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Subcellular distribution of azithromycin and clarithromycin in rat alveolar macrophages (NR8383) in vitro
Kohei Togami1, Sumio Chono, Kazuhiro Morimoto
1Division of Pharmaceutics, Hokkaido Pharmaceutical University School of Pharmacy.
Abstract:
Azithromycin (AZM), a 15-membered ring macrolide antimicrobial agent, has an antibacterial spectrum that includes intracellular parasitic pathogens that survive or intracellularly multiply in alveolar macrophages (AMs). The subcellular distribution of AZM in AMs was evaluated in vitro in comparison with clarithromycin (CAM). AZM and CAM (50 µM) were applied to the NR8383 cells, used as an in vitro model of AMs, followed by incubation at 37°C or 4°C. The total amount of AZM in cells and subcellular distribution (cell fractionation) was determined after incubation. High level of AZM accumulation was observed in the NR8383 cells at 37°C, and the equilibrium intracellular to extracellular concentration ratio (I/E ratio) was approximately 680, which was remarkably higher than that of CAM (equilibrium I/E ratio=28). The intracellular accumulation of AZM and CAM was temperature dependent. In addition, AZM distributed to the granules fraction including organelles and soluble fraction including cytosol in the NR8383 cells, whereas CAM mainly distributed in soluble fraction. The amount of AZM in the granules fraction was markedly reduced in the presence of ammonium chloride for increase in intracellular pH. These results indicate that AZM is distributed in acidic compartment in AMs. This study suggests that high AZM accumulation in the NR8383 cells is due to the trapping and/or binding in acidic organelles, such as lysosomes.
Insights
Azithromycin (AZM) shows high accumulation in alveolar macrophages (AMs) compared to clarithromycin (CAM), concentrating within acidic cellular compartments like lysosomes. This explains AZM's effectiveness against intracellular pathogens.
Area of Science:
- Pharmacology
- Cell Biology
- Microbiology
Background:
- Azithromycin (AZM) is a macrolide antibiotic effective against intracellular pathogens residing in alveolar macrophages (AMs).
- Understanding the subcellular distribution of AZM in AMs is crucial for explaining its efficacy.
Purpose of the Study:
- To compare the in vitro subcellular distribution of Azithromycin (AZM) and clarithromycin (CAM) in alveolar macrophages (AMs).
- To elucidate the mechanisms behind AZM's high intracellular accumulation.
Main Methods:
- NR8383 cells (an in vitro model of AMs) were incubated with AZM and CAM (50 µM) at 37°C or 4°C.
- Cellular uptake and subcellular fractionation were analyzed.
- The effect of ammonium chloride on AZM distribution was assessed.
Main Results:
- AZM exhibited significantly higher intracellular accumulation in AMs than CAM (I/E ratio ~680 vs. 28).
- AZM accumulated in both granular (organelles) and soluble fractions, while CAM primarily localized to the soluble fraction.
- AZM's presence in the granular fraction decreased with increased intracellular pH, suggesting localization in acidic compartments.
Conclusions:
- AZM demonstrates remarkable concentration within alveolar macrophages, significantly exceeding that of clarithromycin.
- The high accumulation of AZM is attributed to trapping and binding within acidic intracellular organelles, such as lysosomes.
- This characteristic enhances AZM's potential for treating infections caused by intracellular pathogens within macrophages.
