Clin

Clin. Microbiol. transmembrane proton gradients are the driving force causing the accumulation of AZM in both cases. Yet, VE did not alter the pH of the lysosomes (approximately 5) or of the cytosol (approximately 7.1). P-glycoprotein was detected by immunostaining at the cell surface as well as in intracellular vacuoles (endosomes and lysosomes). The data suggest that the influx of AZM, ERY, TEL, and ROX is adversely influenced by the activity of P-glycoprotein in J774 macrophages, resulting in suboptimal drug accumulation. Active drug transporters have been described in both procaryotic and eucaryotic cells. Originally described as conferring resistance to anticancer agents in cancer cells, antibiotics in bacteria, or antifungal agents in fungi, these proteins appear today to be part of a very general mechanism that cells have developed to protect themselves from invasion by diffusible, foreign molecules (for a review, see reference 37). In this context, the occurrence of antibiotic transporters in eucaryotic cells has become a common observation (7, 33). More specifically, P-glycoprotein (also referred to as MDR1) and MRP, which are expressed in most cell types and which transport a large variety of drugs, have received much attention. These two types of transporters belong to the superfamily of ATP binding cassette transporters and use ATP hydrolysis as an energy source (28). They play a key role in drug disposition by modulating drug transport through epithelia and other biological barriers to an extent that was completely unsuspected only a few years ago (1). Focusing on macrolides, erythromycin has been shown to be transported by P-glycoprotein in Caco-2 intestinal cells (29, 34). In parallel, erythromycin and azithromycin are capable of inhibiting the transport of various substrates of the P-glycoprotein in epithelial cells in vitro as well as in vivo (9, 12, 13, 23, 30, 31, 39). Yet, little is known about the role of efflux transporters in the handling of macrolides by macrophages, in which these drugs are known to accumulate in large amounts (2, 3, 20, 24). In the present study, we have examined directly in macrophages the potential influence of P-glycoprotein and MRP on the accumulation and efflux of five macrolides of clinical interest. We used both broad-spectrum, nonspecific inhibitors of P-glycoprotein (verapamil and cyclosporine) and MRP (probenecid and gemfibrozil) and the specific P-glycoprotein modulator GF120918 (11, 15). We selected the murine J774 murine macrophage line since much is already known about the dispositions of macrolides in these cells (2, 3, 36). MATERIALS AND METHODS Cells. We used J774 murine macrophages, which were cultivated as described previously (25). Cell viability was assessed by measurement of lactate dehydrogenase release (19). Determination of cellular antibiotic accumulation. Studies of cellular antibiotic accumulation were performed by the general procedure described in previous publications (3, 25). Antibiotic assays were performed with cell lysates by the diffusion disk method (17) with antibiotic medium 2 (Difco, Becton Dickinson & Co., Sparks, Md.) seeded with ATCC 9341. The pH of the medium was adjusted to 9.5 for all drugs except azithromycin (for which the pHs were adjusted to 9.5 for samples with drug concentrations 0.5 mg/liter and 8.0 for higher drug concentrations). The lowest limits of detection and the typical ranges of drug concentrations measured were 0.2 and 0.9 to 4 mg/liter, respectively, for erythromycin; 0.2 and 0.25 to 0.4 mg/liter, respectively, for roxithromycin;.P-glycoprotein inhibitor erythromycin increases oral bioavailability of talinolol in humans. (probenecid [2.5 mM] and gemfibrozil [0.25 mM]) had no effect. Monensin (a proton ionophore) completely suppressed the accumulation of AZM in control cells as well as in cells incubated in the presence of VE, demonstrating that transmembrane proton gradients are the driving force causing the accumulation of AZM in both cases. Yet, VE did not alter the pH of the lysosomes (approximately 5) or of the cytosol (approximately 7.1). P-glycoprotein was detected by immunostaining at the cell