The molecular mechanism of action of the antiresorptive and antiinflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis

Julie Clare Crockett, J. Monkkonen, S. Auriola, H. Monkkonen, Michael John Rogers

Research output: Contribution to journalArticle

201 Citations (Scopus)

Abstract

Objective. The primary aims of this study were to determine whether clodronate and liposome-encapsulated clodronate are metabolized to adenosine 5'-(beta,gamma -dichloromethylene) triphosphate (AppCCl(2)p) by osteoclasts and macrophages in vivo, and to determine whether intracellular accumulation of this metabolite accounts for the antiresorptive and antimacrophage effects of clodronate. To compare the mechanism of action of clodronate and alendronate, effects on protein prenylation in osteoclasts and macrophages in vivo were also assessed.

Methods. High-performance liquid chromatography-mass spectrometry was used to determine whether rabbit osteoclasts (purified ex vivo with immunomagnetic beads) metabolize clodronate, and whether rat peritoneal macrophages metabolize liposome-encapsulated clodronate, following in vivo administration. The effects of clodronate and AppCCl(2)p on bone resorption, osteoclast number, and apoptosis in vitro were compared. Using an antibody to the unprenylated form of Rap1A, effects on protein prenylation were assessed by Western blot analysis of osteoclast and peritoneal macrophage lysates from bisphosphonate-treated animals.

Results. AppCCl(2)p could be detected in extracts from osteoclasts purified from clodronate-treated rabbits. Intracellular accumulation of AppCCl(2)p caused a reduction in the number of osteoclasts, increased osteoclast apoptosis, and inhibited bone resorption in vitro. These effects were indistinguishable from those of clodronate. Liposome-encapsulated clodronate was also metabolized to AppCCl(2)p by rat peritoneal macrophages in vivo. Liposome-encapsulated clodronate caused an increase in peritoneal macrophage apoptosis in ex vivo cultures that was indistinguishable from the increase in apoptosis caused by liposome-encapsulated AppCCl(2)p. Unlike alendronate, clodronate and its metabolite did not affect prenylation of the small GTPase Rap1A in osteoclasts or macrophages in vivo.

Conclusion. These results provide the first direct evidence that the antiinflammatory and antiresorptive effects of clodronate on macrophages and osteoclasts in vivo occur via the intracellular formation of AppCCl(2)p.

Original languageEnglish
Pages (from-to)2201-2210
Number of pages9
JournalArthritis & Rheumatism
Volume44
Issue number9
DOIs
Publication statusPublished - Sep 2001

Keywords

  • ENCAPSULATED DICHLOROMETHYLENE DIPHOSPHONATE
  • IN-VITRO
  • BISPHOSPHONATE DRUGS
  • CONTAINING LIPOSOMES
  • ALENDRONATE
  • RAT
  • ACTIVATION
  • ETIDRONATE
  • DEPLETION
  • CELLS

Cite this

The molecular mechanism of action of the antiresorptive and antiinflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis. / Crockett, Julie Clare; Monkkonen, J.; Auriola, S.; Monkkonen, H.; Rogers, Michael John.

In: Arthritis & Rheumatism, Vol. 44, No. 9, 09.2001, p. 2201-2210.

Research output: Contribution to journalArticle

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abstract = "Objective. The primary aims of this study were to determine whether clodronate and liposome-encapsulated clodronate are metabolized to adenosine 5'-(beta,gamma -dichloromethylene) triphosphate (AppCCl(2)p) by osteoclasts and macrophages in vivo, and to determine whether intracellular accumulation of this metabolite accounts for the antiresorptive and antimacrophage effects of clodronate. To compare the mechanism of action of clodronate and alendronate, effects on protein prenylation in osteoclasts and macrophages in vivo were also assessed.Methods. High-performance liquid chromatography-mass spectrometry was used to determine whether rabbit osteoclasts (purified ex vivo with immunomagnetic beads) metabolize clodronate, and whether rat peritoneal macrophages metabolize liposome-encapsulated clodronate, following in vivo administration. The effects of clodronate and AppCCl(2)p on bone resorption, osteoclast number, and apoptosis in vitro were compared. Using an antibody to the unprenylated form of Rap1A, effects on protein prenylation were assessed by Western blot analysis of osteoclast and peritoneal macrophage lysates from bisphosphonate-treated animals.Results. AppCCl(2)p could be detected in extracts from osteoclasts purified from clodronate-treated rabbits. Intracellular accumulation of AppCCl(2)p caused a reduction in the number of osteoclasts, increased osteoclast apoptosis, and inhibited bone resorption in vitro. These effects were indistinguishable from those of clodronate. Liposome-encapsulated clodronate was also metabolized to AppCCl(2)p by rat peritoneal macrophages in vivo. Liposome-encapsulated clodronate caused an increase in peritoneal macrophage apoptosis in ex vivo cultures that was indistinguishable from the increase in apoptosis caused by liposome-encapsulated AppCCl(2)p. Unlike alendronate, clodronate and its metabolite did not affect prenylation of the small GTPase Rap1A in osteoclasts or macrophages in vivo.Conclusion. These results provide the first direct evidence that the antiinflammatory and antiresorptive effects of clodronate on macrophages and osteoclasts in vivo occur via the intracellular formation of AppCCl(2)p.",
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T1 - The molecular mechanism of action of the antiresorptive and antiinflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis

AU - Crockett, Julie Clare

AU - Monkkonen, J.

