Inhibition of Growth of Dictyostelium discoideum Amoebae by Bisphosphonate Drugs Is Dependent on Cellular Uptake

Purpose. The aim of the study was to determine whether bisphosphonates are internalised by Dictyosteliumamoebae and whether cellular uptake is required for their growth-inhibitory effects. Bisphosphonates inhibit growth of amoebae of the slime mould Dictyostelium discoideum, by mechanisms that appear to be similar to those that cause inhibition of osteoclastic bone resorption. Methods. Cell-free extracts prepared from amoebae that had been incubated with bisphosphonates were analysed by ^3lP-n.m.r. spectroscopy or ion-exchange f.p.l.c., to identify the presence of bisphosphonates or bisphosphonate metabolites respectively. The growth-inhibitory effect of bisphosphonates towards Dictyostelium amoebae was also examined under conditions in which pinocytosis was inhibited. Results. All of the bisphosphonates studied were internalised by Dictyostelium amoebae, probably by fluid-phase pinocytosis, and could be detected in cell-free extracts. Amoebae that were prevented from internalising bisphosphonates by pinocytosis were markedly resistant to the growth-inhibitory effects of these compounds. In addition, bisphosphonates encapsulated within liposomes were more potent growth inhibitors of Dictyostelium owing to enhanced intracellular delivery of bisphosphonates. Conclusions. All bisphosphonates inhibit Dictyostelium growth by intracellular mechanisms following internalisation of bisphosphonates by fluid-phase pinocytosis. It is therefore likely that bisphosphonates also affect osteoclasts by interacting with intracellular, rather than extracellular, processes.     

Fluorescent risedronate analogues reveal bisphosphonate uptake by bone marrow monocytes and localization around osteocytes in vivo

Bisphosphonates are effective antiresorptive agents owing to their bone‐targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non‐osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein‐labeled risedronate (FAM‐RIS) and Alexa Fluor 647–labeled risedronate (AF647‐RIS), were used to address this question. Twenty‐four hours after injection into 3‐month‐old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM‐RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14^high bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14⁺ cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone‐resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo. © 2010 American Society for Bone and Mineral Research     

Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells

Bisphosphonates (BPs) target bone due to their high affinity for calcium ions. During osteoclastic resorption, these drugs are released from the acidified bone surface and taken up by osteoclasts, where they act by inhibiting the prenylation of small GTPases essential for osteoclast function. However, it remains unclear exactly how osteoclasts internalise BPs from bone and whether other cells in the bone microenvironment can also take up BPs from the bone surface. We have investigated this using a novel fluorescently-labelled alendronate analogue (FL-ALN), and by examining changes in protein prenylation following treatment of cells with risedronate (RIS). Confocal microscopic analysis showed that FL-ALN was efficiently internalised from solution or from the surface of dentine by resorbing osteoclasts into intracellular vesicles. Accordingly, unprenylated Rap1A accumulated to the same extent whether osteoclasts were cultured on RIS-coated dentine or with RIS in solution. By contrast, J774 macrophages internalised FL-ALN and RIS from solution, but took up comparatively little from dentine, due to their inability to resorb the mineral. Calvarial osteoblasts and MCF-7 tumour cells internalised even less FL-ALN and RIS, both from solution and from the surface of dentine. Accordingly, the viability of J774 and MCF-7 cells was drastically reduced when cultured with RIS in solution, but not when cultured on dentine pre-coated with RIS. However, when J774 macrophages were co-cultured with rabbit osteoclasts, J774 cells that were adjacent to resorbing osteoclasts frequently internalised more FL-ALN than J774 cells more distant from osteoclasts. This was possibly a result of increased availability of BP to these J774 cells due to transcytosis through osteoclasts, since FL-ALN partially co-localised with trancytosed, resorbed matrix protein within osteoclasts. In addition, J774 cells occupying resorption pits internalised more FL-ALN than those on unresorbed surfaces. These data demonstrate that osteoclasts are able to take up large amounts of BP, due to their ability to release the BP from the dentine surface during resorption. By contrast, non-resorbing cells take up only small amounts of BP that becomes available due to natural desorption from the dentine surface. However, BP uptake by non-resorbing cells can be increased when cultured in the presence of resorbing osteoclasts.     

