Fluorescently labeled macrolides as a tool for monitoring cellular and tissue distribution of azithromycin.

Exceptional therapeutic effects of macrolides in treating various infections and inflammatory conditions can be significantly contributed to their unique pharmacokinetic properties. Macrolides accumulate in cells and tissues, with concentrations usually 10 to more than 100 times higher of those measured in plasma. Intracellular distribution of macrolides has so far been examined using extensive subcellular fractionation techniques, radiolabeled compounds and conventional pharmacokinetic methods. In this study we evaluated four fluorescently labeled macrolides on their applicability to monitor azithromycin distribution in vitro and in vivo. 9-Deoxo-9a-{3-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]propyl}-9a-aza-9a-homoerythromycin A (9a-NBD-azithromycin) was selected as a compound with most similar cellular pharmacokinetics to azithromycin. 9a-NBD-azithromycin demonstrated antimicrobial properties comparable to azithromycin, displayed the same biological activity profile in LPS-stimulated J774A.1 murine macrophage cells and, even though it accumulated in cells almost 50% more than azithromycin, it showed same rate of retention. Identical to azithromycin, 9a-NBD-azithromycin was localized in lysosomes of J774A.1 cells. Two hours after 9a-NBD-azithromycin was administered intraperitonally to mice, a strong fluorescent signal was located in kidneys and liver and slightly weaker in the spleen. In kidneys, the signal was concentrated in tubuli, and glomeruli were negative. Patchy florescence in hepatocytes supports lysosomal cellular localization. Weaker staining of white pulp compared to red pulp of spleen is in agreement with lower accumulation of azithromycin in lymphocytes compared to other cell types present. We conclude that 9a-NBD-azithromycin can be used as a fluorescent analog of azithromycin to visualize its distribution in in vitro systems, and is also suitable for in vivo studies.