Influence of efflux transporters on the accumulation and efflux of four quinolones (ciprofloxacin, levofloxacin, garenoxacin, and moxifloxacin) in J774 macrophages

Abstract

Ciprofloxacin is subject to efflux from J774 macrophages through a multidrug resistance-related protein-like transporter (J. M. Michot, F. Van Bambeke, M. P. Mingeot-Leclercq, and P. M. Tulkens, Antimicrob. Agents Chemother. 48:2673-2682, 2004). Here, we compare ciprofloxacin to levofloxacin, garenoxacin, and moxifloxacin for transport. At 4 mg/liter, an apparent steady state in accumulation was reached after 30 to 60 min for all quinolones but to quite different levels (approximately 3, 5, 10, and 16 fold). Accumulation of ciprofloxacin was increased (to about 16 to 20 fold) by ATP depletion, increase in extracellular concentration, and the addition of probenecid, gemfibrozil, or MK571 (but not verapamil or GF120918). These treatments did not affect the accumulation of moxifloxacin. Levofloxacin and garenoxacin showed an intermediate behavior. Efflux of ciprofloxacin was slowed down by probenecid (half-life, 7.2 versus 1.6 min). Moxifloxacin efflux was faster and unaffected by probenecid (half-lifes, 0.27 versus 0.33 min). Efflux of levofloxacin and garenoxacin was modestly decreased by probenecid (1.5 and 2.1 fold). Accumulation of 14C-labeled ciprofloxacin was increased by unlabeled ciprofloxacin and moxifloxacin, but moxifloxacin was two times less potent. Accumulation of moxifloxacin at 4 degrees C was almost identical to that at 37 degrees C, whereas that of ciprofloxacin was minimal (levofloxacin and garenoxacin showed intermediate behaviors). Cells subjected to thermal shock (56 degrees C; 10 min) accumulated all quinolones at a similar level (16 to 23 fold). We conclude that moxifloxacin is apparently not subject to efflux from J774 macrophages, even though it can interact with the ciprofloxacin transporter. Levofloxacin and garenoxacin are partially effluxed. Data suggest that efflux plays an important role in the differential accumulation of quinolones by J774 macrophages.

FIG. 1.

Comparative kinetics of the accumulation of the four quinolones at an extracellular concentration of 5 mg/liter and at 37°C in J774 murine macrophages (circles, ciprofloxacin; inverted triangles, levofloxacin; triangles, garenoxacin; squares, moxifloxacin). All data are means ± standard deviation (SD) of three experiments (for symbols without bars, the SD is smaller than the symbol size). Results are expressed as the ratio of the apparent cellular concentration of each quinolone to its extracellular concentration. Data obtained for each drug were fitted to one-phase exponential association equations; regression parameters are as follows: ciprofloxacin, R² = 0.817; y_max = 2.775 [95% CI, 2.259 to 3.289]; levofloxacin, R² = 0.950; y_max = 4.987 [95% CI, 4.600 to 5.373]; garenoxacin, R² = 0.983; y_max = 9.50 [95% CI, 9.95 to 10.06]; moxifloxacin, R² = 0.967; y_max = 16.48 [95% CI, 15.61 to 17.35]).

FIG. 2.

Comparative short-term kinetics of the accumulation of ciprofloxacin (left) and moxifloxacin (right) at two different extracellular concentrations (open symbols, 5 mg/liter; closed symbols, 17 mg/liter). All data are the mean ± SD of three experiments (for symbols without bars, the SD is smaller than the symbol size). Results are expressed as the ratio of the apparent cellular concentration of each quinolone to its extracellular concentration. Data obtained for each drug were fitted to a two-phase exponential association; regression parameters are as follows: ciprofloxacin, 5 mg/liter and R² = 0.987; ciprofloxacin, 17 mg/liter and R² = 0.992; moxifloxacin, 5 mg/liter and R² = 0.999; moxifloxacin, 17 mg/liter and R² = 0.991. The parameters best describe the biphasic character of the uptake of these two quinolones.

FIG. 3.

Influence of the extracellular concentration of quinolones on their accumulation at equilibrium (2 h) in J774 murine macrophages in control conditions (open symbols) or under conditions of ATP depletion (closed symbols). All data are expressed as the percentages of the maximal ratio of apparent cellular to extracellular concentration observed for the corresponding drug in these experiments (ciprofloxacin, 23.05 ± 0.71; levofloxacin, 9.33 ± 0.45; garenoxacin, 15.00 ± 0.50; moxifloxacin, 16.73 ± 0.31) and are shown as means ± the 95% confidence interval (for three experiments). For ciprofloxacin and levofloxacin (controls), data were fitted to a variable-slope sigmoidal dose-response equation. For all other conditions, the figure shows straight lines corresponding to arbitrary linear regression equations.

FIG. 4.

Influence of MRP inhibitors (probenecid [5 mM], gemfibrozil [500 μM], MK571 [100 μM]) and P-glycoprotein inhibitors (verapamil [100 μM] and GF120918 [2 μM]) on the accumulation of quinolones. Cells were incubated for 2 h in the presence of 5 mg/liter of each quinolone in the absence (control) or in the presence of the corresponding inhibitors. All values are the means of three independent determinations ± SD. Statistical analysis (one-way analysis of variance): P < 0.01 for ciprofloxacin, levofloxacin, or garenoxacin alone versus these antibiotics in the presence of probenecid, gemfibrozil, or MK571, respectively; P < 0.05 for moxifloxacin alone versus moxifloxacin in the presence of verapamil. All differences between each antibiotic alone and the same antibiotic in the presence of other inhibitors are not significant (P > 0.05).

