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Antimicrobial Agents and Chemotherapy, August 2007, p. 2748-2757, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00090-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Modulation of the Cellular Accumulation and Intracellular Activity of Daptomycin towards Phagocytized Staphylococcus aureus by the P-Glycoprotein (MDR1) Efflux Transporter in Human THP-1 Macrophages and Madin-Darby Canine Kidney Cells

Unité de Pharmacologie Cellulaire et Moléculaire, Université Catholique de Louvain, B-1200 Brussels, Belgium

Received 20 January 2007/ Returned for modification 18 March 2007/ Accepted 27 May 2007

P-glycoprotein (P-gp; MDR1), a major efflux transporter, recognizes various antibiotics and is present in macrophages. We have examined its effect on the modulation of the intracellular accumulation and activity of daptomycin towards phagocytized Staphylococcus aureus (ATCC 25923) in human THP-1 macrophages, in comparison with MDCK epithelial cells (wild type and MDCK-MDR1 overexpressing P-gp; the bulk of the protein was immunodetected at the surface of all three cell types). Daptomycin displayed concentration-dependent intracellular activity (Hill equation pattern) in THP-1 and MDCK cells with (i) 50% effective drug extracellular concentration (EC₅₀; relative potency) and static concentrations at 9 to 10 times the MIC and (ii) maximal efficacy (E_max; CFU decrease at infinite extracellular drug concentration) at 1.6 to 2 log compared to that of the postphagocytosis inoculum. Verapamil (100 µM) and elacridar (GF 120918; 0.5 µM), two known inhibitors of P-gp, decreased daptomycin EC₅₀ (about threefold) in THP-1 and MDCK cells without affecting E_max. Daptomycin EC₅₀ was about three- to fourfold higher and accumulation in MDCK-MDR1 commensurately lower than in wild-type cells. In THP-1 macrophages, (i) verapamil and ATP depletion increased, and ouabain (an inducer of mdr1 [the gene encoding P-gp] expression) decreased the accumulation of daptomycin in parallel with that of DiOC₂ (a known substrate of P-gp); (ii) silencing mdr1 with duplex human mdr1 siRNAs reduced the cell content in immunoreactive P-gp to 15 to 30% of controls and caused an eight- to 13-fold increase in daptomycin accumulation. We conclude that daptomycin is subject to efflux from THP-1 macrophages and MDCK cells by P-gp, which reduces its intracellular activity against phagocytized S. aureus.

Published ahead of print on 4 June 2007.

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