ↵*Corresponding author. Mailing address: Unité de Pharmacologie cellulaire et moléculaire, Université catholique de Louvain, UCL 73.70, Avenue E. Mounier 73, B-1200 Brussels, Belgium. Phone: 3227647378. Fax: 3227647373. E-mail: francoise.vanbambeke{at}uclouvain.be
  • ↵▿ Published ahead of print on 12 April 2010.

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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 ∼60% of the drug colocalizing to the cytosol and ∼40% 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.

Antibiotic accumulation in phagocytic cells has been the subject of numerous studies over the last 20 years. These studies have examined to what extent drugs accumulate and where they distribute in cells and have also tried to address the mechanisms of entry and efflux. Several antibiotics have been profiled in this way, including beta-lactams, macrolides, fluoroquinolones, aminoglycosides, and glycolipopeptides (see references 2, 21, and 41 for recent key examples). Little is known so far, however, about oxazolidinones (30), although recent work showed that significant differences in accumulation can be observed between apparently closely related derivatives (19). Yet, oxazolidinones deserve special interest in this context, as they represent a useful alternative for treatment of infections caused by multidrug-resistant Gram-positive organisms, especially methicillin-resistant Staphylococcus aureus (MRSA) (46, 48), which we know to thrive and persist intracellularly (10, 23). Several new oxazolidinones are currently undergoing preclinical evaluation to assess potential improvements in activity and pharmacokinetic profile (see reference 44 for a review).

In the present study, we have focused our interest on radezolid (RX-1741), the first molecule brought to clinical evaluation in the subclass of biaryloxazolidinones (49, 50). Biaryloxazolidinones combine into a single molecular design the most important interactions defined by sparsomycin and linezolid with the 50S subunit of the ribosome. This confers to them an improved antimicrobial activity, including against linezolid-resistant strains (17, 35, 50). Within this family, radezolid was selected for further development and has shown appropriate efficacy and tolerability in ongoing phase 2 clinical trials for community-acquired pneumonia and uncomplicated skin and skin structure infections (12).

At the structural level, the presence of a secondary amine coupled with the triazole heterocycle confers to radezolid a dibasic character which markedly increases the ionization and hydrophilicity of the molecule at physiological pH. In contrast, linezolid can be considered a weak monobasic compound (Fig. 1 presents the chemical structure and Table 1 the pertinent physicochemical properties). These properties suggest potentially major differences in the way the two drugs could be processed by cells. This has triggered us to examine the cellular pharmacokinetics of radezolid in eukaryotic cells, using three types of phagocytes (human and murine macrophages and human polymorphonuclear neutrophils [PMN]). We provide a detailed description of uptake, subcellular distribution, and efflux and address the underlying mechanisms of these processes. Our studies use linezolid and azithromycin as comparator compounds. Azithromycin shares with radezolid an amphiphilic, dibasic character and is known to accumulate to high levels in phagocytic cells by a mechanism of diffusion through membranes and segregation in acidic compartments (6, 13). Although less extensively studied, linezolid is known to accumulate only modestly in cells (19).

FIG. 1.

Chemical structure and pertinent physicochemical properties of radezolid {N-[[(5S)-3-[2-fluoro-4′-[[(1H-1,2,3-triazol-5-ylmethyl)amino]methyl][1,1′-biphenyl]-4-yl]-2-oxo-5-oxazolidinyl]methyl]acetamide} and its parent compound linezolid. Gray circles show basic functions, and the light-gray box highlights the biaryl part of the molecule that is characteristic of the subfamily.

Our studies show that radezolid accumulates about 11-fold in phagocytic cells, with no evidence of active efflux. The accumulation is rapid and concentration and energy independent. In addition, radezolid displays a dual subcellular distribution, colocalizing to the cytosol and lysosomes with no specific association with mitochondria. These observations are compatible with a mechanism of diffusion/partial segregation in lysosomes by proton trapping.

