Effect of risedronate on osteocyte viability and bone turnover in paired iliac bone biopsies from early postmenopausal women.

It is unclear whether standard clinical doses of risedronate affect osteocyte viability. This study examined osteocyte viability and bone remodeling rate in early postmenopausal women (1-5 years after menopause) who were treated with a standard clinical dose of risedronate (5 mg/day, orally) for 1 year. Paired transiliac bone biopsies were obtained from 19 postmenopausal women at baseline and after 1-year treatment with placebo (n = 8, mean age 52.9 ± 3.4 years) or risedronate 5 mg/day (n = 11, mean age 52.5 ± 3.4 years). In these samples, we measured osteocyte- and bone remodeling-related variables in trabecular bone. In both the placebo and risedronate groups, empty lacunae were significantly decreased after 1-year treatment compared to baseline. There were no significant differences in osteocyte-related variables between placebo and risedronate. Risedronate significantly reduced bone-remodeling indices including mineralizing surface (MS/BS), bone formation rate (BFR/BS), and activation frequency (Ac.f). Risedronate treatment caused significantly lower MS/BS and Ac.f than placebo administration. In conclusion, risedronate 5 mg/day effectively inhibited bone remodeling but did not significantly reduce osteocyte viability in trabecular bone.

Bisphosphonates: molecular mechanisms of action and effects on bone cells, monocytes and macrophages.

Bisphosphonates are widely used in the treatment of diseases involving excessive bone resorption, such as osteoporosis, cancer-associated bone disease, and Paget's disease of bone. They target to the skeleton due to their calcium-chelating properties, where they primarily act by inhibiting osteoclast-mediated bone resorption. The simple bisphosphonates, clodronate, etidronate and tiludronate, are intracellularly metabolised to cytotoxic ATP analogues, while the more potent, nitrogen-containing bisphosphonates act by inhibiting the enzyme FPP synthase, thereby preventing the prenylation of small GTPases that are necessary for the normal function and survival of osteoclasts. In recent years, these concepts have been refined, with an increased understanding of the exact mode of inhibition of FPP synthase and the consequences of inhibiting this enzyme. Recent studies further suggest that the R2 side chain, as well as determining the potency for inhibiting the target enzyme FPP synthase, also influences bone mineral binding, which may influence distribution within bone and duration of action. While bisphosphonates primarily affect the function of resorbing osteoclasts, it is becoming increasingly clear that bisphosphonates may also target the osteocyte network and prevent osteocyte apoptosis, which could contribute to their anti-fracture effects. Furthermore, increasing evidence implicates monocytes and macrophages as direct targets of bisphosphonate action, which may explain the acute phase response and the anti-tumour activity in certain animal models. Bone mineral affinity is likely to influence the extent of any such effects of these agents on non-osteoclast cells. While alternative anti-resorptive therapeutics are becoming available for clinical use, bisphosphonates currently remain the principle drugs used to treat excessive bone resorption.

Differences between bisphosphonates in binding affinities for hydroxyapatite.

Bisphosphonates (BPs) inhibit bone resorption and are widely used for the treatment of bone diseases, including osteoporosis. BPs are also being studied for their effects on hydroxyapatite (HAP)-containing biomaterials. There is a growing appreciation that there are hitherto unexpected differences among BPs in their mineral binding affinities that affect their pharmacological and biological properties. To study these differences, we have developed a method based on fast performance liquid chromatography using columns of HAP to which BPs and other phosphate-containing compounds can adsorb and be eluted by using phosphate buffer gradients at pH 6.8. The individual compounds emerge as discrete and reproducible peaks for a range of compounds with different affinities. For example, the peak retention times (min; mean +/- SEM) were 22.0 +/- 0.3 for zoledronate, 16.16 +/- 0.44 for risedronate, and 9.0 +/- 0.28 for its phosphonocarboxylate analog, NE10790. These results suggest that there are substantial differences among BPs in their binding to HAP. These differences may be exploited in the development of biomaterials and may also partly explain the extent of their relative skeletal retention and persistence of biological effects observed in both animal and clinical studies.

Synergistic effect of bisphosphonate and docetaxel on the growth of bone metastasis in an animal model of established metastatic bone disease.

