| Sign In to gain access to subscriptions and/or personal tools. |
Clarithromycin Transport by Gingival Fibroblasts and Epithelial CellsSection of Periodontology, College of Dentistry, The Ohio State University Health Sciences Center, 305 West 12th Avenue, PO Box 182357, Columbus, OH 43218-2357, USA Correspondence: * corresponding author, walters.2{at}osu.edu
Macrolide antibiotics penetrate cells, but the mechanism by which this occurs is unclear. The objective of this study was to characterize the mechanisms of clarithromycin uptake by gingival fibroblasts and oral epithelium. Cultured human gingival fibroblasts and SCC-25 cells were incubated with [3H]-clarithromycin. We assayed clarithromycin transport by measuring cell-associated radioactivity over time. Fibroblasts and epithelial cells rapidly accumulated clarithromycin, attaining steady-state intracellular concentrations within 15 minutes. Incubation in medium containing 2 µg/mL clarithromycin yielded steady-state intracellular concentrations of 75.8 µg/mL in fibroblasts and 6.6 µg/mL in SCC-25 cells. Clarithromycin transport exhibited Michaelis-Menten kinetics and was inhibited below 37°C. The Michaelis constants for fibro-blasts and SCC-25 cells were 78.4 and 227 µg/mL, respectively, while the maximum transport velocities were 264 and 381 ng/min/106 cells, respectively. Thus, both types of cells take up clarithromycin via a concentrative active transport system. By increasing intracellular clarithromycin levels, this system may enhance the effectiveness of clarithromycin against invasive periodontal pathogens.
Key Words: macrolides • antimicrobial chemotherapy • aggressive periodontitis
The goals of non-surgical periodontal therapy (scaling and root planing) are debridement of bacterial plaque and removal of bacterial products from root surfaces. While this approach is usually successful in arresting periodontal attachment loss, periodontal breakdown may continue to progress in some individuals. This unsatisfactory outcome is often related to persistent infection by invasive subgingival bacteria (Bragd et al., 1987). The periodontal pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis are exceptionally difficult to eliminate by debridement alone (Chen and Slots, 1993). A. actinomycetemcomitans is strongly associated with aggressive periodontitis and recurrent adult periodontitis, while P. gingivalis is associated with severe chronic periodontitis, failing guided tissue regeneration, and acute periodontal abscesses. Both pathogens are capable of evading the body’s immune/defense system by invading epithelial cells lining periodontal pockets (Saglie et al., 1986). A. actinomycetemcomitans can invade epithelial cells and pass into the underlying connective tissue (Christersson et al., 1987; Fives-Taylor et al., 1995), while P. gingivalis can invade epithelial cells and linger inside (Lamont et al., 1995). Antimicrobial chemotherapy is frequently used as an adjunct to enhance the elimination of pathogens from persons who undergo progressive periodontal attachment loss after scaling and root planing (Chadwick and Mellersh, 1987; van Winkelhoff et al., 1996). The use of systemic antibiotics in conjunction with scaling and root planing significantly enhances gains in clinical attachment level, in comparison with treatment with scaling and root planing alone (Haffajee et al., 2003). Penicillins do not readily cross the plasma membrane, which limits their effectiveness in the treatment of intracellular infections (Schentag et al., 1985). In contrast, macrolides penetrate cells to gain access to intracellular pathogens (Bosnar et al., 2005). The newer macrolide agents azithromycin and clarithromycin are highly effective against A. actinomycetemcomitans and P. gingivalis (Pajukanta et al., 1992; Pajukanta, 1993; Piccolomini et al., 1998; Goldstein et al., 1999). Both agents exhibit good activity against Eikenella corrodens, Prevotella species, fusobacteria, and other anaerobic and facultative oral pathogens ( Sefton et al., 1996; Goldstein et al., 1999; Merriam et al., 2006). In addition to their favorable antimicrobial spectrum, both agents have a low incidence of gastrointestinal toxicity and are administered with a once- or twice-daily dosing interval that promotes patient compliance (Moore, 1999). There have been no previous studies to characterize mechanisms of clarithromycin uptake by human gingival fibroblasts and oral epithelial cells. However, previous studies have shown that azithromycin and clarithromycin attain significantly higher concentrations in gingiva than in serum (Malizia et al., 1997; Blandizzi et al., 1999; Burrell and Walters, 2008). Moreover, clarithromycin reportedly reaches significantly higher steady-state concentrations at human gingivitis sites than at healthy sites (Burrell and Walters, 2008). These reports suggest that active transporters could mediate the cellular uptake of clarithromycin. This issue is clinically relevant, because clarithromycin transport into pocket epithelial cells could facilitate the elimination of invasive periodontal pathogens. Clarithromycin transport and accumulation by fibroblasts, which are abundant in gingival connective tissue, could potentially help sustain clarithromycin levels in the gingiva. In this report, cultured gingival fibroblasts and an oral epithelial cell line were studied to test the hypothesis that these cells possess an active transport system for accumulating clarithromycin.