Novel insights into actions of bisphosphonates on bone: differences in interactions with hydroxyapatite

Bisphosphonates are now the most widely used drugs for diseases associated with increased bone resorption, such as osteoporosis. Although bisphosphonates act directly on osteoclasts, and interfere with specific biochemical processes such as protein prenylation, their ability to adsorb to bone mineral also contributes to their potency and duration of action. The aim of the present study was to compare the binding affinities for hydroxyapatite (HAP) of 6 bisphosphonates currently used clinically and to determine the effects of these bisphosphonates on other mineral surface properties including zeta potential and interfacial tension. Affinity constants (K(L)) for the adsorption of bisphosphonates were calculated from kinetic studies on HAP crystal growth using a constant composition method at 37 degrees C and at physiological ionic strength (0.15 M). Under conditions likely to simulate bisphosphonate binding onto bone, there were significant differences in K(L) among the bisphosphonates for HAP growth (pH 7.4) with a rank order of zoledronate > alendronate > ibandronate > risedronate > etidronate > clodronate. The measurements of zeta potential show that the crystal surface is modified by the adsorption of bisphosphonates in a manner best explained by molecular charges related to the protonation of their side-chain moieties, with risedronate showing substantial differences from alendronate, ibandronate, and zoledronate. The studies of the solid/liquid interfacial properties show additional differences among the bisphosphonates that may influence their mechanisms for binding and inhibiting crystal growth and dissolution. The observed differences in kinetic binding affinities, HAP zeta potentials, and interfacial tension are likely to contribute to the biological properties of the various bisphosphonates. In particular, these binding properties may contribute to differences in uptake and persistence in bone and the reversibility of effects. These properties, therefore, have potential clinical implications that may be important in understanding differences among potent bisphosphonates, such as the apparently more prolonged duration of action of alendronate and zoledronate compared with the more readily reversible effects of etidronate and risedronate.     

Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy

Summary Bisphosphonates (BPs) are well established as the leading drugs for the treatment of osteoporosis. There is new knowledge about how they work. The differences that exist among individual BPs in terms of mineral binding and biochemical actions may explain differences in their clinical behavior and effectiveness. Introduction The classical pharmacological effects of bisphosphonates (BPs) appear to be the result of two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Discussion There is new information about both properties. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The antiresorptive effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) appear to result from their inhibition of the enzyme farnesyl pyrophosphate synthase (FPPS) in osteoclasts. FPPS is a key enzyme in the mevalonate pathway, which generates isoprenoid lipids utilized for the post-translational modification of small GTP-binding proteins that are essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. BPs share several common properties as a drug class. However, as with other families of drugs, there are obvious chemical, biochemical, and pharmacological differences among the individual BPs. Each BP has a unique profile that may help to explain potential clinical differences among them, in terms of their speed and duration of action, and effects on fracture reduction.     

Synthesis and biological evaluation of prostaglandin-F alkylphosphinic acid derivatives as bone anabolic agents for the treatment of osteoporosis.

A series of novel C(1) alkylphosphinic acid analogues of the prostaglandin-F family have been evaluated at the eight human prostaglandin receptors for potential use in the treatment of osteoporosis. Using molecular modeling as a tool for structure-based drug design, we have discovered that the phosphinic acid moiety (P(O)(OH)R) behaves as an isostere for the C(1) carboxylic acid in the human prostaglandin FP binding assay in vitro and possesses enhanced hFP receptor selectivity when compared to the parent carboxylic acid. When evaluated in vivo, the methyl phosphinic acid analogue (4b) produced a bone anabolic response in rats, returning bone mineral volume (BMV) [corrected], to intact levels in the distal femur in the ovariectomized rat (OVX) model. These results suggest that prostaglandins of this class may be useful agents in the treatment of diseases associated with bone loss.     