Bisphosphonates decrease bone resorption and reduce significantly the rate of skeletal complications in patients with metastatic bone disease. Bisphosphonates have also been shown to exhibit antitumor activity in vitro but in vivo results have been equivocal. In the present study, we investigated the effects of bisphosphonate treatment alone or in combination with the cytostatic docetaxel on the growth of breast cancer cells in bone. Tumor gowth was studied in an athymic nude mice model inoculated with MDA-231-B/luc+ breast cancer cells. Two days after the inoculation, mice were treated with risedronate, zolendronate or docetaxel alone or with a combination of risedronate and docetaxel. Bone destruction and tumor growth were evaluated radiographically, histologically and by whole-body bioiluminescent reporter imaging (BLI). Five week treatment with high doses risedronate or zoledronate (37.5-150 microg/kg, 5 times/week), fully protected the bones from osteolysis, but did not affect tumour growth. Docetaxel (2, 4, and 8 mg/kg, 2 times/week) inhibited tumour growth dose-dependently and after 5 weeks treatment with the highest dose, there was no detectable tumour in bone. The combination of a dose of docetaxel (4 mg/kg) that demonstrated only a minimal effect on tumour growth, with risedronate (150 microg/kg), protected bone integrity and nearly completely inhibited the growth of the cancer cells. Risedronate and docetaxel act synergistically to protect bone and decrease tumour burden in an animal model of established bone metastases from breast cancer cells.

Fluorescently labeled risedronate and related analogues: "magic linker" synthesis.

We report synthesis of the first fluorescently labeled conjugates of risedronate (1), using an epoxide linker strategy enabling conjugation of 1 via its pyridyl nitrogen with the label (carboxyfluorescein). Unlike prior approaches to create fluorescent bisphosphonate probes, the new linking chemistry did not abolish the ability to inhibit protein prenylation in vitro, while significantly retaining hydroxyapatite affinity. The utility of a fluorescent 1 conjugate in visualizing osteoclast resorption in vitro was demonstrated.

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

UNLABELLED: 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.

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.

End-capping of the modified melanocortin tetrapeptide (p-Cl)Phe-D-Phe-Arg-Trp-NH2 as a route to hMC4R agonists.

Of the 42 R'-X-(p-Cl)Phe-D-Phe-Arg-Trp-NH(2) (X=CO, SO(2), PO, PS) tested at the human (h)MC1, hMC3, and hMC4 receptors (R), the most potent MC4R agonists (EC(50) of 8-20 nM) were obtained by end-capping with R'=CH(2)CHCH(2) (9), NCCH(2) (16), NH(2)COCH(2) (17), HCONHCH(2) (18), CH(3)NH (19), CH(2)CHCH(2)NH (21), 2-Th (23), PhCH(2) (30) and X=CO. These compounds possess 35-60-fold hMC4 versus hMC1Rs selectivity with urea LK-71 (19) being the most potent at hMC4R and MC4/1R selective (EC(50)=8.5 nM, MC4/1R=100). LK-75 (16) combines high potency at hMC4R and MC4/3R selectivity (EC(50)=10.5 nM, MC4/3R=290). SAR is discussed.

Analogs of sub-nanomolar hMC1R agonist LK-184 [Ph(CH2)3CO-His-D-Phe-Arg-Trp-NH2]. An additional binding site within the human melanocortin receptor 1?

Twenty nine analogs of a superpotent MC1R agonist LK-184 (1) were tested at human melanocortin receptors (hMC1, hMC3, and hMC4Rs). All derivatives with the spacer between the N-terminus and the aromatic ring longer or shorter than C(3) were much less potent at hMC1R than 1. Only LK-312 PhCO(CH(2))(3)CO-His-d-Phe-Arg-Trp-NH(2) (3), partially mimicking the pi-system of 1, had an EC(50) of 0.05 nM at hMC1R, which confirms the localization of the pi-binding zone of the receptor. Truncation of 1 to Ph(CH(2))(3)CO-His-d-Phe-Arg-NH(2) gave a full MC1 agonist, LK-394 (30), with an EC(50) of 5 nM and a weak partial agonism at MC3/4Rs. This suggests the existence of an additional binding site within hMC1R next to that for the core sequence His-d-Phe-Arg-Trp-NH(2).

Differentiating the mechanisms of antiresorptive action of nitrogen containing bisphosphonates.

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.

Sub-nanomolar hMC1R agonists by end-capping of the melanocortin tetrapeptide His-D-Phe-Arg-Trp-NH(2).

