Cellular uptake and subcellular distribution of roxithromycin and erythromycin in phagocytic cells

169

140

In contrast to currently marketed fluoroquinolones, which are zwitterionic, delafloxacin is an investigational fluoroquinolone with an anionic character that is highly active against Gram-positive bacteria. We have examined the effect of acidic pH on its accumulation in Staphylococcus aureus and in human THP-1 cells, in parallel with its activity against extracellular and intracellular S. aureus. Moxifloxacin was used as a comparator. Delafloxacin showed MICs 3 to 5 log 2 dilutions lower than those of moxifloxacin for a collection of 35 strains with relevant resistance mechanisms and also proved to be 10-fold more potent against intracellular S. aureus ATCC 25923. In medium at pH 5.5, this difference was further enhanced, with the MIC decreasing by 5 log 2 dilutions. In infected cells incubated in acidic medium, the relative potency was 10-fold higher than that at neutral pH and the maximal relative efficacy reached a bactericidal effect at 24 h. These results can be explained by a 10-fold increase in delafloxacin accumulation in both bacteria and cells at acidic pH, making delafloxacin one of the most efficient drugs tested in this model. Opposite effects were seen for moxifloxacin with respect to both activity and accumulation. As reported for zwitterionic fluoroquinolones, delafloxacin was found associated with the soluble fraction in homogenates of eukaryotic cells. Taken together, these properties may confer to delafloxacin an advantage for the eradication of S. aureus in acidic environments, including intracellular infections.Fluoroquinolones are one of the first families of fully synthetic antibiotics with large clinical use, and they represent a wide range of molecules, which permits the establishment of in-depth structure-activity relationships (16,17). Over the last 20 years, most fluoroquinolone research efforts have been focused on new molecules with improved activity toward Grampositive cocci because of the increasing spread of multiresistant staphylococci and streptococci. The 8-methoxy fluoroquinolone moxifloxacin is the first example of this new generation to reach the commercial market. It combines many desirable microbiological and pharmacokinetic properties (rapid bactericidal activity, a spectrum covering pertinent pathogens, excellent oral bioavailability, a large tissue distribution, and a propensity to accumulate within eukaryotic cells [see reference 45 for a review]). Moxifloxacin shows extensive activity toward bacteria thriving in the cytosol (Listeria monocytogenes [14]), phagosomes (Legionella pneumophila, Mycobacterium avium, and Mycobacterium tuberculosis [5,9,46]), and phagolysosomes (Staphylococcus aureus [6]), suggesting that it easily diffuses between different subcellular compartments. Yet moxifloxacin activity is partially defeated by the acidic pH prevailing in vacuolar subcellular compartments (6).Delafloxacin [RX-3341; 1-(6-amino-3,5-difluoropyridin-2-yl)-8-chloro-6-fluoro-7-(3-hydroxyazetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] is an investigational fluoro...

Section: Discussionmentioning

confidence: 72%

Contrasting Effects of Acidic pH on the Extracellular and Intracellular Activities of the Anti-Gram-Positive Fluoroquinolones Moxifloxacin and Delafloxacin againstStaphylococcus aureus

Lemaire

Tulkens

Bambeke

2011

169

140

“…**, P < 0.01; *, P < 0.05. of antibiotics taken up and concentrated by leukocytes; as a result, they can reach intracellular concentrations far higher than those attained in the extracellular medium (8,21,28). Furthermore, their different pharmacokinetic properties, especially with respect to their biological half-lives and intracellular concentrations at a given time (9,16,27,28), could explain their differential effects on IL-6 production within the same group. Indeed, differences in their interactions with phagocytes have already been shown (24,32).…”

Section: Resultsmentioning

confidence: 99%

Differential modulation of cytokine production by macrolides: interleukin-6 production is increased by spiramycin and erythromycin

Bailly

Pocidalo

Fay

et al. 1991

Antibiotics do not act alone but act in conjunction with the host defense system. In pairticular, it has been shown that some antibiotics can modify cytokine production. We compared the in vitro effiects of three macrolides (roxithroinycin, spiramycin, and erythromycin) actively concentrated by leukocytes on interleukinla, (IL-la), IL-1p, , and tumor necrosis factor alpha. production by human monocytes stimulated with lipopolysaccharide. Our results show that the three macrolides tested have different effects on production of these cytokines. Spiramycin and, to a lesser extent, erythromycin increased total IL-6 production without affecting IL-la, IL-1j0, or tumor necrosis factor alpha production, whereas roxithromycin had no effect. To our knowledge, this is the first time that an antibiotic has been shown to increase IL-6 production.Over the last 20 years, a growing interest in the study of interactions between antimicrobial agents and host defense systems has arisen. In effect, a wide range of antimicrobial agents administered in vivo or in vitro have been reported to modify host immune responses (14,17,31), including cytokine production (3,4,20,29,30).Interleukin-la (IL-la), and IL-1p, IL-6, and tumor necrosis factor alpha (TNF-a) are the major cytokines produced by mononuclear phagocytes (6, 10, 23, 34). They mediate a wide range of biological activities that play a critical role in the induction of inflammatory and immune responses. Although these cytokines are biochemically and immunologically distinct proteins transcribed from different genes, they share a number of activities, such as the induction of fever and acute-phase protein synthesis by the liver (5,12,15,26). They have also been found to exhibit synergy, and each can modulate the production of the others (1,25,33).Macrolides are a class of antibiotics taken up and concentrated by leukocytes; as a result, they can reach intracellular concentrations far higher than those attained in the extracellular medium (8,21,28 Cytokine production. Following adherence, the cells were washed, and 1 ml of fresh serum-free medium was added to the monolayers with 10 p,g of lipopolysaccharide (LPS) (Escherichia coli O.55:B5; Difco) per ml and with or without various concentrations of the antibiotics (1, 10, and 50 ,ug/ml). Cultures were incubated for 18 h at 37°C in a humidified atmosphere of 95% air and 5% CO2. To determine extracellular cytokine concentrations, cell-free supernatants were collected, centrifuged at 800 x g for 10 min, and stored at -70°C until assay. To determine cell-associated cytokine concentrations, the cells remaining in the wells were covered with 1 ml of fresh medium, lysed with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) (9 mM; Sigma) for 30 min, and stored at -70°C until assay. Addition of CHAPS to any of the recombinant cytokines used in this experiment did not modify the immunoassays.

Section: ) P-glycoprotein Was Detected By Immunostaining At the Celmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 98%

See 1 more Smart Citation

Influence of P-Glycoprotein Inhibitors on Accumulationof Macrolides in J774 MurineMacrophages

Seral

Michot

Chanteux

et al. 2003

The influence of inhibitors of P-glycoprotein (verapamil [VE], cyclosporine [CY], and GF120918 [GF]) on the cell handling of macrolides (erythromycin [ERY], clarithromycin [CLR], roxithromycin [ROX], azithromycin [AZM], and telithromycin [TEL]) was examined in J774 murine macrophages. The net influx rates of AZM and TEL were increased from 2-to 3.5-fold in the presence of these inhibitors, but their efflux was slowed only marginally. At 3 h, the inhibitors increased the levels of AZM, ERY, and TEL accumulation approximately three-to fourfold (the effect of VE, however, was lower) but did not influence CLR accumulation ( 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 J77...