Nitrogen-containing bisphosphonates can inhibit angiogenesis in vivo without the involvement of farnesyl pyrophosphate synthase

Nitrogen-containing bisphosphonates (N-BPs) are widely used to block bone destruction associated with bone metastasis because they are effective inhibitors of osteoclast-mediated bone resorption. More specifically, once internalized by osteoclasts, N-BPs block the activity of farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway. In addition to their antiresorptive activity, preclinical evidence shows that N-BPs have antiangiogenic properties. However, the exact reasons for which N-BPs inhibit angiogenesis remain largely unknown. Using different angiogenesis models, we examined here the effects of zoledronate, risedronate and three structural analogs of risedronate (NE-58025, NE-58051 and NE-10790) with lower potencies to inhibit FPPS activity. Risedronate and zoledronate were much more potent than NE-compounds at inhibiting both endothelial cell proliferation in vitro and vessel sprouting in the chicken egg chorioallantoic membrane (CAM) assay. In addition, only risedronate and zoledronate inhibited the revascularization of the prostate gland in testosterone-stimulated castrated rats. Moreover, as opposed to NE-compounds, risedronate and zoledronate induced intracellular accumulation of isopentenyl pyrophosphate (IPP) in endothelial cells by blocking the activity of the IPP-consuming enzyme FPPS. Thus, these results indicated that N-BPs inhibited angiogenesis in a FPPS-dependent manner. However, drug concentrations used to inhibit angiogenesis, both in vitro and in the CAM and prostate gland assays, were high. In contrast, a low concentration of risedronate (1 μM) was sufficient to inhibit blood vessel formation in the ex vivo rat aortic ring assay. Moreover, NE-58025 (which had a 7-fold lower potency than risedronate to inhibit FPPS activity) was as effective as risedronate to reduce angiogenesis in the rat aortic ring assay. In conclusion, our results suggest that low concentrations of N-BPs inhibit angiogenesis in a FPPS-independent manner, whereas higher drug concentrations were required to inhibit FPPS activity in vivo.     

Bisphosphonate binding affinity as assessed by inhibition of carbonated apatite dissolution in vitro

Bisphosphonates (BPs), which display a high affinity for calcium phosphate surfaces, are able to selectively target bone mineral, where they are potent inhibitors of osteoclast-mediated bone resorption. The dissolution of synthetic hydroxyapatite (HAP) has been used previously as a model for BP effects on natural bone mineral. The present work examines the influence of BPs on carbonated apatite (CAP), which mimics natural bone more closely than does HAP. Constant composition dissolution experiments were performed at pH 5.50, physiological ionic strength (0.15M) and temperature (37 degrees C). Selected BPs were added at (0.5 x 10(-6)) to (50.0 x 10(-6))M, and adsorption affinity constants, K(L), were calculated from the kinetics data. The BPs showed concentration-dependent inhibition of CAP dissolution, with significant differences in rank order zoledronate > alendronate > risedronate. In contrast, for HAP dissolution at pH 5.50, the differences between the individual BPs were considerably smaller. The extent of CAP dissolution was also dependent on the relative undersaturation, sigma, and CAP dissolution rates increased with increasing carbonate content. These results demonstrate the importance of the presence of carbonate in mediating the dissolution of CAP, and the possible involvement of bone mineral carbonate in observed differences in bone affinities of BPs in clinical use.     

Cancer‐induced bone loss and associated pain‐related behavior is reduced by risedronate but not its phosphonocarboxylate analog NE‐10790

Prostate, breast and lung cancers readily develop bone metastases which lead to fractures, hypercalcemia and pain. Malignant growth in the bones depends on osteoclast‐mediated bone resorption and in this regard bisphosphonate compounds, which have high‐bone affinity and inhibit osteoclast activity, have been found to alleviate bone cancer symptoms. In this study, the bisphosphonate risedronate and its phosphonocarboxylate derivative NE‐10790 was tested in a murine bone cancer pain model. Risedronate decreased bone cancer‐related bone destruction and pain‐related behavior and decreased the spinal expression of glial fibrillary acidic protein, whereas NE‐10790 had no effect on these parameters. Furthermore, risedronate but not NE‐10790 induced dose‐dependent toxicity in NCTC‐2472 cells in vitro. Furthermore, the direct toxic effect of risedronate on tumor cells observed in vitro opens the possibility that a direct toxic effect on tumor cells may also be present in vivo and be related to the efficacy of bisphosphonate compounds. In conclusion, these results suggest that risedronate treatment may lead to an increased life quality, in patient suffering from bone cancer, in terms of decreased osteolysis and pain, and merits further study. © 2009 UICC