Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high-and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that ''recycling'' had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton. ß

Alpha-halogenated analogues of the anti-resorptive bisphosphonate risedronate (5, Ris) and its phosphonocarboxylate cognate (7, 3-PEHPC) were synthesized and compared with 5, 7, and the corresponding desoxy analogues in bone mineral affinity and mevalonate pathway inhibition assays. The Ris (5e-h) and 3-PEHPC (7e-h) analogues had decreased bone mineral affinity, confirming that the alpha-OH group in 5 and 7 enhances bone affinity. The 5 alpha-halo-analogues potently inhibited farnesyl pyrophosphate synthase (FPPS) with IC50 values from 16 (alpha-F) to 340 (alpha-Br) nM (5, 6 nM). In contrast, 7 alpha-halo-analogues were ineffective versus FPPS (IC50 > 600 microM), but inhibited Rab geranylgeranyl transferase (RGGT) (IC50 = 16-35 microM) similarly to 7 itself (IC50 = 24 microM). The alpha-F analogue 7e was 1-2 times as active as 7 in J774 cell viability and Rab11 prenylation inhibition assays.