In Vitro Uptake of SCH 27899 (Evernimicin) by Rat Alveolar Macrophages

Abstract

The in vitro uptake of [¹⁴C]evernimicin ([¹⁴C]SCH 27899) by primary cultures of rat alveolar macrophages and hepatocytes was determined. Both cell populations exhibited linear rates of uptake. However, the initial rate of drug uptake by alveolar macrophages was about threefold higher than that by hepatocytes. These findings demonstrate that [¹⁴C]evernimicin is taken up by rat alveolar macrophages, supporting the likelihood that the drug is able to reach sites of infection.

Alveolar phagocytic cells, through uptake and release of antibiotics, play a key role in delivery of drugs to the lung during pneumonia. Previous in vitro studies have demonstrated that the oligosaccharide antibiotic evernimicin (SCH 27899) (Fig. 1) is active against gram-positive pathogens, including Streptococcus pneumoniae (R. S. Hare, F. J. Sabatelli, and the Ziracin Susceptibility Testing Group, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-119, 1998), which causes pneumonia. Phagocytic cells are involved in delivering compounds to the site of infection (3). However, uptake of evernimicin by phagocytic cells has not been thoroughly assessed. The purpose of this study was to evaluate uptake of evernimicin by rat pulmonary alveolar macrophages. Drug uptake by rat hepatocytes was also assessed for comparison purposes.

FIG. 1.

SCH 27899 (Ziracin) structure with carbon numbering system.

Rat alveolar macrophages were obtained from bronchoalveolar lavage fluid from normal rats by using previously described methods (1). Recovered alveolar cells were resuspended in RPMI medium (3 × 10⁶ cells/ml). Hepatocytes were prepared using buffers (aerated with 95% O₂–5% CO₂) purchased from Gibco BRL (Rockville, Md.) in accordance with the company's recommended procedures, with minor modifications. The viability of alveolar macrophage and hepatocyte preparations was assessed by trypan blue exclusion and determined to be 85 to 100% and 80 to 95%, respectively. Red blood cell (RBC) contamination of alveolar macrophage preparations was found to be 5 × 10⁴ cells/ml. Consequently, RBCs were similarly purified from peripheral blood and resuspended in RPMI medium at a concentration of 5 × 10⁴ cells/ml. All studies were carried out in accordance with the National Institutes of Health Guide to the Care and Use of Laboratory Animals and the Animal Welfare Act in an AAALAC-accredited program.

To determine uptake of evernimicin by rat alveolar macrophages and hepatocytes, [¹⁴C]evernimicin was supplied by the Department of Radiochemistry at Schering-Plough Research Institute (specific activity of 28.9 μCi/mg [578 μCi/ml]). The compound, which is minimally soluble in water, was formulated as an aqueous solution with [¹⁴C]evernimicin, meglumine, hydroxypropyl-β-cyclodextrin (molar ratio, 1:3:5), Tween, and mannitol (4). The excipients provided a solution of evernimicin suitable for intravenous administration used in the clinic. Hepatocytes and alveolar macrophages (final concentration, 3 × 10⁶/ml) and RBCs (final concentration, 3 × 10⁴/ml) were added to flasks containing a solution of [¹⁴C]evernimicin (2.5 and/or 25 μg/ml) in Waymouth buffer (liver cells) or RPMI medium (alveolar macrophages and RBCs) at 37°C in a shaker bath. The higher concentration was extrapolated from the in vivo dose (15 mg/kg of body weight) for rat, monkey, and human (P. A. Krieter, M. Thonoor, M. Wirth, S. Gupta, J. Patrick, and M. Cayen, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-112, 1997; P. A. Krieter, C. Banfield, M. Thonoor, N. Blumenkrantz, S. Chowdhury, T. Musick, S. Pai, V. Batra, M. Cayen, and J. Patrick, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-1196, 1999), and the lower was based on the concentration in plasma and tissue distribution levels (Krieter et al., 37th ICAAC; Krieter et al., 39th ICAAC). Addition of the cells to the uncapped flasks containing ¹⁴C-drug initiated drug uptake (i.e., T = 0). At specific time points, the cells (200-μl aliquots in duplicate or triplicate for each time point) were removed and placed in 400-μl polyethylene tubes previously layered with 50 μl of 3 M potassium hydroxide and 100 μl of silicone oil (density, 1.05 g/ml for hepatocytes; 1.02 g/ml for alveolar macrophages). The samples were centrifuged (12,000 × g, 1 min) in a tabletop microcentrifuge. The polyethylene tubes were then cut at the oil-aqueous alkaline interface. An aliquot (30 μl) of the aqueous bottom portion (containing alkaline cell lysate) was placed in 10 ml of scintillation cocktail and was analyzed by liquid scintillation spectroscopy for 5 min. Uptake of [¹⁴C]evernimicin by RBCs was subtracted from drug uptake by alveolar macrophage preparations to quantify the alveolar macrophage specific uptake of evernimicin. The average values at zero time were also subtracted to account for nonspecific binding. Calculations were performed using Microsoft Excel 97.

