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The Cellular Uptake and Metabolism of Clodronate in RAW 264 Macrophages

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Abstract

Purpose. Non-nitrogen-containing bisphosphonates, such as clodronate (dichloromethylene bisphosphonate), appear to act as prodrugs, their active form being the AppCp-type analogues of ATP. To further elucidate this, we examined the cellular uptake of clodronate and intracellular accumulation of the metabolite of clodronate (AppCCl2p) in RAW 264 macrophages, the influence of clodronate metabolism on the intracellular ATP concentration, and the time course of clodronate metabolism and the effects of clodronate on cytokine secretion from macrophages.

Methods. The cellular uptake of clodronate was measured using 14C-labeled clodronate. AppCCl2p was determined in cell extracts by using an ion-pairing HPLC-ESI-MS. The cytokine concentrations in the culture supernatants were measured with time-resolved fluoroimmunoassay. Intracellular ATP concentration was measured with a luminometer using a luciferin-luciferase assay.

Results. Of the clodronate internalized by macrophages in vitro, 30-55% is metabolized to AppCCl2p, which accumulates to high intracellular concentrations during the first 12 h of exposure. This accumulation does not affect the ATP levels in the cells. The time course of metabolite appearance in the cells and the inhibition of cytokine secretion were very similar.

Conclusions. These results strongly support the idea that clodronate acts as a prodrug, the active form being its intracellular AppCCl2p metabolite.

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Authors and Affiliations

  1. Department of Pharmaceutics, University of Kuopio, Kuopio, Finland

    Hannu Mönkkönen, Niina Makkonen, Sanna Niva & Jukka Mönkkönen

  2. Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK

    Michael J. Rogers

  3. Department of Pharmaceutical Chemistry, University of Kuopio, Kuopio, Finland

    Seppo Auriola

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  1. Hannu Mönkkönen
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Mönkkönen, H., Rogers, M.J., Makkonen, N. et al. The Cellular Uptake and Metabolism of Clodronate in RAW 264 Macrophages. Pharm Res 18, 1550–1555 (2001). https://doi.org/10.1023/A:1013026313647

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