Twenty three derivatives of the core fragment His(6)-D-Phe(7)-Arg(8)-Trp(9)-NH(2) end-capped with carboxylic and sulfonic acids were synthesized and evaluated at human melanocortin receptors (hMC1, hMC3, and hMC4Rs). The SAR within this series allowed us to map the hMCRs near the His(6) binding site and design a superpotent MC1R agonist, LK-184, Ph(CH(2))(3)CO-His-D-Phe-Arg-Trp-NH(2) (19) with EC(50) 0.01 nM (5 nM at MC3 and MC4Rs).

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.

Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages.

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.

Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates.

It has long been known that small changes to the structure of the R(2) side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo, although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently, several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase, thereby preventing protein prenylation in osteoclasts. In this study, we examined the potency of a wider range of nitrogen-containing bisphosphonates, including the highly potent, heterocycle-containing zoledronic acid and minodronate (YM-529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro, to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro, and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations > or = 1 nM zoledronic acid or minodronate, the order of potency (zoledronic acid approximately equal to minodronate > risedronate > ibandronate > incadronate > alendronate > pamidronate) closely matching the order of antiresorptive potency. Furthermore, minor changes to the structure of the R(2) side chain of heterocycle-containing bisphosphonates, giving rise to less potent inhibitors of bone resorption in vivo, also caused a reduction in potency up to approximately 300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo, and that small changes to the structure of the R(2) side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase.

Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases.

The molecular mechanisms by which tumor cells metastasize to bone are likely to involve invasion, cell adhesion to bone, and the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are, therefore, used in the treatment of patients with osteolytic metastases. However, an added beneficial effect of BPs may be direct antitumor activity. We previously reported that BPs inhibit breast and prostate carcinoma cell adhesion to bone (Boissier et al., Cancer Res., 57: 3890-3894, 1997). Here, we provided evidence that BP pretreatment of breast and prostate carcinoma cells inhibited tumor cell invasion in a dose-dependent manner. The order of potency for four BPs in inhibiting tumor cell invasion was: zoledronate > ibandronate > NE-10244 (active pyridinium analogue of risedronate) > clodronate. In addition, NE-58051 (the inactive pyridylpropylidene analogue of risedronate) had no inhibitory effect, whereas NE-10790 (a phosphonocarboxylate analogue of risedronate in which one of the phosphonate groups is substituted by a carboxyl group) inhibited tumor cell invasion to an extent similar to that observed with NE-10244, indicating that the inhibitory activity of BPs on tumor cells involved the R2 chain of the molecule. BPs did not induce apoptosis in tumor cells, nor did they inhibit tumor cell migration at concentrations that did inhibit tumor cell invasion. However, although BPs did not interfere with the production of matrix metalloproteinases (MMPs) by tumor cells, they inhibited their proteolytic activity. The inhibitory effect of BPs on MMP activity was completely reversed in the presence of an excess of zinc. In addition, NE-10790 did not inhibit MMP activity, suggesting that phosphonate groups of BPs are responsible for the chelation of zinc and the subsequent inhibition of MMP activity. In conclusion, our results provide evidence for a direct cellular effect of BPs in preventing tumor cell invasion and an inhibitory effect of BPs on the proteolytic activity of MMPs through zinc chelation. These results suggest, therefore, that BPs may be useful agents for the prophylactic treatment of patients with cancers that are known to preferentially metastasize to bone.

Heterocycle-containing bisphosphonates cause apoptosis and inhibit bone resorption by preventing protein prenylation: evidence from structure-activity relationships in J774 macrophages.

