Intracellular penetration and bactericidal activity of the novel des-fluoro(6) quinolone, BMS-284756

Sir, Many organisms of clinical importance, such as mycobacterium, chlamydia, legionella and Staphylococcus aureus, are intracellular pathogens. These intracellular organisms are inhibited only by antimicrobial agents that can achieve high levels in phagocytic cells. Quinolones generally have good intracellular uptake into phagocytes, with a cellular to extracellular ratio (C/E) of c. 5–10. In this study, the penetration of the novel des-fluoro(6) quinolone BMS284756 into macrophages and its ability to kill intracellular staphylococci were examined. BMS-284756 was compared with ciprofloxacin (positive control) and cefepime (negative control) for intracellular uptake into phagocytes. C/E ratios were determined using the RAW 264.7 murine macrophage cell line and A549 human type II pneumocytes (Table). Quinolone uptake was determined by mixing 50 mg/L of the antimicrobial agent and 1–5 10 phagocytic cells (RAW 264.7 or A549) in Hanks’ balanced salt solution (HBSS) and incubated at 37°C for 1 h. Extracellular drug was removed by centrifugation (10 000g, 10 s) and two HBSS washes. Intracellular drug was released by disrupting the phagocytes by suspending the cellular pellet in distilled water and repeated freeze–thawing. The antimicrobial concentration was determined using a standard bioassay. The bioassay strain utilized for assessing BMS-284756 and ciprofloxacin concentrations was S. aureus A20698, and for cefepime was Micrococcus luteus A9547. Antimicrobial standard curves were generated by dispensing 20 L of known antimicrobial concentrations onto paper discs, which were air-dried for 30 min before placement on to agar plates seeded with the bioassay strain. Similarly, 20 L of neat and two-fold serial diluted samples of the freeze–thawed phagocytic cells were dispensed onto paper discs, air-dried, and placed onto seeded bioassay plates. The bioassay plates were incubated at 37°C for 18 h, and the inhibition zone diameters were measured. Intracellular antimicrobial agent concentrations were determined by extrapolation from the antimicrobial standard curves. Killing of intracellular staphylococci was done by incubating J744.1 murine macrophage cells with 10 cfu of opsonized S. aureus A20698 (at 20:1 bacteria:macrophage ratio) at 37°C for 1 h. Opsonization of the staphylococci was achieved by incubation of the bacteria with fetal calf serum for 15 min at 35°C. The extracellular staphylococci were removed by centrifugation (10 000g, 3 min), lysostaphin treatment (5 U/mL, 15 min at 37°C) and two washes in Dulbecco’s minimum essential medium. The quinolone (at 2–6 mg/L) was added to the resuspended macrophages and incubated at 37°C for 3 h. Extracellular quinolone was removed by centrifugation, followed by two washes. The intracellular staphylococci were released by suspending the macrophages in distilled water for cfu determination. BMS-284756 and ciprofloxacin were comparable in their uptake by murine and human phagocytes (Table). Their C/E ratios ranged from c. 4 to 5.9 and are comparable to those reported for other quinolones. In contrast, as anticipated for -lactams, cefepime uptake into macrophages was poor. The rate of BMS-284756 uptake into RAW 264.7 cells was examined, by C/E determination at 1, 5, 10, 20, 30 and 60 min of incubation. The C/E ratios obtained at 1 min and 1 h were the same, indicating the rapid uptake of BMS284756 intracellularly. Intracellular killing was carried out in the murine J774.1 macrophage cell line because of its enhanced phagocytic activity. After 3 h of exposure to BMS-284756 (at 2–6 mg/L), c. 60–66% of the intracellular S. aureus A20698 cells were killed, compared with a 46% reduction in cell count