The cellular uptake and metabolism of clodronate in RAW 264 macrophages

H Monkkonen, M J Rogers, M Makkonen, S Niva, S Auriola, J Monkkonen

Research output: Contribution to journalArticle

35 Citations (Scopus)

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 (AppCCl(2)p) 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 C-14-labeled clodronate. AppCCl(2)p 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 AppCCl(2)p, 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 AppCCl(2)p metabolite.

Original languageEnglish
Pages (from-to)1550-1555
Number of pages6
JournalPharmaceutical Research
Volume18
Publication statusPublished - 2001

Keywords

  • clodronate
  • metabolism
  • cellular uptake
  • macrophage
  • CELLS IN-VITRO
  • MOLD DICTYOSTELIUM-DISCOIDEUM
  • CONTAINING LIPOSOMES
  • FREE BISPHOSPHONATES
  • ADENINE-NUCLEOTIDES
  • ARTHRITIS
  • GROWTH
  • AMEBAS
  • OSTEOCLASTS
  • ALENDRONATE

Cite this

Monkkonen, H., Rogers, M. J., Makkonen, M., Niva, S., Auriola, S., & Monkkonen, J. (2001). The cellular uptake and metabolism of clodronate in RAW 264 macrophages. Pharmaceutical Research, 18, 1550-1555.

The cellular uptake and metabolism of clodronate in RAW 264 macrophages. / Monkkonen, H ; Rogers, M J ; Makkonen, M ; Niva, S ; Auriola, S ; Monkkonen, J .

In: Pharmaceutical Research, Vol. 18, 2001, p. 1550-1555.

Research output: Contribution to journalArticle

Monkkonen, H, Rogers, MJ, Makkonen, M, Niva, S, Auriola, S & Monkkonen, J 2001, 'The cellular uptake and metabolism of clodronate in RAW 264 macrophages', Pharmaceutical Research, vol. 18, pp. 1550-1555.
Monkkonen H, Rogers MJ, Makkonen M, Niva S, Auriola S, Monkkonen J. The cellular uptake and metabolism of clodronate in RAW 264 macrophages. Pharmaceutical Research. 2001;18:1550-1555.
Monkkonen, H ; Rogers, M J ; Makkonen, M ; Niva, S ; Auriola, S ; Monkkonen, J . / The cellular uptake and metabolism of clodronate in RAW 264 macrophages. In: Pharmaceutical Research. 2001 ; Vol. 18. pp. 1550-1555.
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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 (AppCCl(2)p) 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 C-14-labeled clodronate. AppCCl(2)p 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 AppCCl(2)p, 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 AppCCl(2)p metabolite.",
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TY - JOUR

T1 - The cellular uptake and metabolism of clodronate in RAW 264 macrophages

AU - Monkkonen, H

AU - Rogers, M J

AU - Makkonen, M

AU - Niva, S

AU - Auriola, S

AU - Monkkonen, J

PY - 2001

Y1 - 2001

N2 - 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 (AppCCl(2)p) 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 C-14-labeled clodronate. AppCCl(2)p 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 AppCCl(2)p, 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 AppCCl(2)p metabolite.

AB - 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 (AppCCl(2)p) 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 C-14-labeled clodronate. AppCCl(2)p 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 AppCCl(2)p, 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 AppCCl(2)p metabolite.

KW - clodronate

KW - metabolism

KW - cellular uptake

KW - macrophage

KW - CELLS IN-VITRO

KW - MOLD DICTYOSTELIUM-DISCOIDEUM

KW - CONTAINING LIPOSOMES

KW - FREE BISPHOSPHONATES

KW - ADENINE-NUCLEOTIDES

KW - ARTHRITIS

KW - GROWTH

KW - AMEBAS

KW - OSTEOCLASTS

KW - ALENDRONATE

M3 - Article

VL - 18

SP - 1550

EP - 1555

JO - Pharmaceutical Research

JF - Pharmaceutical Research

SN - 0724-8741

ER -