(Parts of this study were presented at the 19th European Congress of Clinical Microbiology and Infectious Diseases, Helsinki, Finland, May 2009, as oral presentation O29, and at the 26th International Conference on Chemotherapy, Toronto, Ontario, Canada, June 2009, as an oral presentation.)

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MATERIALS AND METHODS

Antibiotics and main reagents.Radezolid (RX-1741, supplied as microbiological standard powder with a potency of 93%) and [14C]RX-1741 (25 mCi/mmol; labeled on the C of the methylacetamide replacing the oxazolidinone ring) were obtained from Rib-X Pharmaceuticals (New Haven, CT). [14C]RX-1741 was diluted with the cold drug to obtain a stock solution at 1 mg/ml (4 μCi/ml) that was used for all experiments. Linezolid was obtained as the corresponding branded product (Zyvoxid) distributed in Belgium for human use by Pfizer SA/NV (Brussels, Belgium), and azithromycin as the microbiological standard (potency of 94.4%) from Pfizer, Groton, CT. Verapamil, gemfibrozil, and monensin were purchased from Sigma-Aldrich (St. Louis, MO). Cell culture media and sera were from Invitrogen Corp. (Carlsbad, CA), and other reagents from Sigma-Aldrich or Merck KGaA (Darmstadt, Germany).

Cell lines.Most of the experiments were performed with 2 macrophage cell lines, namely, (i) human THP-1 cells (ATCC TIB-202 [American Tissue Culture Collection, Manassas, VA], a myelomonocytic cell line displaying macrophage-like activity [38]), and (ii) murine J774.1 cells (ATCC TIB-67). These cells were maintained in our laboratory as previously described (9, 27). Additional experiments were conducted with PMN, which were isolated from buffy coat samples obtained from healthy volunteers using the Histopaque (1007 and 1119; Sigma-Aldrich) gradient centrifugation technique (700 rpm, 30 min) (24). The purity of the preparation was estimated to be 85%, based on microscopic examination of cells stained with a Hemacolor staining kit (Merck KGaA, Darmstadt, Germany). The viability of the cells was checked by the trypan blue exclusion test and found to be >95%.

Accumulation and release experiments.Antibiotic accumulation and release were determined exactly as described earlier (32) for adherent cells, with adaptations for cells growing in suspension. In brief, cells incubated in the presence of antibiotics were washed three times in ice-cold phosphate-buffered saline (PBS) after suitable incubation times (THP-1 cells and PMN, which grow in suspension, were previously harvested by low-speed centrifugation). They were thereafter collected by centrifugation (THP-1 cells and PMN) or scraping (J774 cells) in distilled water. When measuring the kinetics of release, cells incubated with radezolid were washed, reincubated in a drug-free medium, and collected as described above. When studying the influence of extracellular pH, cells were incubated with buffered medium adjusted to specific pH values ranging from 5.0 to 7.4. The exact pH of each medium was measured before and after incubation and was found to not vary by more than 0.1 pH unit during the experiment.

Assay of cell-associated antibiotics.Cells lysates (obtained by sonication) were used for determination of antibiotic content and protein assay. Radezolid was assayed by liquid scintillation counting, cells having been incubated with the radiolabeled drug (lowest limit of detection, 0.003 mg/liter; linear response between 0.01 and 0.78 mg/liter; R2 = 0.999). This method has been fully validated with respect to specificity, reproducibility, and linearity under the conditions of our assays. We also checked in pilot experiments that the antibiotic cell content measured by the disc plate method gave similar values (the P value was >0.4 when comparing concentrations determined by the two methods). Because the corresponding radiolabeled compounds were not available to us, linezolid and azithromycin were assayed by a microbiological method (disc plate assay), using S. aureus ATCC 25923 as test organism (linear response between 16 and 500 mg/liter [linezolid] and between 8 and 500 mg/liter [azithromycin]; R2 values of 0.989 [linezolid] and 0.963 [azithromycin]). All cellular drug contents were expressed by reference to the total cell protein content (determined by the Lowry method) and converted into apparent total cell concentrations using a conversion factor of 5 μl per mg of cell protein (3).

