[show abstract][hide abstract] ABSTRACT: The gap junction protein, connexin43 (Cx43) controls both bone formation and osteoclastogenesis via osteoblasts and/or osteocytes. Cx43 has also been proposed to mediate an anti-apoptotic effect of bisphosphonates, potent inhibitors of bone resorption. We studied whether bisphosphonates are effective in protecting mice with a conditional Cx43 gene deletion in osteoblasts and osteocytes (cKO) from the consequences of ovariectomy on bone mass and strength. Ovariectomy resulted in rapid loss of trabecular bone followed by a slight recovery in wild type (WT) mice, and a similar degree of trabecular bone loss, albeit slightly delayed, occurred in cKO mice. Treatment with either risedronate (20 μg/kg) or alendronate (40 μg/kg) prevented ovariectomy-induced bone loss in both genotypes. In basal conditions, bones of cKO mice have larger marrow area, higher endocortical osteoclast number, and lower cortical thickness and strength relative to WT. Ovariectomy increased endocortical osteoclast number in WT but not in cKO mice. Both bisphosphonates prevented these increases in WT mice, and normalized endocortical osteoclast number, cortical thickness and bone strength in cKO mice. Thus, lack of osteoblast/osteocyte Cx43 does not alter bisphosphonate action on bone mass and strength in estrogen deficiency. These results support the notion that one of the main functions of Cx43 in cortical bone is to restrain osteoblast and/or osteocytes from inducing osteoclastogenesis at the endocortical surface.
[show abstract][hide abstract] ABSTRACT: We used Raman and Fourier transform infrared microspectroscopy (FTIRM) analysis to examine the intrinsic bone material properties at actively bone-forming trabecular surfaces in iliac crest biopsies from women with postmenopausal osteoporosis (PMO) who were treated with either alendronate (ALN) or risedronate (RIS). At eight study sites, women were identified who had postmenopausal osteoporosis (PMO), were at least 5 years postmenopause, and had been on long-term therapy (either 3-5 years or >5 years) with daily or weekly ALN or RIS. Following standard tetracycline labeling, biopsies were collected from 102 women (33 treated with ALN for 3-5 years [ALN-3], 35 with ALN for >5 years [ALN-5], 26 with RIS for 3-5 years [RIS-3], and 8 with RIS for >5 years [RIS-5]) and were analyzed at anatomical areas of similar tissue age in bone-forming areas (within the fluorescent double labels). The following outcomes were monitored and reported: mineral to matrix ratio (corresponding to ash weight), relative proteoglycan content (regulating mineralization commencement), mineral maturity (indicative of the mineral crystallite chemistry and stoichiometry, and having a direct bearing on crystallite shape and size), and the ratio of two of the major enzymatic collagen cross-links (pyridinoline/divalent). In RIS-5 there was a significant decrease in the relative proteoglycan content (-5.83% compared to ALN-5), while in both RIS-3 and RIS-5 there was significantly lower mineral maturity/crystallinity (-6.78% and -13.68% versus ALN-3 and ALN-5, respectively), and pyridinoline/divalent collagen cross-link ratio (-23.09% and -41.85% versus ALN-3 and ALN-5, respectively). The results of the present study indicate that ALN and RIS exert differential effects on the intrinsic bone material properties at actively bone-forming trabecular surfaces.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 02/2012; 27(5):995-1003. · 6.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 01/2012; 27(4):835-47. · 6.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ability of bisphosphonates ((HO)(2)P(O)CR(1)R(2)P(O)(OH)(2)) to inhibit bone resorption has been known since the 1960s, but it is only recently that a detailed molecular understanding of the relationship between chemical structures and biological activity has begun to emerge. The early development of chemistry in this area was largely empirical and based on modifying R(2) groups in a variety of ways. Apart from the general ability of bisphosphonates to chelate Ca(2+) and thus target the calcium phosphate mineral component of bone, attempts to refine clear structure-activity relationships had led to ambiguous or seemingly contradictory results. However, there was increasing evidence for cellular effects, and eventually the earliest bisphosphonate drugs, such as clodronate (R(1)=R(2)=Cl) and etidronate (R(1)=OH, R(2)=CH(3)), were shown to exert intracellular actions via the formation in vivo of drug derivatives of ATP. The observation that pamidronate, a bisphosphonate with R(1)=OH and R(2)=CH(2)CH(2)NH(2), exhibited higher potency than previously known bisphosphonate drugs represented the first step towards the later recognition of the critical importance of having nitrogen in the R(2) side chain. