engleski

Interaction of macrolide antibiotics with P-glycoprotein

Macrolides are known to accumulate in cells but the exact mechanism is not fully understood. Intensity of accumulation as well as affinity for different cell types varies among macrolide compounds (Bosnar M. et al. 2005). Clarithromycin, erythromycin, azithromycin and josamycin have been reported to inhibit the function of P-glycoprotein (P-gp) (Wakasugi H. et al 1998 ; Wang L. et al. 2000 ; Asakura E. et al. 2004). Erythromycin and azithromycin have also been shown to be substrates for P-gp (Schuetz E.G. et al. 1998 ; Sugie M. et al. 2004). The aim of our study was to compare five commonly used macrolide antibiotics (azithromycin, erythromycin, clarithromycin, roxithromycin and telithromycin) with respect to their ability to inhibit P-gp function, as well as to determine how P-gp affects intracellular accumulation of these macrolides. Studies were performed on MES-SA/Dx5 cells which overexpress P-gp and the parent cell line MES-SA without this transporter expressed. Expression of P-gp was confirmed by quantitative RT-PCR and by flow cytometry using a specific antibody for P-gp. P-gp function was assessed by flow cytometric assay of rhodamine-123 efflux from MES-SA/Dx5 cells. Verapamil was used as standard P-gp inhibitor (IC50= 9.1 uM). Accumulation of macrolides was studied in MES-SA/Dx5 cells with or without 50uM verapamil as well as in the MES-SA parent cell line. Intracellular concentrations were determined by the microbiological agar diffusion method. IC50 values obtained revealed that tested macrolides can be clustered into two groups with respect to P-gp inhibition: potent inhibitors - clarithromycin (83.4± ; 5.8uM), roxithromycin (204.1± ; 11.0uM), telithromycin (210.2± ; 14.1uM) ; and less potent inhibitors - erythromycin (1920.3± ; 295.4 uM), azithromycin (>1500 uM). All tested macrolides accumulate to a greater extent in MES-SA than in MES-SA/Dx5 cells and more in MES-SA/Dx5 cells treated with verapamil than in vehicle treated cells. These results indicate that all five tested macrolides are clearly substrates for P-gp. Inhibition of P-gp by verapamil had the most pronounced effect on accumulation of azithromycin and erythromycin which displayed more than 16-fold higher intracellular concentrations when added together with the P-gp inhibitor. Increase in cellular accumulation of other tested macrolides (clarithromycin, telithromycin and roxithromycin) was 2-10 fold. Furthermore, expression of P-gp affects the relative affinity of cells for different macrolides. MES-SA/Dx5 cells which overexpress P-gp accumulated azithromycin, clarithromycin, telithromycin and roxithromycin to about the same intensity (accumulation of erythromycin was much lower). On the other hand, cells without P-gp expressed (MES-SA), as well as cells with inhibited P-gp (MES-SA/Dx5 with verapamil), accumulated azithromycin 4-7 fold better than all other tested macrolides (although more than 20-fold higher than baseline in the presence of verapamil, uptake of erythromycin was still well below that of roxithromycin, telithromycin and clarithromycin). Our results reveal differences between five macrolide antibiotics in their interaction with P-gp, showing that clarithromycin, roxithromycin and telithromycin display a 10-20-fold higher potential to inhibit P-gp function than azithromycin or erythromycin. However, P-gp most strikingly influences accumulation of azithromycin and erythromycin in cells, whereas the uptake of the other three compounds is affected by this transporter to a lesser extent. Finally, our results demonstrate how the expression of a single transporter can dramatically change the relative affinity of the cell for structurally very similar compounds, which may at least in part explain differences in their pharmacokinetic behaviour as well as in the extent of their pharmacodynamic effects. References: Asakura E. et al. Eur.J. Pharmacol. 26 (2004), 484(2-3) ; 333-339. Bosnar M. et el. Antimicrob. Agents Chemother. 49 (2005) ; 1-6. Schuetz E.G. et al. Arch.Biochem. Biophys. 350(1998), 2 ; 340-347. Sugie M. et al. Antimicrob. Agents Chemother. 48(2004), 3 ; 809-814. Wakasugi H. et al. Clin. Pharm. Ther. 64.(1998) ; 123-128. Wang L. et al. Clinical and Exp. Pharm. Physiol. 27(2000) ; 587-593.

macrolide; P-glycoprotein

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