Cell Culture SCC-25 epithelial cells (CRL-1628, obtained from ATCC, Manassas, VA, USA), originally derived from oral mucosa (Rheinwald and Beckett, 1981), were seeded into 75 cm2 tissue culture flasks (Corning International, Corning, NY, USA) and cultured in a 37°C incubator containing 5% CO2. Cells were grown in 50% Dulbecco’s modified Eagle’s medium/50% Ham’s F12 medium (Invitrogen Corp., Grand Island, NY, USA) containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA), and 0.4 µg/mL hydrocortisone (Sigma Chemical Co., St. Louis, MO, USA). They were fed every 3 days until they formed a confluent monolayer. A fibroblast strain previously isolated from healthy (non-edematous, non-bleeding, pink) interproximal papillae under an IRB-approved protocol (Mariotti and Cochran, 1990) was seeded into 75-cm2 tissue culture flasks and cultured in a 37°C incubator containing 5% CO2. Cells were grown in minimal essential medium (Invitrogen Corp.) containing 2 mM L-glutamine and 10% heat-inactivated fetal bovine serum (Atlanta Biologicals). They were fed every 3 days until the cells formed a confluent monolayer.
Assay of Clarithromycin Transport
Assay of Clarithromycin Efflux
Determination of Intracellular Clarithromycin Concentration
SCC-25 cells and gingival fibroblasts rapidly accumulated clarithromycin, but exhibited signs of saturation after 10 min (Fig. 1  ). In comparison with fibroblasts, SCC-25 cells appeared to saturate earlier and at a lower level of clarithromycin content per cell. Clarithromycin transport activity by SCC-25 cells and fibroblasts was directly correlated with temperature between 4° and 37°C (Fig. 2, upper panel). The slope of the regression line was steeper for fibroblasts, suggesting that transport by these cells is more sensitive to temperature.
The kinetics of clarithromycin transport yielded a linear Lineweaver-Burk plot in SCC-25 cells as well as fibroblasts (Fig. 2  , lower panel). The estimated Km values for transport by SCC-25 cells and gingival fibroblasts were 227 µg/mL and 78.4 µg/mL, respectively, and the Vmax values were 381 ng/min/106 cells and 264 ng/min/106 cells, respectively (Table). Differences between the two types of cells were statistically significant (P < 0.05, t test). At steady state, after 20 min of cell incubation in medium containing 2 µg/mL clarithromycin, the ratio of intracellular clarithromycin concentration to extracellular concentration was approximately 3.3 in SCC-25 cells and 38 in gingival fibroblasts (Table).
When SCC-25 cells and fibroblasts were loaded to steady state with 10 µg/mL clarithromycin, and clarithromycin in the extracellular medium was then diluted to 0.9 µg/mL, clarithromycin efflux from the cells was observed (Fig. 3 ). Loaded SCC-25 cells lost approximately half of their clarithromycin content within 5 min. Thereafter, the rate of efflux decreased significantly. Under similar conditions, fibroblasts lost less than half of their clarithromycin content over a 20-minute period. Clarithromycin efflux from fibroblasts was consistently slower than efflux from SCC-25 cells.