surface as well as Sivelestat sodium hydrate (ONO-5046 sodium hydrate) in intracellular vacuoles (endosomes and lysosomes). The data suggest that the influx of AZM, ERY, TEL, and ROX is adversely influenced by the activity of P-glycoprotein in J774 macrophages, resulting in suboptimal drug accumulation. Active drug transporters have been described in both procaryotic and eucaryotic cells. Originally described as conferring resistance to anticancer agents in cancer cells, antibiotics in bacteria, or antifungal agents in fungi, these proteins appear today to be part of a very general mechanism that cells have developed to protect themselves from invasion by diffusible, foreign molecules (for a review, see reference 37). In this context, the occurrence of antibiotic transporters in eucaryotic cells has become a common observation (7, 33). More specifically, P-glycoprotein (also referred to as MDR1) and MRP, which are expressed in most cell types and which transport a large variety of drugs, have received much attention. These two types of transporters belong to the superfamily of ATP binding cassette transporters and use ATP hydrolysis as an energy source (28). They play a key role in drug disposition by modulating drug transport through epithelia and other biological barriers to an extent that was completely unsuspected only a few years ago (1). Focusing on macrolides, erythromycin has been shown to be transported by P-glycoprotein in Caco-2 intestinal cells (29, 34). In parallel, erythromycin and azithromycin are capable of inhibiting the transport of various substrates of the P-glycoprotein in epithelial cells in vitro as well as in vivo (9, 12, 13, 23, 30, 31, 39). Yet, little is known about the role of efflux transporters in the handling of macrolides by macrophages, in which these drugs are known to accumulate in large amounts (2, 3, 20, 24). In the present study, we have examined directly in macrophages the potential influence of P-glycoprotein and MRP on the accumulation and efflux of five macrolides of clinical interest. We used both broad-spectrum, nonspecific inhibitors of P-glycoprotein (verapamil and cyclosporine) and MRP (probenecid and gemfibrozil) and the specific P-glycoprotein Rabbit polyclonal to PHF7 modulator GF120918 (11, 15). We selected the murine J774 murine macrophage line since much is already known about the dispositions of macrolides in these cells (2, 3, 36). MATERIALS AND METHODS Cells. We used J774 murine macrophages, which were cultivated as described previously (25). Cell viability was assessed by measurement of lactate dehydrogenase release (19). Determination of cellular antibiotic accumulation. Studies of cellular antibiotic build up were performed by the general procedure explained in previous publications (3, 25). Antibiotic assays were performed with cell lysates from the diffusion disk method (17) with antibiotic medium 2 (Difco, Becton Dickinson & Co., Sparks, Md.) seeded with ATCC 9341. The pH of the medium was modified to 9.5 for those drugs except azithromycin (for which the pHs were modified to 9.5 for samples with drug concentrations 0.5 mg/liter and 8.0 for higher drug concentrations). The lowest limits of detection and the typical ranges of drug concentrations Sivelestat sodium hydrate (ONO-5046 sodium hydrate) measured were 0.2 and 0.9 to 4 mg/liter, respectively, for erythromycin; 0.2 and 0.25 to 0.4 mg/liter, respectively, for roxithromycin; 0.08 and 0.25 to 0.9 mg/liter, respectively, for azithromycin; 0.2 and 0.4 Sivelestat sodium hydrate (ONO-5046 sodium hydrate) to 0.8 mg/liter, respectively, for clarithromycin; and 0.08 and 0.3 to 1 1.3 mg/liter, respectively, for telithromycin. Linearity was acquired up to a concentration of 2 mg/liter for those drugs (the concentration at which linearity was acquired for azithromycin at pH 8 was 32 mg/liter), with test, one-way analysis of variance) were done with Instat Prism software (version 3.01; GraphPad Prism Software). RESULTS Influence of P-glycoprotein inhibitors within the kinetics of macrolide build up and efflux. Figure ?Number1A1A demonstrates azithromycin (5.

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