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates took place particularly in the 1980s, but still with an incomplete understanding of their structure-activity relationships. A major advance was the discovery that the anti-resorptive effects of the nitrogen-containing bisphosphonates (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their potency as inhibitors of the enzyme farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids utilized in sterol synthesis and for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of bisphosphonates have been synthesized and studied. Interest in expanding the structural scope of the bisphosphonate class has also motivated new approaches to the chemical synthesis of these compounds. Recent chemical innovations include the synthesis of fluorescently labeled bisphosphonates, which has enabled studies of the biodistribution of these drugs. As a class, bisphosphonates share common properties. However, as with other classes of drugs, there are chemical, biochemical, and pharmacological differences among the individual compounds. Differences in mineral binding affinities among bisphosphonates influence their differential distribution within bone, their biological potency, and their duration of action. The overall pharmacological effects of bisphosphonates on bone, therefore, appear to depend upon these two key properties of affinity for bone mineral and inhibitory effects on osteoclasts. The relative contributions of these properties differ among individual bisphosphonates and help determine their clinical behavior and effectiveness.
[show abstract][hide abstract] ABSTRACT: To establish whether the combination of anti-resorptive therapy with mechanical loading has a negative, additive or synergistic effect on bone structure, we assessed the separate and combined effects of risedronate and non-invasive dynamic loading on trabecular and cortical bone. Seventeen-week-old female C57BL/6 mice were given daily subcutaneous injections of vehicle (n=20) or risedronate at a dose of 0.15, 1.5, 15 or 150 μg/kg/day (n=10 in each) for 17 days. From the fourth day of treatment, the right tibiae were subjected to a single period of axial loading (40 cycles/day) for three alternate days per week for two weeks. The left tibiae were used as internal controls. Trabecular and cortical sites in the tibiae were analyzed by high-resolution micro-computed tomography and imaging of fluorochrome labels. In the non-loaded tibiae, treatment with the higher doses of risedronate at 15 or 150 μg/kg/day resulted in higher trabecular bone volume and trabecular number than in vehicle-treated controls, whereas such treatment was associated with no differences in cortical bone volume at any dose. In the loaded tibiae, loading induced increases in trabecular and cortical bone volume compared with contra-lateral controls primarily through increased trabecular thickness and periosteal expansion, respectively, independently of risedronate treatment. In conclusion, the response to mechanical loading in both trabecular and cortical bone in mice is therefore not impaired by short-term treatment with risedronate, even over a 1000-fold dose range. In considering the optimization of treatments for osteoporosis, it is reassuring that anti-resorptive therapy and mechanical loading can exert independent beneficial effects.
[show abstract][hide abstract] ABSTRACT: Under certain conditions, the phosphonocarboxylate analogue (3) of the bisphosphonate drug minodronate (4) in contact with borosilicate glassware reversibly forms an isolable dimer complex of boron, as revealed by the X-ray crystallographic structure of the (R,R/S,S) complex and supported by NMR and HRMS data.