Recent evidence suggests that bisphosphonates (BPs) may inhibit bone resorption by mechanisms that lead to osteoclast apoptosis. We have previously shown that BPs also reduce cell viability and induce apoptosis in the macrophage-like cell line J774. To determine whether BPs inhibit osteoclast-mediated bone resorption and affect J774 macrophages by the same molecular mechanism, we examined the potency to reduce J774 cell viability of pairs of nitrogen-containing BPs that differ slightly in the structure of the heterocycle-containing side chain but that differ markedly in antiresorptive potency. In all cases, the most potent antiresorptive BP of each pair also caused the greatest loss of J774 viability, while the less potent antiresorptive BPs were also less potent at reducing J774 cell viability. Similarly, the bisphosphinate, phosphonoalkylphosphinate and monophosphonate analogs of BPs (in which one or both phosphonate groups are modified, giving rise to much less potent or inactive antiresorptive agents) were much less potent or inactive at reducing J774 cell viability. Thus, the structure-activity relationships of BPs for inhibiting bone resorption match those for causing loss of cell viability in J774 cells, indicating that BPs inhibit osteoclast-mediated bone resorption and reduce J774 macrophage viability by the same molecular mechanism. Loss of J774 cell viability after treatment with BPs was associated with a parallel increase in apoptotic cell death. We have recently proposed that nitrogen-containing BPs reduce cell viability and cause J774 apoptosis as a consequence of inhibition of enzymes of the mevalonate pathway and hence loss of prenylated proteins. In this study, the BPs that were potent inducers of J774 apoptosis and potent antiresorptive agents were also found to be effective inhibitors of protein prenylation in J774 macrophages, whereas the less potent BP analogs did not inhibit protein prenylation. This provides strong evidence that BPs with a heterocyclic, nitrogen-containing side chain, such as risedronate, inhibit osteoclast-mediated bone resorption and induce J774 apoptosis by preventing protein prenylation.

Binding and antiresorptive properties of heterocycle-containing bisphosphonate analogs: structure-activity relationships.

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 inhibit prostate and breast carcinoma cell adhesion to unmineralized and mineralized bone extracellular matrices.

The molecular mechanisms by which tumor cells induce osteolytic metastases are likely to involve tumor cell adhesion to bone as well as the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are, therefore, used in the treatment of cancer-associated osteolytic metastases. Here, we investigated the effect of BPs on breast and prostate carcinoma cell adhesion to unmineralized and mineralized bone extracellular matrices. BP pretreatment of tumor cells inhibited tumor cell adhesion to unmineralized and mineralized osteoblastic extracellular matrices in a dose-dependent manner. In contrast, BP did not affect adhesion of normal cells (fibroblasts) to extracellular matrices. The order of potency for four BPs in inhibiting tumor cell adhesion to extracellular matrices was found to be: ibandronate > NE-10244 (antiresorptive active pyridinium analogue of risedronate) > pamidronate > clodronate. BP did not affect [3H]thymidine incorporation by tumor cells, as assessed by a mitogenesis assay, indicating that BP did not exert any cytotoxic effect at concentrations used to inhibit tumor cell adhesion. NE-58051, the inactive pyridylpropylidene analogue of risedronate, had no inhibitory effect on tumor cell adhesion compared to that observed with its active counterpart NE-10244, suggesting that the mechanism of action of BP on tumor cells involved a stereospecific recognition step. Although integrins mediate cell-matrix interactions, BP recognition by tumor cells did not modulate cell surface integrin expression. In conclusion, our results provide evidence for a direct cellular effect of BP in preventing tumor cell adhesion to bone, suggesting that BPs may be useful agents for the prophylactic treatment of patients with cancer that is known to preferentially metastasize to bone.

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 Dictyostelium amoebae 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 31P-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.

Structure-activity relationships of new heterocycle-containing bisphosphonates as inhibitors of bone resorption and as inhibitors of growth of Dictyostelium discoideum amoebae.

The mechanisms by which bisphosphonate drugs inhibit osteoclast-mediated bone resorption are unclear. Effects of bisphosphonates on cellular enzymes, metabolic pathways, and osteoclast morphology have previously been described and could culminate in a generalized cytotoxic effect or a decreased capacity of osteoclasts to resorb bone. Recent studies of the structure-activity relationship for the bisphosphonate side chain indicate, however, that at least the newer generations of nitrogen-containing bisphosphonates probably act by binding to a specific target at a site that is complementary in structure to the bisphosphonate side chain. We have previously proposed that such a target for bisphosphonates is also present in amoebae of the cellular slime mold Dictyostelium discoideum, because growth of this microorganism is inhibited by a wide range of bisphosphonates in a manner that closely reflects the antiresorptive potencies of the bisphosphonates in vivo. We have added support for this view by examining the potency towards Dictyostelium of bisphosphonates in which slight changes in the structure of the side chain or conformational restrictions to the side chain have marked effects on antiresorptive potency. The changes in the side chain that affected the in vivo antiresorptive potency of the bisphosphonates consistently affected in a similar manner the potency of the bisphosphonates as inhibitors of the growth of Dictyostelium amoebae. These observations confirm that bisphosphonate drugs have a molecular target that is common to both Dictyostelium amoebae and osteoclasts.