Differentiating the mechanisms of antiresorptive action of nitrogen containing bisphosphonates E.R. van Beek, a, * L.H. Cohen, b I.M. Leroy, b F.H. Ebetino, c C.W.G.M. Lo ¨wik, a and S.E. Papapoulos a a Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands b Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands c Procter & Gamble Pharmaceuticals, Health Care Research Center, Mason, OH, USA Received 24 March 2003; revised 9 July 2003; accepted 31 July 2003 Abstract Bisphosphonates (BPS) inhibit bone resorption and are divided into two classes according to their chemical structure and mechanism of action: nonnitrogen containing BPS such as etidronate and clodronate that are of low potency and inhibit osteoclast function via metabolism into toxic ATP-metabolites and nitrogen-containing BPS (NBPS), such as alendronate and risedronate that inhibit the enzyme of the mevalonate biosynthetic pathway farnesyl pyrophosphate synthase (FPPS), resulting in inhibition of the prenylation of small GTP-binding proteins in osteoclasts and disruption of their cytoskeleton. Previously, studies in various cell types suggested, however, that pamidronate functions by mechanism(s) additional or independent of the mevalonate pathway. To examine if such mechanism(s) are also involved in the action of NBPS on osteoclastic bone resorption, we examined the action of alkyl and heterocyclic NBPS with close structural homology on FPPS/isopentenyl pyrophosphate isomerase (IPPI) activity, on osteoclastic resorption, and on reversibility of this effect with GGOH. As expected, both pamidronate and alendronate suppressed bone resorption and FPPS/IPPI activity, the latter with greater potency than the first. Surprisingly, however, unlike alendronate, the antiresorptive effect of pamidronate was only partially reversible with GGOH, indicating the involvement of mechanism(s) of action additional to that of suppression of FPPS. Comparable results were obtained with the heterocyclic NBP NE-21650, a structural analog of risedronate. Thus, despite an effect on FPPS, the actions on bone resorption of some NBPS may involve mechanisms additional to suppression of FPPS. These findings may lead to identification of additional pathways that are important for bone resorption and may help to differentiate among members of the NBP class which are currently distinguished only according to their potency to inhibit bone resorption. © 2003 Elsevier Inc. All rights reserved. Keywords: Bisphosphonates; Geranylgeranylation; Bone resorption; FPP synthase; Osteoclasts Introduction Bisphosphonates (BPS) suppress osteoclast-mediated bone resorption and are widely used in the management of patients with skeletal disorders. BPS are distinguished into two classes according to their chemical structure and mo- lecular mechanism of action [1–3]. First, BPS with no nitrogen functionality in their structure, such as etidronate, clodronate and tiludronate, have relatively low antiresorp- tive potency and inhibit osteoclast function via intracellular metabolism to toxic ATP-metabolites [4,5]. Second, nitro- gen-containing BPS (NBPS), such as alendronate, iban- dronate, olpadronate, pamidronate, risedronate, and zole- dronate are more potent inhibitors of osteoclastic bone resorption and inhibit farnesyl pyrophosphate synthase (FPPS), a key enzyme of the mevalonate biosynthetic route [1–3,6 –11]. Suppression of this enzyme in osteoclasts by NBPS causes inhibition of the synthesis of FPP and GGPP and thereby of the prenylation of small GTP-binding pro- teins like CdC42, rho, and rab and disruption of the orga- nization of the cytoskeleton of these cells [10 –14]. These * Corresponding author. Fax: +31-715248136. E-mail address: E.R.van_Beek@lumc.nl (E.R. van Beek). Bone 33 (2003) 805– 811 www.elsevier.com/locate/bone 8756-3282/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2003.07.007