Chemical Communications 06/2011; 47(22):6395-7. · 6.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Phosphonocarboxylate (PC) analogues of bisphosphonates are of interest due to their selective inhibition of a key enzyme in the mevalonate pathway, Rab geranylgeranyl transferase (RGGT). The dextrarotatory enantiomer of 2-hydroxy-3-(imidazo[1,2-a]pyridin-3-yl)-2-phosphonopropanoic acid (3-IPEHPC, 1) is the most potent PC-type RGGT inhibitor thus far identified. The absolute configuration of (+)-1 in the active site complex has remained unknown due to difficulties in obtaining RGGT inhibitor complex crystals suitable for X-ray diffraction analysis. However, we have now succeeded in crystallizing (-)-1 and here report its absolute configuration (AC) obtained by X-ray crystallography, thus also defining the AC of (+)-1. An Autodock Vina 1.1 computer modeling study of (+)-1 in the active site of modified RGGT binding GGPP (3DSV) identifies stereochemistry-dependent interactions that could account for the potency of (+)-1 and supports the hypothesis that this type of inhibitor binds at the TAG tunnel, inhibiting the second geranylgeranylation step. We also report a convenient (31)P NMR method to determine enantiomeric excess of 1 and its pyridyl analogue 2, using α- and β-cyclodextrins as chiral solvating agents, and describe the synthesis of a small series of 1 α-X (X = H, F, Cl, Br; 7a-d) analogues to assess the contribution of the α-OH group to activity at enzyme and cellular levels. The IC(50) of 1 was 5-10× lower than 7a-d, and the LED for inhibition of Rab11 prenylation in vitro was 2-8× lower than for 7a-d. However, in a viability reduction assay with J774 cells, 1 and 7b had similar IC(50) values, ~10× lower than those of 7a and 7c-d.
European journal of medicinal chemistry 05/2011; 46(10):4820-6. · 3.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: Progress in the synthesis of novel fluorescent conjugates of N-heterocyclic bisphosphonate drugs and related analogues, together with some recent applications of these compounds as imaging probes, are briefly discussed.
Phosphorus Sulfur and Silicon and the Related Elements 04/2011; 186(4):970-971. · 0.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and have demonstrated clinical utility in the treatment of patients with osteolytic bone metastases. They also exhibit direct antitumor activity in vitro and can reduce skeletal tumor burden and inhibit the formation of bone metastases in vivo. However, whether such effects are caused by a direct action of bisphosphonates on tumor cells or indirectly through inhibition of bone resorption remains unclear. To address this question, we used here a structural analog of the bisphosphonate risedronate, NE-58051, which has a bone mineral affinity similar to that of risedronate, but a 3000-fold lower bone antiresorptive activity. In vitro, risedronate and NE-58051 inhibited proliferation of breast cancer and melanoma cell lines. In vivo, risedronate and NE-58051 did not inhibit the growth of subcutaneous B02 breast tumor xenografts or the formation of B16F10 melanoma lung metastasis. In contrast to NE-58051, risedronate did inhibit B02 breast cancer bone metastasis formation by reducing both bone destruction and skeletal tumor burden, indicating that the antitumor effect of bisphosphonates is achieved mainly through inhibition of osteoclast-mediated bone resorption.
Neoplasia (New York, N.Y.) 07/2010; 12(7):571-8. · 5.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: 3-(3-Pyridyl)-2-hydroxy-2-phosphonopropanoic acid (3-PEHPC, 1) is a phosphonocarboxylate (PC) analogue of 2-(3-pyridyl)-1-hydroxyethylidenebis(phosphonic acid) (risedronic acid, 2), an osteoporosis drug that decreases bone resorption by inhibiting farnesyl pyrophosphate synthase (FPPS) in osteoclasts, preventing protein prenylation. 1 has lower bone affinity than 2 and weakly inhibits Rab geranylgeranyl transferase (RGGT), selectively preventing prenylation of Rab GTPases. We report here the synthesis and biological studies of 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC, 3), the PC analogue of minodronic acid 4. Like 1, 3 selectively inhibited Rab11 vs. Rap 1A prenylation in J774 cells, and decreased cell viability, but was 33-60x more active in these assays. After resolving 3 by chiral HPLC (>98% ee), we found that (+)-3-E1 was much more potent than (-)-3-E2 in an isolated RGGT inhibition assay, approximately 17x more potent (LED 3 microM) than (-)-3-E2 in inhibiting Rab prenylation in J774 cells and >26x more active in the cell viability assay. The enantiomers of 1 exhibited a 4-fold or smaller potency difference in the RGGT and prenylation inhibition assays.
