Vol. 61, Issue 5, 1255-1262, May 2002

Further Insight into Mechanism of Action of Clodronate: Inhibition of Mitochondrial ADP/ATP Translocase by a Nonhydrolyzable, Adenine-Containing Metabolite

Petri P. Lehenkari, Maarit Kellinsalmi, Juha P. Näpänkangas, Kari V. Ylitalo, Jukka Mönkkönen, Michael J. Rogers, Alex Azhayev, H. Kalervo Väänänen, and Ilmo E. Hassinen

Departments of Surgery (P.P.L.), Anatomy (P.P.L., M.K.), and Medical Biochemistry (J.N.N., K.V.Y., I.E.H.), University of Oulu, Oulu, Finland; Departments of Pharmaceutics (J.M.) and Pharmaceutical Chemistry (A.A.), University of Kuopio, Kuopio, Finland; Department of Medicine and Therapeutics, University of Aberdeen Medical School, Foresterhill, United Kingdom (M.J.R.); and Department of Anatomy, University of Turku, Turku, Finland (H.K.V.)

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. Recent studies have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R² side chain. Alendronate, like other nitrogen-containing bisphosphonates, inhibits bone resorption and causes apoptosis of osteoclasts and other cells in vitro by preventing post-translational modification of GTP-binding proteins with isoprenoid lipids. Clodronate, a bisphosphonate that lacks a nitrogen, does not inhibit protein isoprenylation but can be metabolized intracellularly to a --methylene (AppCp-type) analog of ATP, which is cytotoxic to macrophages in vitro. The detailed molecular basis for the cytotoxic effects of adenosine-5'-[,-dichloromethylene]triphosphate (AppCCl₂p) has not been determined yet. We addressed this question by studying the effects of alendronate, clodronate, and the clodronate metabolite AppCCl₂p on isolated mitochondria, mitochondrial fractions, and mitochondrial membrane potential in isolated human osteoclasts. We found that AppCCl₂p inhibits mitochondrial oxygen consumption by a mechanism that involves competitive inhibition of the ADP/ATP translocase. Alendronate or the native form of clodronate did not have any immediate effect on mitochondria. However, longer treatment with liposome-encapsulated clodronate caused collapse of the mitochondrial membrane potential, although prominent apoptosis was a late event. Hence, inhibition of the ADP/ATP translocase by the metabolite AppCCl₂p is a likely route by which clodronate causes osteoclast apoptosis and inhibits bone resorption.