Determination of protein binding in culture medium.The proportion of radezolid bound to serum proteins in our experimental conditions was evaluated after 2 h of incubation with [14C]radezolid in culture medium containing increasing amounts of fetal calf serum. Bound and free fractions were separated using a Centrifree ultrafiltration device from Millipore (Carrigtwohill, Cork, Ireland) with a regenerated cellulose membrane (molecular mass cutoff, 30 kDa). Amounts of 200 μl of samples were transferred to the ultrafiltration device and centrifuged for 10 min (2,000 × g, Eppendorf centrifuge 5810R equipped with an A-4-62 rotor) as previously described for linezolid (4). Radezolid was then quantified in the ultrafiltrate by scintillation counting; the bound concentration was calculated as the difference between the total concentration and the free concentration.

Cell fractionation studies of J774 cells.The major subcellular organelles were separated by combined differential and isopycnic centrifugations as previously described (42). In brief, cells were incubated with [14C]radezolid for 2 h, washed free of antibiotics in 0.25 M sucrose, 1 mM EGTA, 3 mM imidazole (pH 7.4), and finally collected by gentle scraping in the same medium. The cells were then homogenized with a Dounce tissue grinder, and a cytoplasmic extract free of nuclei was obtained after three successive low-speed centrifugations (770, 625, and 500 × g, 10 min). The resulting cytoplasmic extract was further fractionated into a “granule” fraction (containing the bulk of the cells' organelles and membranes [MLP fraction]) and a final supernatant fraction (S fraction) by high-speed centrifugation (145,000 × g) for 30 min (Ti50 rotor; Beckman instruments, Inc., Fullerton, CA). The MLP fraction was further analyzed by isopycnic centrifugation in a linear sucrose gradient (10.9 ml, density limits 1.10 to 1.24, resting on a 600-μl 1.34 g/cm3 cushion). After equilibration, 12 fractions of approximately 1 ml each were collected and weighed, and their densities were determined by refractometry. The amounts of protein and [14C]radezolid were determined in each fraction in parallel with the activity of marker enzymes of the main organelles, namely, cytochrome c oxidase (for mitochondria), N-acetyl-β-glucosaminidase (for lysosomes), and lactate dehydrogenase (LDH; for cytosol). The results are expressed as the relative frequency of enzyme activity, protein, or drug recovered in each fraction as a function of the density of the fraction (standardized in sections of equal increments), the area of each histogram being equal to 1 (7, 32).

Assessment of cell viability.The viability of cells in the different experimental conditions (exposure to oxazolidinones [up to 50 mg/liter] or incubation in media at different pH, in the presence of monensin, or at low temperature) was evaluated by measuring the release of lactate dehydrogenase, which is a cytosolic enzyme (31), or the formation of purple formazan crystal dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (29)]. Using both procedures, no significant differences (<10%) were detected between treated and control cells.

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RESULTS

Cellular accumulation and release of radezolid in phagocytic cells.In a first series of experiments, we examined the kinetics of radezolid uptake and efflux in three types of phagocytic cells, namely, human macrophage-like THP-1 cells, murine J774 macrophages, and human PMN (Fig. 2). These experiments were performed with an extracellular concentration of radezolid of 4 mg/liter. In contrast to linezolid, which only reached a cellular concentration close to the extracellular one in THP-1 cells, radezolid accumulated quickly and markedly in these cells, reaching cellular levels ∼12-fold higher than linezolid. The kinetics and extent of accumulation of radezolid seen with THP-1 cells was also observed with the other cell types investigated, with an accumulation half-life (t1/2) of ∼6 min and a maximal cellular-to-extracellular concentration ratio of ∼11 (analysis of variance [ANOVA] of individual curves did not evidence any significant difference between cell types with respect to plateau values [P = 0.957] and accumulation constant rates [k = 0.693/t1/2; P = 0.252]). Release was then examined in the three cell types after loading with radezolid for 2 h. The release rates (k = 0.693/t1/2) were not significantly different (P = 0.067) between cell types, with a mean half-life of ∼8.7 min.

