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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.

Nitrogen-containing bisphosphonates (NBps) are taken up by osteoclasts and inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway. There is evidence, however, that cells other than mature osteoclasts, like osteoclast precursors and osteoblasts, are also involved in the action of Bps on bone resorption in vitro. To examine this issue further, we developed a new in vitro model, which allows the study of the effects of additives on early osteoclast precursors. In this model, osteogenic cells are essential for osteoclastogenesis. The model consists of 15-day-old fetal mouse metatarsals. At time of explantation, these bone rudiments do not yet contain a mineralized matrix or osteoclasts; only early osteoclast precursors are present in the perichondrium. During culture and after the addition of Nabeta-glycerolphosphate, the bones form a mineralized matrix that is consequently resorbed by osteoclasts that develop from their precursors. Short treatment of these explants with Bps, before the formation of a mineralized matrix, resulted in a subsequent dose-dependent inhibition of bone resorption. The relative potencies of eight Bps to suppress resorption were comparable with those observed after the addition of Bps after the formation of a mineralized matrix, the natural target of Bps. In addition, the effects of the NBp olpadronate, but not of clodronate, on osteoclastic resorption, could be partly reversed by geranylgeraniol. Results indicate that Bps can suppress osteoclastic resorption in vitro by a direct action on very early osteoclast precursors at the bone surface, and not by affecting the osteoclastogenic capacity of osteogenic cells. Moreover, the mechanism of action of the NBp olpadronate, but not clodronate, on early tartrate-resistant acid phosphatase-negative osteoclast precursors involves inhibition of protein geranylgeranylation, indicating a molecular mechanism similar to that established for mature osteoclasts.

To define structure-activity relationships for bisphosphonate activity, we examined the bone binding and antiresorptive properties of heterocycle-containing analogs of risedronate, a pyridylbisphosphonate, in cultures of mouse fetal bone explants. Our studies indicated that hydroxybisphosphonates with the nitrogen molecule in the pyridyl ring were very potent inhibitors of osteoclastic resorption. Changing the place of the nitrogen in the ring structure of risedronate or its methylation did not significantly alter antiresorptive potency in relation to risedronate. Extension of the R2 chain, however, reduced efficacy. In binding experiments, we found that all heterocyclic bisphosphonates with a hydroxyl group in R1 had comparable affinity for bone mineral and inhibited calcium incorporation into bone explants to a similar extent. The affinity of a risedronate analog without R1 was markedly reduced. We also examined the properties of a risedronate analog (NE-10790) belonging to the group of phosphonocarboxylates in which one of the phosphonate groups is substituted by a carboxyl group. NE-10790 had strongly reduced binding affinity, but still retained some antiresorptive activity. Interestingly, the continuous presence of NE-10790 in cultures of fetal mouse metacarpal bones increased its antiresorptive efficacy by about 40-fold compared with 24 h preincubation, whereas, under the same conditions, the potency of high-affinity hydroxybisphosphonates did not change or only slightly increased. This may be explained by the differences in pharmacokinetic behavior between compounds of high and of low affinity for bone mineral. These data show that, as with alkylbisphosphonates, heterocycle-containing bisphosphonates with a nitrogen functionality in the R2 chain are potent antiresorptive agents and a hydroxyl substitution in the R1 chain confers high affinity for bone mineral, probably due to tridentate configuration. The group of phosphonocarboxylates, with strongly reduced bone affinity, provides an interesting therapeutic option.

Bisphosphonates suppress bone resorption by inhibiting the activity of mature osteoclasts as well as the formation of osteoclasts from bone marrow-derived precursor cells. It is not yet known at which level of osteoclast development this latter action is exerted and whether this is due to a systemic effect of circulating bisphosphonate or to an action at the bone surface, given the property of these compounds to concentrate specifically at active bone sites. We addressed these questions in an ex vivo study in mice. The animals were treated with the bisphosphonate alendronate for various periods, and the central compartment of the bone marrow was isolated and cultured together with osteoclast-free fetal mouse bone explants. In this way the capacity of bone marrow cells, exposed previously to alendronate in vivo, to generate osteoclasts and induce resorption in vitro was examined. Alendronate (0.25 mg/kg, subcutaneously) given to mice for periods up to 4 weeks suppressed bone resorption, as expected, but did not affect the capacity of bone marrow cells to develop into mature osteoclasts and resorb the calcified matrix of bone explants. In addition, using monoclonal antibodies specific for different macrophage-granulocyte precursor subsets, we found that alendronate treatment did not affect the pattern of antigen expression of bone marrow cells, which is in line with the lack of an effect of the bisphosphonate on the ability of bone marrow cells to induce resorption in vitro. In contrast, in control experiments, lipopolysaccharide (0.1 mg) treatment induced marked changes in the pattern of antigen expression of bone marrow cells. In conclusion, our studies demonstrate that the inhibition of bone resorption by alendronate does not involve alteration of the osteoclastogenic potential of osteoclast progenitors (precursors) from the central bone marrow compartment. Moreover, this treatment did not alter the expression of markers specific for monocyte-macrophage precursors as well as mature macrophages. These results suggest that the effects of alendronate are exerted at the bone surface at late osteoclast precursors (and mature osteoclasts).