Unité de Pharmacologie Cellulaire et Moléculaire, Université Catholique de Louvain, UCL 73.70 Avenue E. Mounier 73, B-1200 Brussels, Belgium
Received 20 January 2003; returned 3 February 2003; revised 8 February 2003; accepted 20 February 2003
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Abstract |
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Keywords: transporters, intracellular, accumulation, verapamil, gemfibrozil
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Introduction |
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Materials and methods |
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Azithromycin (dihydrate salt; potency 94.4%) was obtained from Pfizer Inc. (Groton, CT, USA) as a laboratory sample for microbiological investigations. Unlabelled ciprofloxacin (potency 85%) and 14C-labelled ciprofloxacin (6.96 MBq/mg) were kindly donated by Bayer AG (Wuppertal, Germany). Gentamicin was obtained from GlaxoSmithKline-Belgium as Geomycin (the commercial form for clinical usage in Belgium), on behalf of Schering-Plough Corp. Verapamil was supplied by Fluka Chemie (Buchs, Switzerland) and gemfibrozil by SigmaAldrich Chemie (Steinheim, Germany). Cell culture media and serum were from Gibco Biocult (Paisley, Scotland, UK). All other reagents were obtained from E. Merck AG (Darmstadt, Germany).
Bacteria
S. aureus (ATCC 25923) and L. monocytogenes (haemolysin-producing strain EGD serotype 1/2a) were used for all the experiments, with MICs determined in broth by standard methods.
Cell infection and assessment of intracellular activity of antibiotics
We used the general methods described for L. monocyto genes6 (with adaptation for adherent cells) and for S. aureus (C. Seral, F. Van Bambeke & P. M. Tulkens, unpublished results). In brief, J774 mouse macrophages were cultivated in RPMI medium supplemented with 10% fetal calf serum. The lack of toxic effect exerted by verapamil or gemfibrozil (at the concentrations used) was checked by measuring the release of lactate dehydrogenase7 (release <10% in excess of control values). Infection with L. monocytogenes was obtained using untreated bacteria at an initial bacteria/macrophage ratio of 5 and with incubation carried out for 1 h. Extracellular bacteria were removed by extensive washing and intracellular growth evaluated after 5 h, in the absence of antibiotic (control) or in the presence of azithromycin or ciprofloxacin. Infection by S. aureus was made with opsonized bacteria (0.5 h incubation in fresh human serum). Phagocytosis was initiated at a bacteria/macrophage ratio of 0.5 and with incubation carried out for 1 h. Extracellular bacteria were eliminated by incubation for 1 h in the presence of 50 µg/mL gentamicin followed by extensive washing with PBS. Intracellular growth was evaluated after 24 h of incubation in the absence (controls) or in the presence of azithromycin or ciprofloxacin. Extracellular growth of S. aureus in control cultures (no antibiotic added) was prevented by addition of 0.5 mg/L gentamicin (1 x MIC) (C. Seral, F. Van Bambeke & P. M. Tulkens, unpublished results). Cells were collected by gentle scraping in distilled water after extensive in situ washing with PBS. The number of cell-associated viable bacteria was then measured by plating aliquots on tryptic soy agar (TSA) after vigorous mixing and suitable dilution and by counting the number of colonies after 24 h incubation at 37°C. Activity was defined as the change in the number of cfu recovered from cells at the time considered compared with the value found at time = 0 h (post-phagocytosis). The incubation periods were systematically 5 h for L. monocytogenes and 24 h for S. aureus. These time periods were selected to obtain an intracellular bacterial growth of 10-fold in each case without evidence of growth of extracellular bacteria (based on direct microscopic examination and of plating of the culture media and washing fluids). To allow for direct pharmacodynamic comparisons between the two antibiotics under study, all extracellular concentrations were adjusted to multiples of their MIC for the corresponding organisms. All antibiotics were also compared at an extracellular concentration corresponding to the maximal serum concentration achievable in patients during conventional treatment (Cmax; values taken from literature data6).