FIG. 2.

Kinetics of radezolid uptake and release within THP-1 and J774 cell lines and PMNs. Left, uptake. Cells were incubated for up to 5 h in the presence of 4 mg/liter radezolid (RDZ) or 250 mg/liter linezolid (LZD). The ordinate shows the apparent cellular-to-extracellular concentration ratio. Data are fitted to one-phase exponential association (R2 = 0.837 for RDZ, 0.824 for linezolid). Right, release. Cells were incubated with 4 mg/liter radezolid during 2 h before being transferred to drug-free medium. Values are expressed as the percentage of the accumulated amount at 2 h (time zero). Data are fitted to one-phase exponential decay (R2 = 0.988). Results are given as means ± standard deviations (n = 3).

Influence of radezolid's extracellular concentration and of serum concentration in the culture medium on cellular accumulation.We then examined the effect of increasing the extracellular concentration on the accumulation of radezolid in 2-h uptake experiments (Fig. 3, upper panel). The cellular concentration increased linearly with no sign of saturation up to 50 mg/liter, with an apparent cellular-to-extracellular concentration ratio of approximately 8.5-fold over the entire range of extracellular concentrations investigated (R2 = 0.977 for data pooled from THP-1 cells and PMN; there is no significant difference [t test] between linear regressions when data are plotted per cell line [P = 0.438]).

FIG. 3.

Upper panel, cellular concentration of radezolid in cells incubated for 2 h with increasing extracellular concentrations in medium supplemented with 10% fetal calf serum. Cellular concentrations are expressed either as ng/mg cell protein (prot) (left) or in μg/ml cell volume (conversion factor, 5 μl cell volume/mg of protein) (right). Data are fitted to a linear regression (R2 = 0.997; slope, 8.5 for cellular concentration expressed in μg/ml cell volume, corresponding to the mean accumulation factor). Lower panel, influence of the fetal calf serum concentration in the culture medium on cellular accumulation of radezolid. Cells were incubated for 2 h with 4 mg/liter radezolid in medium supplemented with increasing concentrations of fetal calf serum. Left y axis, cellular accumulation factor of radezolid; right y axis, % radezolid bound to serum proteins. Data for cellular accumulation are fitted to one-phase exponential decay (R2 = 0.904). A statistically significant difference (P < 0.01; one-way ANOVA with Dunnet multiple comparison test [comparison with 10% fetal calf serum]) is shown by an asterisk. Data for protein binding are fitted to sigmoidal regression (R2 = 0.974). Results are given as means ± standard deviations (n = 3).

As the in vitro model used implies the presence of fetal bovine serum in the culture medium, we investigated whether its concentration would influence the capacity of radezolid to accumulate in cells. This was tested with human and murine macrophages incubated for 2 h with 4 mg/liter radezolid in medium supplemented with fetal calf serum from 2 to 20%, these limits being imposed by the maintenance of the viability of the cells. The data presented in Fig. 3 show that decreasing the serum concentration below the standard 10% caused a commensurate increase in radezolid accumulation, whereas increasing it above this value did not cause any significant decrease. The fraction of radezolid bound to serum proteins was determined in parallel for the culture medium; it increased from 0 to 28% when the content of serum was increased from 2.5% to 20%.

