Kg Thompson

Molecular mechanisms of action of bisphosphonates: current status.

Purpose: Bisphosphonates are currently the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor-associated osteolysis and hypercalcemia. These compounds have high affinity for calcium ions and therefore target bone mineral, where they are internalized by bone-resorbing osteoclasts and inhibit osteoclast function. Experimental Design: This article reviews the pharmacology of bisphosphonates and the relationship between chemical structure and antiresorptive potency. We also describe new insights into their intracellular molecular mechanisms of action, methods for assessing the effects of bisphosphonates on protein prenylation, and their potential as direct antitumor agents. Results: Nitrogen-containing bisphosphonates act intracellularly by inhibiting farnesyl diphosphate synthase, an enzyme of the mevalonate pathway, thereby preventing prenylation of small GTPase signaling proteins required for normal cellular function. Inhibition of farnesyl diphosphate synthase also seems to account for their antitumor effects observed in vitro and for the activation of γ,δ T cells, a feature of the acute-phase response to bisphosphonate treatment in humans. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that seems to involve primarily the formation of cytotoxic metabolites in osteoclasts. Conclusions: Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect osteoclasts in vivo but could also have direct effects on other cell types, such as tumor cells. After >30 years of clinical use, their molecular mechanisms of action on osteoclasts are finally becoming clear but their exact antitumor properties remain to be clarified.

Statins Prevent Bisphosphonate‐Induced γ,δ‐T‐Cell Proliferation and Activation In Vitro

The acute phase response is the major adverse effect of intravenously administered N‐BPs. In this study we show that N‐BPs cause γ,δ‐T‐cell activation and proliferation in vitro by an indirect mechanism through inhibition of FPP synthase, an effect that can be overcome by inhibiting HMG‐CoA reductase with a statin. These studies clarify the probable initial cause of the acute phase response to N‐BP drugs and suggest a possible way of preventing this phenomenon.

Peripheral blood monocytes are responsible for γδ T cell activation induced by zoledronic acid through accumulation of IPP/DMAPP

Nitrogen‐containing bisphosphonates indirectly activate Vγ9Vδ2 T cells through inhibition of farnesyl pyrophosphate synthase and intracellular accumulation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but the cells responsible for Vγ9Vδ2 T cell activation through IPP/DMAPP accumulation are unknown. Treatment of human peripheral blood mononuclear cells (PBMCs) with a pharmacologically relevant concentration of zoledronic acid induced accumulation of IPP/DMAPP selectively in monocytes, which correlated with efficient drug uptake by these cells. Furthermore, zoledronic acid‐pulsed monocytes triggered activation of γδ T cells in a cell contact‐dependent manner. These observations identify monocytes as the cell type directly affected by bisphosphonates responsible for Vγ9Vδ2 T cell activation.

Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis.

Bisphosphonates such as alendronate and zoledronate are blockbuster drugs used to inhibit osteoclast-mediated bone resorption. Although the molecular mechanisms by which bisphosphonates affect osteoclasts are now evident, the exact route by which they are internalized by cells is not known. To clarify this, we synthesized a novel, fluorescently labeled analog of alendronate (AF-ALN). AF-ALN was rapidly internalized into intracellular vesicles in J774 macrophages and rabbit osteoclasts; uptake of AF-ALN or [14C]zoledronate was stimulated by the presence of Ca2+ and Sr2+ and could be inhibited by addition of EGTA or clodronate, both of which chelate calcium ions. Both EGTA and clodronate also prevented the bisphosphonate-induced inhibition of Rap1A prenylation, an effect that was reversed by addition of Ca2+. In J774 cells and osteoclasts, vesicular AF-ALN colocalized with dextran (but not wheat germ agglutinin or transferrin), and uptake of AF-ALN or [14C]zoledronate was inhibited by dansylcadaverine, indicating that fluid-phase endocytosis is involved in the initial internalization of bisphosphonate into vesicles. Endosomal acidification then seems to be absolutely required for exit of bisphosphonate from vesicles and entry into the cytosol, because monensin and bafilomycin A1, both inhibitors of endosomal acidification, did not inhibit vesicular uptake of AF-ALN or internalization of [14C]zoledronate but prevented the inhibitory effect of alendronate or zoledronate on Rap1A prenylation. Taken together, these results demonstrate that cellular uptake of bisphosphonate drugs requires fluid-phase endocytosis and is enhanced by Ca2+ ions, whereas transfer from endocytic vesicles into the cytosol requires endosomal acidification.