Antibiotic accumulation and subcellular distribution
Cells were incubated with azithromycin or [14C]ciprofloxacin, collected and homogenized in 0.25 M sucrose, 1 mM EGTA, 3 mM imidazole pH 7.4, as described previously.11 The homogenate was separated into a nuclear fraction (containing mostly nuclei and unbroken cells) and a cytosolic extract by three successive low-speed centrifugations (2000 rpm, 10 min; 1800 rpm, 10 min; 1600 rpm, 10 min). This cytosolic extract was then separated by high-speed centrifugation (40 000 rpm; 30 min) into a sedimentable fraction (henceforth referred to as granules fraction and containing the bulk of the intracellular organelles) and a supernatant (henceforth referred to as soluble fraction and containing soluble proteins and free ribosomes). Details concerning these procedures are given in Renard et al.11 and used the methods described previously for cultured fibroblasts12. The activity of lactate dehydrogenase and N-acetyl-ß-hexosaminidase was assayed in all fractions, as markers of the soluble proteins (cytosol) and of lysosomes, respectively.11 Azithromycin was assayed by the disc diffusion method using Micrococcus luteus ATCC 9341 as test organism7 and [14C]ciprofloxacin by scintillation counting. The total drug cell content was expressed with reference to the protein content of the unfractionated homogenate, and the apparent cellular to extracellular concentration ratio calculated using a conversion factor of 5 µL of cell volume per mg of cell protein as in previous publications.2,3,7
Statistical analyses
Group comparisons (Students t-test) were carried out with Instat Prism (V.3.01) from GraphPad Prism Software, San Diego, CA, USA.
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Results |
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Figure 1 shows the activity of azithromycin and ciprofloxacin against intracellular bacteria in macrophages incubated with increasing extracellular concentrations of these antibiotics (expressed as multiples of their respective MIC). Considering the L. monocytogenes model first, we see that azithromycin was poorly active against the intracellular form of this bacterium, becoming bacteriostatic only at an extracellular concentration corresponding to 10 x MIC. Addition of verapamil allowed for the same levels of activity to be obtained at extracellular concentrations of about 12 x MIC. No increase in activity, however, was noted at higher extracellular concentrations demonstrating that azithromycin was only bacteriostatic in this model. Addition of gemfibrozil did not affect azithromycin activity towards L. monocytogenes (data not shown). In contrast to azithromycin, ciprofloxacin showed a strong concentration-dependent activity towards intracellular L. monocytogenes over the whole range of concentrations examined, being bacteriostatic at an extracellular concentration equivalent to its MIC and becoming markedly bactericidal at higher concentrations. As shown in Figure 1, addition of gemfibrozil made the drug more effective at all concentrations examined, decreasing by about two-fold the extracellular concentration needed to obtain a bacteriostatic effect while increasing the bactericidal effect observed at higher concentrations in comparison with controls. No effect of verapamil was seen on ciprofloxacin activity for L. monocytogenes (data not shown).
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Figure 2 shows the influence of the efflux pump inhibitors on the cellular accumulation and subcellular distribution of azithromycin and ciprofloxacin in uninfected cells after 24 h of incubation with these antibiotics. The figure also shows the distribution of N-acetyl-ß-hexosaminidase (used as a marker of lysosomes) and of lactate dehydrogenase (used as a marker of soluble proteins). No change in the distribution of these markers was seen, demonstrating that neither verapamil nor gemfibrozil grossly affected the biophysical properties and integrity of lysosomes or the distribution of soluble proteins. As anticipated from the results of previous studies,3,7 azithromycin was accumulated to a large extent by cells, and predominantly localized in the granules fraction. Verapamil increased the cellular accumulation of azithromycin by about 2.4-fold, but without marked change in its subcellular distribution. These experiments needed the use of large extracellular concentrations of azithromycin (50 mg/L) for detection in the subcellular fractions. Yet, verapamil had a similar effect on azithromycin total accumulation in cells exposed to only 5 mg/L antibiotic (data not shown). Gemfibrozil (250 µM) was without effect on azithromycin accumulation (whether tested at 5 or 50 mg/L; data not shown). In contrast to azithromycin, ciprofloxacin was mostly found in the soluble fraction of control cells, as observed in previous studies (B. Scorneaux, unpublished data). Addition of gemfibrozil increased the total drug accumulation also by 2.4-fold. In contrast with what was seen with azithromycin, however, most of the ciprofloxacin accumulated in excess was found in the soluble fraction. Verapamil was without effect on ciprofloxacin accumulation (data not shown). These experiments combining drug accumulation and drug distribution studies were not carried out on infected cells. Yet, in independent experiments, we checked that infection did not affect the antibiotic accumulation whether in control conditions (no inhibitor added) or in cells exposed to verapamil or gemfibrozil.