Mechanistic studies: influence of temperature, ATP-depletion, and efflux pump inhibitors.The next series of experiments was designed to achieve an understanding of the mechanisms involved in radezolid accumulation in cells, using THP-1 macrophages as a model (Fig. 4). All data were obtained after 2 h of incubation with 50 mg/liter radezolid, 250 mg/liter linezolid, or 10 mg/liter azithromycin. When cells were incubated at 4°C, the apparent cellular concentrations of radezolid and of linezolid decreased to about 35 to 40% of their control values (Fig. 4, left panel). In parallel experiments, we observed that radezolid's accumulation was not modified by either ATP depletion or efflux pump inhibitors, while that of azithromycin, a substrate of P-glycoprotein (34), was increased 1.7-fold in both situations (Fig. 4, middle and right panels). Additional experiments confirmed an absence of an effect of efflux pump inhibitors on (i) the rate of uptake and efflux of radezolid in THP-1 cells; (ii) its level of accumulation over a wide range of extracellular concentrations (4 to 50 mg/liter) in THP-1 cells; or (iii) its level of accumulation in J774 macrophages and PMN (data not shown).

FIG. 4.

Influence of temperature, ATP depletion, and efflux transporter inhibitors on radezolid accumulation in THP-1 cells (incubation time, 2 h; extracellular concentration of radezolid [RDZ] is 50 mg/liter, of linezolid [LZD] is 250 mg/liter, of azithromycin [AZI] is 10 mg/liter, of verapamil is 100 μM, and of gemfibrozil is 250 μM). ATP depletion was obtained by preconditioning cells during 1 h with 60 mM deoxyglucose and 5 mM NaN3 and performing incubation in the same medium (27). The ordinate shows the apparent cellular-to-extracellular concentration ratio (% of control values). All results are given as means ± standard deviations (n = 3). A statistically significant difference (P < 0.05; t test in comparison with control) is shown by an asterisk.

Influence of the proton ionophore monensin and pH gradients.The dibasic character of radezolid (Fig. 1) suggests that pH gradients between cellular compartments and the ionization state of the molecule could play a determining role in its accumulation, as previously described for macrolides like azithromycin (13, 21). We therefore studied the effect of the proton ionophore monensin (known to dissipate transmembrane pH gradients) on radezolid accumulation in THP-1 cells, again in comparison with linezolid and azithromycin. We also tested how acidification of the extracellular medium or the combination of both conditions would affect these molecules. After incubating cells for 2 h in the presence of 50 μM monensin (Fig. 5, left panel), we observed marked reductions in the apparent cellular concentration of both oxazolidinones (residual value, 20% to 25% of control) and a still-larger reduction in that of azithromycin (residual value, 10% of control). Likewise, acidification of the culture medium (Fig. 5, middle panel) caused progressive decreases in the apparent cellular concentration of both oxazolidinones at 30 min when the pH of the culture medium was reduced from 7.0 to 6.0, to reach again 25% of control values for a pH value of ≤6.0.

FIG. 5.

Influence of the proton ionophore monensin and of extracellular pH on radezolid, linezolid, and azithromycin accumulation in THP-1 macrophages (extracellular concentration of radezolid [RDZ] is 50 mg/liter, of linezolid [LZD] is 250 mg/liter, and of azithromycin [AZI] is 10 mg/liter). Left, cells coincubated with 50 μM monensin during 2 h; middle, cells incubated in pH-adjusted medium during 30 min; right, cells coincubated with 50 μM monensin in pH-adjusted medium during 30 min. The ordinate shows the apparent cellular-to-extracellular concentration ratio (% of control values [no monensin or pH 7.4]). All results are given as means ± standard deviations (n = 3). A statistically significant difference (P < 0.01; Student's t test [left and right panels, comparison of control with monensin-treated cells] or one-way ANOVA with Dunnet multiple comparison test [middle panel, comparison with pH 7.4]) is shown by an asterisk.

The azithromycin accumulation fell to 10% of control values as soon as the pH was brought from 7.0 to ≤6.5. Interestingly enough, monensin did not affect radezolid's accumulation in cells incubated in medium adjusted at a pH value of <7.0 but caused a significant decrease at a pH value of ≥7.0, with the residual accumulation remaining similar to that measured in the absence of monensin at pH 6.5 (Fig. 5, right panel). The same experiments were performed with J774 cells, with similar results (residual accumulation, 30% of control values in the presence of monensin or at pH 5; data not shown).

