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
Jc. Frith et al., 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, ARTH RHEUM, 44(9), 2001, pp. 2201-2210
Objective. The primary aims of this study were to determine whether clodron
ate and liposome-encapsulated clodronate are metabolized to adenosine 5'-(b
eta,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 alendr
onate, effects on protein prenylation in osteoclasts and macrophages in viv
o were also assessed.
Methods. High-performance liquid chromatography-mass spectrometry was used
to determine whether rabbit osteoclasts (purified ex vivo with immunomagnet
ic beads) metabolize clodronate, and whether rat peritoneal macrophages met
abolize liposome-encapsulated clodronate, following in vivo administration.
The effects of clodronate and AppCCl(2)p on bone resorption, osteoclast nu
mber, and apoptosis in vitro were compared. Using an antibody to the unpren
ylated form of Rap1A, effects on protein prenylation were assessed by Weste
rn blot analysis of osteoclast and peritoneal macrophage lysates from bisph
osphonate-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 apopt
osis, and inhibited bone resorption in vitro. These effects were indistingu
ishable 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 apoptos
is in ex vivo cultures that was indistinguishable from the increase in apop
tosis caused by liposome-encapsulated AppCCl(2)p. Unlike alendronate, clodr
onate and its metabolite did not affect prenylation of the small GTPase Rap
1A in osteoclasts or macrophages in vivo.
Conclusion. These results provide the first direct evidence that the antiin
flammatory and antiresorptive effects of clodronate on macrophages and oste
oclasts in vivo occur via the intracellular formation of AppCCl(2)p.