Differentiating the mechanisms of antiresorptive action of nitrogen containing bisphosphonates

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.

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 molecular mechanism of action [1], [2], [3]. First, BPS with no nitrogen functionality in their structure, such as etidronate, clodronate and tiludronate, have relatively low antiresorptive potency and inhibit osteoclast function via intracellular metabolism to toxic ATP-metabolites [4], [5]. Second, nitrogen-containing BPS (NBPS), such as alendronate, ibandronate, olpadronate, pamidronate, risedronate, and zoledronate are more potent inhibitors of osteoclastic bone resorption and inhibit farnesyl pyrophosphate synthase (FPPS), a key enzyme of the mevalonate biosynthetic route [1], [2], [3], [6], [7], [8], [9], [10], [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 proteins like CdC42, rho, and rab and disruption of the organization of the cytoskeleton of these cells [10], [11], [12], [13], [14]. These events result, among others, in disappearance of the ruffled border, leading to inactivity and apoptosis of the osteoclasts [1], [2], [3], [15].

We previously showed that the ability of a number of NBPS to inhibit the activity of FPPS/isopentenyl pyrophosphate isomerase (IPPI) was generally related to their antiresorptive potencies in vitro and in vivo, due to their specific effect on FPPS [16], [6]. More recently, Dunford et al. [9] using NBPS with a wide range of antiresorptive potencies, showed a close relation between the ability of BPS to inhibit recombinant human FPPS in vitro and their potencies to inhibit bone resorption, indicating that this enzyme is the main intracellular target of NBPS. However, effects of NBPS in addition to or independent of inhibition of the mevalonate pathway have been reported to explain some effects of the NBP pamidronate in various cell systems [7], [8], [9], [16], [17], [18], suggesting that the relation between suppression of FPPS and bisphosphonate action may be more complex than is currently thought. Identification of additional relations is particularly important as there is currently no way to differentiate among members of this class of bisphosphonates other than their potency to suppress bone resorption.

In the present study we addressed this issue and we compared pamidronate to the structurally related alkyl NBPS alendronate and NH₂-pamidronate [19]. In further experiments, we examined heterocyclic NBPS with small structural differences that result in major changes in antiresorptive potency. We performed experiments with the following specific aims: assessment of effects on osteoclastic resorption, on enzymatic activity, and on reversibility of resorption with GGOH. The latter experiments are essential for establishing the functional significance of the suppression of enzymatic activity by BPS, as GGOH has been previously shown to rescue osteoclasts and restore their function in cultures of cells and bone explants treated with some NBPS [12], [11]. Our results show that at least two of the NBPS tested, namely pamidronate and the risedronate analog NE-21650, suppress bone resorption by mechanism(s) additional to inhibition of FPPS activity, which in the case of pamidronate appears to be dominant.