Subcellular localization of radezolid in J774 macrophages.These studies were performed with J774 cells because preliminary experiments with THP-1 cells, using both sucrose and Percoll gradients, showed that the distribution of lysosomes and of mitochondria could not be satisfactorily resolved in these cells based on density equilibration. We first separated cell homogenates into an unbroken cell/nucleus fraction and a cytoplasmic extract using low-speed centrifugation. The unbroken cells and nuclei contained about 20% of the total activity of each enzymatic marker (lactate dehydrogenase, N-acetyl-β-glucosaminidase, and cytochrome c oxidase) and about 20% of the radezolid-associated radioactivity (data not shown). As we see the same percentage for all markers in this fraction, this suggests no specific association of radezolid to nuclei. The cytoplasmic fraction was then further separated by high-speed centrifugation into a supernatant (S) and a granular fraction (MLP) containing the bulk of the mitochondria, lysosomes, and endoplasmic reticulum. About 42% of the cell-associated radezolid was recovered in the granular fraction (MLP), while 58% was recovered in the supernatant (S) (Fig. 6, upper panel). As expected, lactate dehydrogenase activity was almost exclusively recovered in the supernatant, while 90% of the N-acetyl-β-glucosaminidase and 98% of the cytochrome c oxidase activities were found in the MLP. The MLP fraction was then further fractionated by isopycnic centrifugation to establish the distribution of radezolid between the organelles present in this fraction (Fig. 6, lower panel). The distribution of radezolid could be superimposed on that of the lysosomal marker (N-acetyl-β-glucosaminidase) and was clearly distinct from that of the mitochondrial marker (cytochrome c oxidase), with modal equilibration densities of 1.15 and 1.17, respectively.

FIG. 6.

Distribution of marker enzymes (N-acetyl-β-glucosaminidase [NAB; lysosomes], cytochrome c oxidase [CytOx; mitochondria], and lactate dehydrogenase [LDH; cytosol]) and radezolid [RDZ] upon fractionation of J774 cells incubated during 2 h in the presence of 50 mg/liter of [14C]radezolid. The upper panel (extract fractionation) summarizes the percentage of each constituent recovered in supernatant (S) or granular (MLP) fractions. The lower panel shows the density distribution of each constituent within the MLP fraction in a linear sucrose gradient. y axis, relative frequency (fractional quantity in each fraction/density increment).

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DISCUSSION

The data presented in this paper show that radezolid accumulates intracellularly to about 10- to 12-fold its extracellular levels in three types of phagocytic cells. This process is rapid, reversible, nonsaturable, and energy independent but pH dependent. It drives the drug to the cytosolic and lysosomal compartments. This level of accumulation contrasts with that of linezolid, for which we found an accumulation factor close to 1. This is consistent with in vivo data describing linezolid as an antibiotic with high diffusibility in body tissues but poor cellular accumulation, reaching, in human alveolar macrophages, a concentration lower than in serum (14).