FIG. 5.

Influence of probenecid on the efflux of ciprofloxacin and moxifloxacin from J774 macrophages. Control (open symbols), cells were loaded by incubation at 37°C for 2 h in the presence of 17 mg/liter of the corresponding quinolone and then transferred to a quinolone-free medium to measure efflux (controls). Probenecid-treated cells are shown by closed symbols. Loading and efflux studies were performed in the presence of 5 mM probenecid; we showed in a previous report (16) that probenecid does not alter the kinetic parameters of the influx of ciprofloxacin. Data are expressed as a percentage of the amount accumulated by the cells at the end of the loading phase (apparent cellular to extracellular concentration ratios: ciprofloxacin, 3.92 ± 0.29, ciprofloxacin plus probenecid, 15.83 ± 0.2.03; moxifloxacin, 17.09 ± 2.26; moxifloxacin plus probenecid, 17.94 ± 2.30). They were fitted to one-phase exponential decay curves (goodness of fit [R²] and half-lives: ciprofloxacin, 0.967 and 1.62 ± 0.25 min; ciprofloxacin plus probenecid, 0.963 and 7.20 ± 0.59 min; moxifloxacin, 0.980 and 0.27 ± 0.03 min; moxifloxacin plus probenecid, 0.993 and 0.33 ± 0.22 min).

FIG. 6.

Influence of the addition of unlabeled ciprofloxacin on the accumulation of ¹⁴C-labeled ciprofloxacin. Cells were maintained for 2 h at 37°C with a constant concentration of 940 nCi (34.8 kBq) of ¹⁴C-labeled ciprofloxacin per ml (corresponding to a weight concentration of 5 mg/liter and a molar concentration of 15.1 μM). Open symbols and the solid line indicate that the medium contained increasing amounts of unlabeled ciprofloxacin, starting from a concentration of 0 mg/liter (the first point on the graph) to a maximum of 195 mg/liter (590 μM, close to the limit of solubility in the culture medium). Grey symbols and the dotted line indicate that the medium contained 5 mg/liter of unlabeled ciprofloxacin plus increasing amounts of moxifloxacin, starting from a concentration of 5 mg/liter (12.5 μM). Data were fitted to sigmoidal dose-response curves (R² = 0.979 and 0.997, respectively) according to a previously described model of the cooperative effect of ciprofloxacin on ciprofloxacin accumulation by J774 macrophages (16).

FIG. 7.

Cellular accumulation of quinolones after 2 h incubation at an extracellular drug concentration of 5 mg/liter. (Left) Hatched bars, controls (incubated at 37°C); black bars, cells were incubated at 4°C. (Right) Hatched bars, controls (incubated at 37°C); grey bars, cells were exposed to 56°C for 10 min in the absence of drug, cooled to 37°C, and then incubated at 37°C in the presence of drug for 2 h. All values are the means of three independent determinations ± SD.

FIG. 8.

Structural formula of the quinolones used in the present study. Atoms are numbered counterclockwise starting from the heteroatom (N) in the main byclic quinolone ring, discounting those with no potential substituents. The figure emphasizes the following. (i) Both garenoxacin (also known as T 3811 or BMS-284756) and moxifloxacin possess bulky substituents in position 7 (indicated by the large black arrows; garenoxacin, 2,3-dihydro-1-methyl-1H-isoindole; moxifloxacin, 4a,7a-octahydro-6H-pyrrolo[3,4-b]pyridine) that confer a more hydrophobic character to these molecules than ciprofloxacin or levofloxacin (for which the corresponding substituent is a piperazine or a 4-methyl-piperazine, respectively). (ii) Both garenoxacin and moxifloxacin possess free rotating substituents in position 8 (indicated by the black arrowheads; garenoxacin, difluoromethoxy; moxifloxacin, methoxy) which contribute to an increase in lipophilicity. In levofloxacin, the substituent in position 8 is part of a more rigid structure (morpholino) that bears some degree of similarity with a methoxy. (iii) Garenoxacin is a desfluoroquinolone (having no F substituent in position 6 [grey arrow]). The reported log P (octanol:water partition coefficient) and log D_pH=7 (octanol/water partition coefficient at pH 7; log D = log P − log[1 + 10^(−charge * ^(pH−pKa))]) values, two accepted measures of the hydrophobic character of organic molecules, are 1.31 ± 0.81 and −1.20, 1.49 ± 0.79 and −1.35, 1.62 ± 0.93 and −0.90, and 1.98 ± 0.82 and −0.53 for ciprofloxacin, levofloxacin, garenoxacin, and moxifloxacin, respectively. The pKa values of the protonable function of the C7 substituent of the four quinolones are 8.76 ± 0.25 (ciprofloxacin), 6.8 ± 0.3 (levofloxacin), 8.4 ± 0.4 (garenoxacin), and 10.8 ± 0.4 (moxifloxacin). This will result in larger proportion of moxifloxacin (and a lower proportion of levofloxacin) under a zwitterionic form at neutral pH. All log P, log D, and pKa data, calculated with the Advanced Chemistry Development software Solaris V4.67, were obtained from Sci Finder Scholar 2004, American Chemical Society, Washington, D.C.