Journal of Medicinal Chemistry 05/2010; 53(9):3454-64. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Abstract Bisphosphonates are effective anti-resorptive agents due to their bone-targeting property and ability to inhibit osteoclasts by preventing protein prenylation. It remains unclear, however, whether any non-osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogs, 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 three-month old mice, fluorescent risedronate analogs were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogs 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 analog 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 analogs 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, BPs may exert direct effects on bone marrow monocytes in vivo.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 10/2009; · 6.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Cancer 04/2009; 125(5):1177-85. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: The local environment plays an important role in osteogenic tissue regeneration. Our previous studies have shown that xenogenic transplantation of human mesenchymal stem cells (hMSCs) alone into immunodeficient mice did not result in long-term bone formation. This study investigates whether bone formation can be prolonged by incorporating human mesenchymal stem cells in mineralized scaffolds together with controlled delivery of a growth factor, BMP2. A composite of coralline hydroxyapatite (CHA) with poly(lactic-co-glycolic acid) (PLGA)-encapsulated rhBMP2 was incorporated with hMSCs in vitro. After 2 weeks in vitro culture the constructs were implanted subcutaneously in CB17 scid beige mice and harvested 10 weeks after implantation. The mineralized tissues were stained by using a fluorescent marker, 5FAM-risedronate, followed by observation with fluorescence microscopy, histology, histomorphometry, mouse-anti-human vimentin immunohistochemistry, and scanning microscopy. The results showed that compared with control materials in which only fibrous tissue formed following implantation of coralline scaffolds, bone-like tissue formed within the CHA composite containing PLGA encapsulated rhBMP2 and hMSCs for up to 10 weeks after implantation. Human cells, identified by the human vimentin-specific monoclonal antibody were seen within the bone-like tissue. In conclusion, incorporation of hMSCs into CHA with controlled delivery of BMP showed prolonged bone formation in immunodeficient mice. Further research is required to optimize the growth factor delivery system and to understand the underlying cellular and molecular mechanisms involved.
Journal of Biomedical Materials Research Part A 04/2009; 92(4):1256-64. · 2.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.
Journal of Biological Chemistry 12/2008; 284(11):6861-8. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bisphosphonates bind avidly to bone mineral and are potent inhibitors of osteoclast-mediated bone destruction. They also exhibit antitumor activity in vitro. Here, we used a mouse model of human breast cancer bone metastasis to examine the effects of risedronate and NE-10790, a phosphonocarboxylate analogue of the bisphosphonate risedronate, on osteolysis and tumor growth. Osteolysis was measured by radiography and histomorphometry. Tumor burden was measured by fluorescence imaging and histomorphometry. NE-10790 had a 70-fold lower bone mineral affinity compared with risedronate. It was 7-fold and 8,800-fold less potent than risedronate at reducing, respectively, breast cancer cell viability in vitro and bone loss in ovariectomized animals. We next showed that risedronate given at a low dosage in animals bearing human B02-GFP breast tumors reduced osteolysis by inhibiting bone resorption, whereas therapy with higher doses also inhibited skeletal tumor burden. Conversely, therapy with NE-10790 substantially reduced skeletal tumor growth at a dosage that did not inhibit osteolysis, a higher dosage being able to also reduce bone destruction. The in vivo antitumor activity of NE-10790 was restricted to bone because it did not inhibit the growth of subcutaneous B02-GFP tumor xenografts nor the formation of B16-F10 melanoma lung metastases. Moreover, NE-10790, in combination with risedronate, reduced both osteolysis and skeletal tumor burden, whereas NE-10790 or risedronate alone only decreased either tumor burden or osteolysis, respectively. In conclusion, our study shows that decreasing the bone mineral affinity of bisphosphonates is an effective therapeutic strategy to inhibit skeletal tumor growth in vivo.
Cancer Research 12/2008; 68(21):8945-53. · 8.65 Impact Factor