To further probe the mechanism(s) involved in radezolid uptake, we first examined influx and efflux mechanisms. Our data are compatible with a passive diffusion process, and this is for four main reasons. First, the rate of uptake and efflux is similar and quite fast in all cell types investigated, with an apparent half-life of 6 to 10 min. These rates are of the same order of magnitude for drugs known to enter cells by diffusion, such as macrolides (8, 13), fluoroquinolones (7, 27), or chloroquine (42). These rates are, however, much faster than those observed for antibiotics accumulating by fluid-phase pinocytosis, such as aminoglycosides in nonrenal cells (39) or oritavancin in macrophages (42). Furthermore, the involvement of a cell-specific transport system is unlikely because (i) the rates of uptake and levels of accumulation of radezolid are quite similar in the three cell types investigated (as well as several types of nonphagocytic cells; see our companion paper [18]) and (ii) its accumulation is nonsaturable over a wide range of extracellular concentrations, as previously described for azithromycin (13). Second, the accumulation of radezolid is not modified by ATP depletion but is markedly reduced by incubating the cells at 4°C. The first observation rules out a potential active intake process, and the second one, also described with azithromycin (13), highlights the importance of membrane fluidity for drug diffusion, as is generally known in most in vitro and in vivo systems (see reference 25 for a review). These data, as well as the absence of effect of efflux pump inhibitors on radezolid accumulation, also suggest that this drug is not subject to active efflux by those multidrug resistance proteins known to decrease the intracellular accumulation of fluoroquinolones (MRP [27]) or macrolides and daptomycin (P-glycoprotein) (20, 34). Third, the higher accumulation of radezolid measured in medium with low serum content suggests that only the free fraction is able to enter the cells, which is what one would expect from a diffusible drug. The plateau of accumulation reached at a higher serum content may reflect a displacement of the protein-bound fraction as the free drug enters the cell. This suggests that a dynamic equilibrium between intracellular and extracellular compartments may take place, with the predominant flux oriented toward the cells as described for azithromycin (33). Finally, it must be emphasized that both the logP and logD values of radezolid are in the range of those considered compatible with drug membrane permeability (5, 22). Also of interest, azithromycin and radezolid display similar logD values calculated at pH 7.4 (Table 1), suggesting comparable capacity to cross the pericellular membrane.

Moving now to the mechanism of accumulation itself, our data are highly suggestive of a proton-driven segregation of radezolid in acidic compartments, somewhat following the model described to explain the cellular accumulation of weakly basic drugs in cells (45) and in lysosomes (11) and best illustrated by macrolides in the field of antiinfective pharmacology (6, 8). We show here that radezolid indeed displays a dual localization, with about 60% recovered in the soluble fraction and 40% distributing together with a lysosomal enzyme in the large-organelle fraction. This means a moderate accumulation in the cytosol (about 6-fold) but a much larger one in the lysosomes (about 80-fold or higher, based on a lysosomal volume of 5% or less than the total cell volume, as estimated in macrophages or fibroblasts [1, 36]). These data may actually reflect the gradients of pH between the extracellular milieu and the cytosol (about 0.4 pH units) or the lysosomes (about 2 pH units), respectively. This is also consistent with our observation of a marked decrease in cellular accumulation at acidic pH (which will reduce the pH gradient between medium and cytosol and lysosomes) and in the presence of monensin (known to collapse the cytosol-lysosomal pH gradient [37]). This is quite similar to what was observed for azithromycin (6, 40). For radezolid, however, the proportion of the drug found in the lysosomes is lower than that reported for azithromycin (40% versus 50 to 70% [6]) and the residual accumulation in the presence of monensin or at acidic pH remains higher than for azithromycin. This probably results from differences in pKa values (Table 1), with azithromycin being more basic and therefore more fully protonated at acidic pH than radezolid. Of interest also, cellular fractionation studies did not show any specific association of radezolid with mitochondria, which are the target organelle for oxazolidinone and chloramphenicol toxicity (26, 47).

The unavailability of radiolabeled linezolid prevented us from performing as detailed experiments with this drug as we were able to do with radezolid, especially with respect to kinetics of uptake and efflux and determination of subcellular distribution. Yet, we observed a globally similar effect of monensin and of acidification of the culture medium on linezolid's accumulation. Actually, the differences in accumulation levels observed between linezolid, radezolid, and azithromycin can easily be explained by commensurate differences in the relative abundances of their charged and uncharged species on the one hand and their lipophilicity (as illustrated by logP and logD values) on the other hand. The model of proton-driven segregation of weak basic drugs in membrane-bounded compartments implies, indeed, that accumulation at equilibrium will be directly proportional (i) to the ratio of the permeability constants of the nonionized to the ionized forms of the molecules and (ii) to the number and the pKa of the basic ionizable functions. Based on what is known about those properties, this clearly rationalizes the ranking in accumulation observed here (namely, linezolid < radezolid < azithromycin), as well as the fact that extracellular pH modulates radezolid's accumulation over a 1-unit range (7.0 to 6.0) instead of the 0.5-unit range (7.0 to 6.5) for azithromycin. For azithromycin, its capacity to bind to negatively charged phospholipids may further enhance its accumulation (28, 43). Of note also, the cellular accumulation of radezolid was not influenced by a variation of pH from 7.4 to 6.5 in the extracellular milieu when monensin was present (Fig. 5, right panel). This is consistent with the facts that (i) monensin acts specifically on the ATP-driven pump responsible for acidifying lysosomes (37) to values as low as pH 5.5 and does not appear affect the cytosol's pH (which is around 7) and (ii) the pKa1 of radezolid is close to this value, making it a turning point for a significant change in the ratio of the monocationic and dicationic forms of the drug.