The results demonstrate that gingival fibroblasts and SCC-25 cells actively accumulate clarithromycin. SCC-25 cells are a malignant variant of oral squamous epithelial cells, but share many of their functions and features. These cells can be grown in culture without an underlying fibroblast feeder layer, which greatly simplifies their use in transport assays. In fibroblasts as well as SCC-25 cells, clarithromycin accumulation exhibited saturable kinetics and was concentrative and temperature-dependent. While the values for the Km of clarithromycin transport suggest that it is taken up with low affinity, there was evidence of substantial concentration of this agent inside SCC-25 cells as well as fibroblasts. When incubated with clarithromycin at a concentration similar to that attained in human gingiva during antimicrobial chemotherapy (2–3 µg/g, Burrell and Walters, 2008), SCC-25 cells achieved steady-state intracellular clarithromycin levels that were approximately 3.3-fold higher than extracellular levels. A similar degree of intracellular concentration has been observed in kidney epithelial cells (Bosnar et al., 2005). Under similar conditions, gingival fibroblasts attained intracellular concentrations that were almost 40-fold higher than extracellular levels. The difference in intracellular concentration between fibroblasts and SCC-25 cells was greater than expected based on the observed kinetic constants for transport. Although fibroblasts transport clarithromycin with significantly higher affinity than SCC-25 cells, their maximum transport velocity is lower. It is possible that some of the differences in uptake kinetics could be related to differences in the transformation states of fibroblasts and SCC-25 cells. The efficiency of clarithromycin transport by fibroblasts, as estimated by the ratio Vmax/Km, is only about twice that of SCC-25 cells. Previous reports suggested that exposure to clarithromycin concentrations of greater than 7.5 µg/mL is cytocidal to human gingival epithelial cells (Inoue et al., 2004), but does not disrupt vital activities in human periodontal ligament fibroblasts (Maizumi et al., 2002). It is possible that the cytocidal effects of clarithromycin could inhibit transport in epithelial cells, thereby limiting maximum steady-state intracellular drug levels. Our findings suggest that clarithromycin accumulation by cells is mediated by an active transport system. As indicated by the linear Lineweaver-Burk plots, the kinetics of clarithromycin transport is saturable and obeys the Michaelis-Menten equation. Clarithromycin is a weak base, so it could potentially be taken up by transporters that accept organic cation substrates. The two major transport systems for organic cations are the organic cation transporter (OCT) family and the organic anion transporting polypeptide (OATP) family. Consistent with a possible role for OATP, a recent study demonstrated that clarithromycin inhibits the uptake of certain drugs that are known substrates for transport by OATP family members OATP1B1 and OATP1B3 (Seithel et al., 2007). While clarithromycin has not been evaluated in clinical trials as an adjunct to periodontal therapy, it is more effective in vitro against P. gingivalis and Prevotella intermedia than is azithromycin (Goldstein et al., 1999), and inhibits A. actinomycetemcomitans with similar potency (Pajukanta et al., 1992; Piccolomini et al., 1998). Several studies have examined the effectiveness of azithromycin in periodontal applications. A placebo-controlled randomized clinical trial has shown that azithromycin enhances the reduction of pocket depth and bleeding on probing by scaling and root planing in adults with severe recurrent periodontitis (Smith et al., 2002). A second randomized trial demonstrated that use of azithromycin in combination with scaling and root planing enhances pocket reduction and attachment gain in smokers with moderate to advanced attachment loss (Mascarenhas et al., 2005). Azithromycin is a recommended therapy for acute periodontal abscesses with systemic manifestations in individuals with an allergy to beta-lactam drugs (Pallasch, 1996). Transporters are capable of moving their substrates in the forward or reverse direction to maintain equilibrium between intracellular and extracellular concentrations. Since gingival fibroblasts comprise a relatively large volume of the gingival connective tissue (Schroeder and Listgarten, 1997), their ability to take up, accumulate, and release clarithromycin could allow them to function as reservoirs for this agent in the gingiva. Consistent with this role, clarithromycin efflux from clarithromycin-loaded fibroblasts and SCC-25 cells was observed when antibiotic levels were experimentally decreased in the extracellular medium. Forward transport into cells presumably occurs in vivo when clarithromycin levels in tissue are increasing. As antibiotic levels in the blood and interstitial fluid decrease from their peak values, the direction of transport may reverse in a manner that maintains relatively high antimicrobial levels in gingival interstitial fluid and in gingival crevicular fluid. It is unclear how long fibroblasts could potentially sustain clarithromycin levels in the gingiva. However, a recent study demonstrated that the concentration of clarithromycin in gingival connective tissue can be severalfold higher than the level in serum approximately 4 hrs after serum levels start to decrease from their peak concentration (Burrell and Walters, 2008). Doxycycline, which is also accumulated by gingival fibroblasts, yields therapeutic concentrations that are more sustained and less variable in gingival crevicular fluid than in blood serum (Lavda et al., 2004). In epithelial cells, forward transport of clarithromycin greatly enhances the intracellular concentration of this antibiotic. In summary, transport by gingival fibroblasts and pocket epithelium could potentially enhance the effectiveness of clarithromycin in periodontal therapy by sustaining their therapeutic levels in gingival connective tissue and inside epithelial cells that have been invaded by A. actinomycetemcomitans or P. gingivalis.
This investigation was supported by USPHS research grants R01 DE012601 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. Received for publication December 7, 2007. Revision received April 16, 2008. Accepted for publication April 18, 2008.
Journal of Dental Research, Vol. 87, No. 8,
777-781 (2008)
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