Dietary conjugated linoleic acids promote fatty streak formation in the C57BL/6 mouse atherosclerosis model

Conjugated linoleic acids (CLA) are positional isomers of linoleic acid which have been suggested by some to possess antiatherosclerotic properties. To test this hypothesis, three groups of twenty C57BL/6 mice were fed on atherogenic diets containing: 5 g CLA/kg, 2.5 g CLA + 2.5 g linoleic acid/kg or 5 g linoleic acid/kg. All diets were fed for 15 weeks and contained (g/kg): triacylglycerol 145, free fatty acids 5, cholesterol 10 and cholic acid 5. At the completion of the experimental period, when data from both groups fed on CLA were combined, dietary CLA did not produce significant differences in body weight, serum total cholesterol concentration or serum HDL-cholesterol concentration. However, mice receiving CLA developed a significantly higher serum HDL-cholesterol: total cholesterol ratio and a significantly lower serum triacylglycerol concentration than controls. Despite causing a serum lipoprotein profile considered to be less atherogenic, the addition of CLA to the atherogenic diet increased the development of aortic fatty streaks. Considering the increased atherogenesis associated with dietary CLA in the present study, and the failure to demonstrate a significant beneficial effect of CLA in other animal studies, there is currently no conclusive evidence to support the hypothesis that CLA protect against atherogenesis.

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.

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 Pagets 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.

Daily supplementation with aged garlic extract, but not raw garlic, protects low density lipoprotein against in vitro oxidation

The oxidation of low density lipoprotein (LDL) is believed to be an important process in the development and progression of atherosclerosis. In this study, human subjects were supplemented daily with one of: 6 g raw garlic; 2.4 g aged garlic extract (AGE); or 0.8 g DL-alpha-tocopherol acetate for 7 days to determine the effect on the susceptibility of LDL particles to Cu2+-mediated oxidation. LDL isolated from subjects given either alpha-tocopherol or AGE, but not raw garlic, was significantly more resistant to oxidation than LDL isolated from subjects receiving no supplements. These results suggest that if antioxidants are proven to be antiatherogenic, AGE may be useful in preventing atherosclerotic disease.

IL-23 Inhibits Osteoclastogenesis Indirectly through Lymphocytes and Is Required for the Maintenance of Bone Mass in Mice

IL-23 stimulates the differentiation and function of the Th17 subset of CD4+ T cells and plays a critical role in chronic inflammation. The IL-23 receptor-encoding gene is also an inflammatory disease susceptibility gene. IL-23 shares a common subunit with IL-12, a T cell-dependent osteoclast formation inhibitor, and we found that IL-23 also dose-dependently inhibited osteoclastogenesis in a CD4+ T lymphocyte-dependent manner. When sufficiently enriched, γδ T cells also mediated IL-23 inhibition. Like IL-12, IL-23 acted synergistically with IL-18 to block osteoclastogenesis but, unlike IL-12, IL-23 action depended on T cell GM-CSF production. IL-23 did not mediate IL-12 action although IL-12 induced its expression. Male mice lacking IL-23 (IL-23p19−/−) had ∼30% lower bone mineral density and tibial trabecular bone mass (bone volume (BV)/total volume (TV)) than wild-type littermates at 12 wk and 40% lower BV/TV at 26 wk of age; male heterozygotes also had lower bone mass. Female IL-23p19−/− mice also had reduced BV/TV. IL-23p19−/− mice had no detectable osteoclast defect in trabecular bone but IL-23p19−/− had thinner growth plate hypertrophic and primary spongiosa zones (and, in females, less cartilage remnants) compared with wild type. This suggests increased osteoclast action at and below the growth plate, leading to reduced amounts of mature trabecular bone. Thus, IL-23 inhibits osteoclast formation indirectly via T cells in vitro. Under nonpathological conditions (unlike inflammatory conditions), IL-23 favors higher bone mass in long bones by limiting resorption of immature bone forming below the growth plate.

Vitamin D Metabolism and Rickets in Domestic Animals A Review

Rickets and osteomalacia are increasing in prevalence in people because of cultural practices, breast-feeding, decreased sun exposure, and increased sunscreen usage. Several hereditary forms of rickets owing to either renal phosphate wasting or defects in vitamin D metabolism are also reported in people. Rickets is well recognized in domestic animals, but published reports are not always supported by microscopic findings, and diagnoses based on clinical signs and radiology are unreliable. Most cases in domestic animals are caused by dietary deficiency of either vitamin D or phosphorus, but occasional inherited forms are reported in pigs, sheep, cats, and dogs. There is variation between species in susceptibility to dietary vitamin D and phosphorus deficiency and in the ability to manufacture vitamin D in their skin. A number of mouse models have been discovered or created to study human skeletal diseases and skeletal homeostasis. With the discovery that vitamin D is involved in not only calcium and phosphorus homeostasis but also in the immune system and cancer, there is great potential for new and existing animal models to generate valuable information about vitamin D and its many functions. This review presents an overview of vitamin D metabolism and rickets in domestic and laboratory animals and makes comparisons where appropriate with the disease in humans.