We recently observed that another oxazolidinone, torezolid (TR700), shows a larger cellular accumulation than linezolid and a greater-than-90% reduction in uptake upon acidification of the culture medium (19). Direct comparison with the radezolid cellular pharmacokinetics described here, however, cannot be made, as (i) we do not know torezolid's subcellular localization and (ii) torezolid's physicochemical properties are quite distinct from those of radezolid (torezolid being not a dicationic drug but being slightly more lipophilic than linezolid, about 0.4 log units for both logP and logD, calculated using QikProp software [Schrõdinger, LLC, Portland, OR]).

Although still far from reproducing the situation prevailing in vivo, the design of our experiments has allowed us to address the impact of protein binding on drug handling by cells. We first see that increasing the protein content above its standard value does not change the level of accumulation of radezolid at equilibrium. We also show that radezolid's accumulation is similar in various cell lines, in freshly isolated human PMNs, and in primary cultures of human keratinocytes (see also the companion paper [18]), suggesting that what we describe is a general property of this drug.

Besides their interest for the knowledge of pharmacological properties of radezolid and other oxazolidinones, our findings may have important clinical implications. First, the fact that radezolid accumulates to higher levels than linezolid may explain its larger volume of distribution (V), as animal data indicate for radezolid a V 1.5- to 1.8-fold higher than that of linezolid in mice, rats, or dogs (see reference 15 and Rib-X data on file). This may help the drug achieve improved tissue penetration and a higher concentration in the infected compartment. High concentration in PMNs may also contribute to the conveyance and delivery of the drug at the site of infection, as previously proposed for azithromycin (33).

Second, although the correlation is extremely variable from one antibiotic class to another, a higher cellular concentration may help the antibiotic to exert useful activity against intracellular bacteria (see reference 41 for a review). This issue is examined in detail in the companion paper (18).

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ACKNOWLEDGMENTS

We are grateful to the Belgian Red Cross for providing us buffy coat samples isolated from healthy volunteers and to Steve Brickner from Pfizer for communicating to us pertinent data on the physicochemical properties of linezolid. M. C. Cambier, C. Misson, and M. Vergauwen provided dedicated technical assistance throughout this work.

S.L. is a Postdoctoral Researcher and F.V.B. a Senior Research Associate of the Belgian Fonds de la Recherche Scientifique (F.R.S.-F.N.R.S.). This work was supported by the Fonds de la Recherche Scientifique Médicale (grants no. 3.4.597.06 and no. 3.8345.08) and by a grant-in-aid from Rib-X pharmaceuticals.

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FOOTNOTES

    • Received 8 December 2009.
    • Returned for modification 21 February 2010.
    • Accepted 5 March 2010.
  • ↵*Corresponding author. Mailing address: Unité de Pharmacologie cellulaire et moléculaire, Université catholique de Louvain, UCL 73.70, Avenue E. Mounier 73, B-1200 Brussels, Belgium. Phone: 3227647378. Fax: 3227647373. E-mail: francoise.vanbambeke{at}uclouvain.be
  • ↵▿ Published ahead of print on 12 April 2010.

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TABLE 1.

Physicochemical properties of drugs

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