Intracellular penetration and activity of BAY Y 3118 in human polymorphonuclear leukocytes

Abstract

The penetration of a new quinolone (BAY Y 3118) into human polymorphonuclear leukocytes (PMNs) was evaluated by a fluorometric assay. The cellular concentration-to-extracellular concentration (C/E) ratio was higher than 6.3 at extracellular concentrations ranging from 2 to 100 mg/liter. The uptake of BAY Y 3118 was rapid, reversible and nonsaturable. The intracellular penetration of BAY Y 3118 was significantly affected by environmental temperature (C/E ratio at 4°C, 5.4 ± 0.5; control, 7.5 ± 0.9; P < 0.05) and cell viability (C/E ratio in dead PMNs, 5.5 ± 0.8; control, 7.5 ± 0.9; P < 0.05), but it was not affected by metabolic inhibitors. supplied by Bayer A.G., Leverkusen, Germany. In these experiments, PMNs were incubated in Hanks balanced salt solution containing different concentrations of antimicrobial agent (2 to 100 mg/liter). After different incubation times at 37°C, cells were separated from extracellular solution by centrifugation through a water-impermeable silicone-oil barrier in a microcentrifuge tube. The entire cell pellet, obtained by cutting off the portion of the microcentrifuge tube containing the pellet, was placed in 2 ml of 0.1 M glycine-HCl buffer (pH 3.0) and agitated vigorously in a vortex shaker. Incubation for 2 h at room temperature was sufficient to release the intracellular antimicrobial agent fully (9). Samples were centrifuged for 5 min at 5,600 x g, and the amount of antimicrobial agent was determined by fluorescence emission of supernatants with an F 2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The fluorescence excitation and emission maxima in 0.1 M glycine-HCl (pH 3.0) were 285 and 459 nm, respectively. Controls without antimicrobial agents were always used to determine the background fluorescence.Intracellular water space was measured by using triated water and the extracellular marker [14C]polyethylene glycol (1.4 mCi/g; New England Nuclear Corp., Boston, Mass.). Cells were incubated with these radiolabeled compounds for 2 min at 37°C, separated from extracellular fluid by velocity gradient centrifugation as described above, and counted in a liquid scintillation counter. Total water content of the cell pellet was corrected for trapped extracellular water, i.e., polyethylene glycol space, to obtain the intracellular water space. From the values obtained by this procedure, cell-associated antimicrobial agent concentrations were calculated and expressed as ratios of the cellular concentration to extracellular concentration (C/E ratios) (7).Characterization of BAY Y 3118 uptake. Further studies to elucidate the mechanism of BAY Y 3118 uptake of PMNs were performed as described previously (9). The importance of cell viability was studied by using PMNs killed by exposure to 10% Formalin for 30 min. These cells were washed and then suspended in fresh medium. Moreover, the influences of environmental temperature, pH, and metabolic inhibitors were evaluated. The influencc of temperature was examined by

Discrepancy between Uptake and Intracellular Activity of Moxifloxacin in a Staphylococcus aureus -Human THP-1 Monocytic Cell Model

“…However, at external concentrations of 0.1 to 5 mg/liter (CIC: 20 to 1,000 ϫ MBC), the antibiotic only gave, at the single sampling time of 3 h, 90% survival relative to 15% for control cells. Similar studies of other fluoroquinolones (10,11,13,14,26,27,37) have led to similar results and interpretation. Indeed, fluoroquinolones certainly cooperate with PMNs for killing bacteria (36), but the very efficient bactericidal mechanisms of these phagocytes render the PMN models poorly sensitive for evaluating the intrinsic cell bactericidal activity of antibiotics (35).…”

“…Passive diffusion is probably the major mechanism, but active transport systems are certainly involved (24,25), which explains why higher C/E ratios can be observed at the lowest extracellular concentrations. S. aureus phagocytosis significantly enhanced the entry of moxifloxacin into THP-1 cells; this finding contrasts with previous reports on this molecule (25) and other quinolones (9,10,13,14,26,27) in PMNs. However, variable effects of S. aureus ingestion on antibiotic uptake have been previously described (16).…”

“…Moreover, quinolones should be, at least partly, inactivated at the low pH of phagosomes (32). Partial binding of the drug to cell components is unlikely, since moxifloxacin (25), like other quinolones (11,13,14,23,26,27,34,37) effluxes rapidly when the extracellular drug is removed. Given the potential of quinolones for emergence of resistance, persisters were examined to establish whether they were selected resistant mutants.…”

“…Moreover, the last set of studies usually evaluated the uptake and intracellular activity of fluoroquinolones in PMNs, whose intrinsic bactericidal properties certainly interfere, and the single sampling times in short-term experiments provide no information on the bacterial killing rate (35). Finally, factors that may alter such evaluation, that is, the number of ingested bacteria per cell, the level of cell contamination by remaining extracellular bacteria, and the percentage of cell recovery, are rarely specified (13,14,(25)(26)(27).…”

“…Thus, the penetration of moxifloxacin into human monocytic THP-1 cells, which are devoid of bactericidal functions, and its intracellular activity against S. aureus, a common bacterial pathogen widely used as test organism in analogous investigations (6,9,10,13,14,(25)(26)(27)37), were studied at several sampling times in a 5-h assay. In addition, the intracellular activity of moxifloxacin was compared with its in vitro activity, as it is classically evaluated by microbiologists, i.e., by means of MICs, minimal bactericidal concentrations (MBCs), and killing curves.…”