AU - Auriola, S.

AU - Monkkonen, H.

AU - Rogers, Michael John

PY - 2001/9

Y1 - 2001/9

N2 - Objective. The primary aims of this study were to determine whether clodronate and liposome-encapsulated clodronate are metabolized to adenosine 5'-(beta,gamma -dichloromethylene) triphosphate (AppCCl(2)p) by osteoclasts and macrophages in vivo, and to determine whether intracellular accumulation of this metabolite accounts for the antiresorptive and antimacrophage effects of clodronate. To compare the mechanism of action of clodronate and alendronate, effects on protein prenylation in osteoclasts and macrophages in vivo were also assessed.Methods. High-performance liquid chromatography-mass spectrometry was used to determine whether rabbit osteoclasts (purified ex vivo with immunomagnetic beads) metabolize clodronate, and whether rat peritoneal macrophages metabolize liposome-encapsulated clodronate, following in vivo administration. The effects of clodronate and AppCCl(2)p on bone resorption, osteoclast number, and apoptosis in vitro were compared. Using an antibody to the unprenylated form of Rap1A, effects on protein prenylation were assessed by Western blot analysis of osteoclast and peritoneal macrophage lysates from bisphosphonate-treated animals.Results. AppCCl(2)p could be detected in extracts from osteoclasts purified from clodronate-treated rabbits. Intracellular accumulation of AppCCl(2)p caused a reduction in the number of osteoclasts, increased osteoclast apoptosis, and inhibited bone resorption in vitro. These effects were indistinguishable from those of clodronate. Liposome-encapsulated clodronate was also metabolized to AppCCl(2)p by rat peritoneal macrophages in vivo. Liposome-encapsulated clodronate caused an increase in peritoneal macrophage apoptosis in ex vivo cultures that was indistinguishable from the increase in apoptosis caused by liposome-encapsulated AppCCl(2)p. Unlike alendronate, clodronate and its metabolite did not affect prenylation of the small GTPase Rap1A in osteoclasts or macrophages in vivo.Conclusion. These results provide the first direct evidence that the antiinflammatory and antiresorptive effects of clodronate on macrophages and osteoclasts in vivo occur via the intracellular formation of AppCCl(2)p.

AB - Objective. The primary aims of this study were to determine whether clodronate and liposome-encapsulated clodronate are metabolized to adenosine 5'-(beta,gamma -dichloromethylene) triphosphate (AppCCl(2)p) by osteoclasts and macrophages in vivo, and to determine whether intracellular accumulation of this metabolite accounts for the antiresorptive and antimacrophage effects of clodronate. To compare the mechanism of action of clodronate and alendronate, effects on protein prenylation in osteoclasts and macrophages in vivo were also assessed.Methods. High-performance liquid chromatography-mass spectrometry was used to determine whether rabbit osteoclasts (purified ex vivo with immunomagnetic beads) metabolize clodronate, and whether rat peritoneal macrophages metabolize liposome-encapsulated clodronate, following in vivo administration. The effects of clodronate and AppCCl(2)p on bone resorption, osteoclast number, and apoptosis in vitro were compared. Using an antibody to the unprenylated form of Rap1A, effects on protein prenylation were assessed by Western blot analysis of osteoclast and peritoneal macrophage lysates from bisphosphonate-treated animals.Results. AppCCl(2)p could be detected in extracts from osteoclasts purified from clodronate-treated rabbits. Intracellular accumulation of AppCCl(2)p caused a reduction in the number of osteoclasts, increased osteoclast apoptosis, and inhibited bone resorption in vitro. These effects were indistinguishable from those of clodronate. Liposome-encapsulated clodronate was also metabolized to AppCCl(2)p by rat peritoneal macrophages in vivo. Liposome-encapsulated clodronate caused an increase in peritoneal macrophage apoptosis in ex vivo cultures that was indistinguishable from the increase in apoptosis caused by liposome-encapsulated AppCCl(2)p. Unlike alendronate, clodronate and its metabolite did not affect prenylation of the small GTPase Rap1A in osteoclasts or macrophages in vivo.Conclusion. These results provide the first direct evidence that the antiinflammatory and antiresorptive effects of clodronate on macrophages and osteoclasts in vivo occur via the intracellular formation of AppCCl(2)p.

KW - ENCAPSULATED DICHLOROMETHYLENE DIPHOSPHONATE

KW - IN-VITRO

KW - BISPHOSPHONATE DRUGS

KW - CONTAINING LIPOSOMES

KW - ALENDRONATE

KW - RAT

KW - ACTIVATION

KW - ETIDRONATE

KW - DEPLETION

KW - CELLS

U2 - 10.1002/1529-0131(200109)44:9<2201::AID-ART374>3.0.CO;2-E

DO - 10.1002/1529-0131(200109)44:9<2201::AID-ART374>3.0.CO;2-E

M3 - Article

VL - 44

SP - 2201

EP - 2210

JO - Arthritis & Rheumatism

JF - Arthritis & Rheumatism

SN - 0004-3591

IS - 9

ER -