Initial uptake of [¹⁴C]evernimicin was linear at both 2.5 μg of drug/ml (y = 132.28x − 167.51; r² = 0.85) (Fig. 2A) and 25 μg/ml (y = 432.46x + 724.31; r² = 0.58; Fig. 2B). The rate of drug uptake was 0.65 ng of equivalent/10⁶ cells/min at 2.5 μg of drug/ml and 2.5 ng of equivalent/10⁶ cells/min at 25 μg/ml.

FIG. 2.

(A) In vitro uptake of drug-derived radioactivity into freshly isolated rat hepatocytes during incubation with 2.5 μg of [¹⁴C]SCH 27899/ml. dpm, disintegrations per minute. Diamonds represent dpm obtained at specific time points for each of the duplicate samples. (B) In vitro uptake of drug-derived radioactivity into freshly isolated rat hepatocytes during incubation with 25 μg of [¹⁴C]SCH 27899/ml. dpm, disintegrations per minute. Triangles represent dpm obtained at specific time points for each of the duplicate samples. (C) In vitro uptake of drug-derived radioactivity into rat alveolar macrophages during incubation with 25 μg of [¹⁴C]SCH 27899/ml. dpm, disintegrations per minute. Circles represent dpm obtained at specific time points for each of the triplicate samples.

Similarly, [¹⁴C]evernimicin was also taken up by rat alveolar macrophages at a concentration of 25 μg/ml (y = 1,097.4x + 3,032.5; r² = 0.61) (Fig. 2C). The rate of uptake was 7.3 ng of equivalent/10⁶ cells/min.

Results of this study demonstrate that evernimicin is taken up by rat alveolar macrophages. Furthermore, the initial rate of drug uptake by alveolar macrophages was about threefold higher than that observed for hepatocytes when the cells were incubated at an equivalent concentration of radioactive drug (i.e., 25 μg of [¹⁴C]evernimicin/ml).

Results of our studies are in agreement with those of Takemura et al. (H. Takemura, H. Ikejima, H. Kunishima, S. Terakubo, K. Kanemitsu, H. Yamamoto, M. Kaku, and J. Shimada, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1927, 1999), who demonstrated that evernimicin is rapidly taken up by the human monocytic THP-1 cell line. Furthermore, they showed that evernimicin reaches sufficient concentrations in the cell to effectively suppress replication of intracellular pathogens (i.e., Legionella pneumophila), thereby demonstrating that intracellular evernimicin exhibits good antimicrobial activity.

Evernimicin has shown activity against gram-positive pathogens, including Streptococcus pneumoniae (Hare et al., 38th ICAAC). While the uptake of other drugs, including grepafloxacin (5), benzylpenicillin (6), cefpiramide (6), cefazolin (6), and roxithromycin (2, 7), by cells has been well documented, uptake of evernimicin by phagocytic cells, including alveolar macrophages, has not been previously assessed. Entry and accumulation of antimicrobial agents into phagocytic cells are important factors for clinical efficacy, as well as trafficking of antimicrobials to sites of infection. The results of our studies demonstrate that evernimicin accumulates in phagocytic cells, supporting the likelihood that evernimicin is able to reach sites of infection (3).