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Discussion |
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The data presented here show unambiguously that ciprofloxacin is a concentration-dependent antibiotic towards a cytosolic bacterium (L. monocytogenes), and that its intracellular activity can, accordingly, be modulated by changes in its intracellular concentration only, as observed in extracellular media and in broth. This implies that the intracellular drug and the bacteria must come partially into direct contact with one another. Such a contact may take place in the cytosol since (i) L. monocytogenes is located in this compartment, and (ii) the data from the fractionation studies demonstrated that ciprofloxacin in most likely present in that compartment also (the results of these studies are entirely consistent with those of previous ones with other fluoroquinolones2). Thus, the present study extends and rationalizes the observations of Rudin and co-workers made with norfloxacin and ciprofloxacin. These authors indeed showed that gemfibrozil enhances the listeriacidal activity of these fluoroquinolones.16 The present data may also offer a satisfactory explanation for the poor activity of ciprofloxacin towards intracellular S. aureus. Indeed, the data indicate that only a minor proportion of the ciprofloxacin will meet these phagosomal bacteria. More importantly, we also see that the increase in total cell concentration of ciprofloxacin (obtained by a decrease in its efflux in the presence of gemfibrozil) does not allow for a commensurate change in the amount of drug associated with the cell granules. This suggests that the constituents of the cytosol which bind ciprofloxacin are saturated under the conditions used here. The consequence should be that gemfibrozil, and other efflux inhibitors, will be unable to increase ciprofloxacin activity against a non-cytosolic bacterium, which is indeed what we observe with S. aureus.
As for ciprofloxacin, the intrinsic pharmacodynamic properties of azithromycin observed towards extracellular bacteria (i.e. a concentration-dependent activity within a narrow range of concentrations only, and no or little bactericidal activity) seem also to prevail against intracellular L. monocytogenes and S. aureus (C. Seral, F. Van Bambeke & P. M. Tulkens, unpublished results).6 Yet, this activity seems to be considerably nullified considering the exceptionally large accumulation of azithromycin. However, we observe here that verapamil makes azithromycin active at lower extracellular concentrations against both cytosolic (L. monocytogenes) and phagosomal (S. aureus) bacteria. Although of limited amplitude, because of the intrinsically bacteriostatic activity of azithromycin,14 this effect develops in parallel with the increase in drug content occurring both in the cytosol and in the cell granules. This can be explained by the mechanism of accumulation of azithromycin, which, like other cationic amphiphiles, diffuses freely through membranes but is trapped under its protonated form in the acidic compartments (i.e. mainly the lysosomes and to a lesser extent the endosomes and related vacuoles of the endocytic system; see data in Reference 3; see also the reviews on this concept in References 17 and 18). The data presented here confirm this behaviour since azithromycin was mainly found in the granules fraction that contains the bulk of lysosomes, as demonstrated by the distribution of the N-acetyl-ß-hexosaminidase (a typical lysosomal enzyme). Blocking P-glycoprotein at the cell surface should therefore increase azithromycin content in the cytosol (indirect support for this hypothesis is provided by the observation that overexpression of P-glycoprotein decreases the activity of macrolides against L. monocytogenes19). Yet, we may anticipate that the drug will then quickly re-equilibrate between cytosol and lysosomes, hence the effect seen towards intracellular S. aureus.
Beyond their direct pharmacological interest, the conclusions of this study may also lead to potentially useful therapeutic applications. The extracellular concentrations at which we observe an effect of efflux inhibitors on both azithromycin and ciprofloxacin are indeed in the range of those reached in the serum of patients treated with conventional doses of these antibiotics. We may therefore suggest that the activities of azithromycin and ciprofloxacin against intracellular bacteria are actually suboptimal due to basal efflux mechanisms operating in macrophages. Even though verapamil and gemfibrozil are clearly not usable in clinics because of their own pharmacological activities, the data indicate that drugs acting more specifically on antibiotic transporters (such as the specific inhibitors raised against P-glycoprotein and MRP20,21) could prove useful in optimizing antimicrobial intracellular therapy.
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Acknowledgements |
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Footnotes |
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References |
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