Osteogenic PTHs and vascular ossification—Is there a danger for osteoporotics?

Inflammation in vascular (mostly arterial) walls and heart valves triggered by the trans-endothelial influx of LDL particles and the action of subsequently modified (e.g., by oxidation) LDL particles can trigger true bone formation by valvar fibroblasts, by a subpopulation of re-differentiation-competent VSMCs (vascular smooth muscle cells) or by vascular pericytes. Vascular ossification can lead to heart failure and death. Elderly osteoporotic women who need osteogenic drugs to restore their lost skeletal bone are paradoxically prone to vascular ossification-the "calcification paradox." The recent introduction into the clinic of a potently osteogenic parathyroid hormone peptide, Lilly's rhPTH-(1-34)OH (Forteotrade mark), to reverse skeletal bone loss raises the question of whether this and other potently osteogenic PTHs still in clinical trial might also stimulate vascular ossification in such osteoporotic women. Indeed the VSMCs in human and rat atherosclerotic lesions hyperexpress PTHrP and the PTHR1 (or PTH1R) receptor as do maturing osteoblasts. And the evidence indicates that endogenous PTHrP with its NLS (nuclear/nucleolar localization sequence) does stimulate VSMC proliferation (a prime prerequisite for atheroma formation and ossification) via intranuclear targets that inactivate pRb, the inhibitory G1/S checkpoint regulator, by stimulating its hyperphosphorylation. But neither externally added full-length PTHrP nor the NLS-lacking PTHrP-(1-34)OH gets into the VSMC nucleus and instead they inhibit proliferation and calcification by only activating the cell's PTHR1 receptors. No PTH has an NLS and, as expected from the observations on the externally added PTHrPs, hPTH-(1-34)OH inhibits calcification by VSMCs and cannot stimulate vascular ossification in a diabetic mouse model. Encouraging though this may be for osteoporotics with their "calcification paradox," more work is needed to be sure that the skeletally osteogenic PTHs do not promote vascular ossification with its cardiovascular consequences.     

A new dinuclear platinum complex with a nitrogen-containing geminal bisphosphonate as potential anticancer compound specifically targeted to bone tissues

The paper describes the synthesis and characterization of a new platinum dinuclear complex (2) bearing a nitrogen-containing geminal bisphosphonate (NBP, 1), structurally related to the commercial drug risedronate. NBPs themselves have shown in quite a few cases to be endowed with anticancer activity, therefore the new platinum complex has two potential antitumor moieties (the NBP ligand and the platinum residue) and could have high affinity for bone tumors or metastases (due to the presence of NBP). The free bisphosphonate (1) has been crystallized by a sol-gel method and characterized by X-ray diffraction analysis. The platinum complex (2) has been found to have a dinuclear structure with the bisphosphonate bridging two platinum moieties in a W conformation.     

Structurally different bisphosphonates exert opposing effects on alkaline phosphatase and mineralization in marrow osteoprogenitors

Bisphosphonates (BPs) are inhibitors of bone resorption and soft tissue calcification. The biological effects of the BPs in calcium-related disorders are attributed mainly to their incorporation in bone, enabling direct interaction with osteoclasts and/or osteoblasts through a variety of biochemical pathways. Structural differences account for the considerable differences in the pharmacological activity of BPs. We compared the effects of two structurally different compounds, alendronate and 2-(3′-dimethylaminopyrazinio)ethylidene-1,1-bisphosphonic acid betaine (VS-6), in an osteoprogenitor differentiation system. The BPs were examined in a bone marrow stromal-cell culture system, which normally results in osteoprogenitor differentiation. The drugs were present in the cultures from days 2 to 11 of osteogenic stimulation, a period estimated as being comparable to the end of proliferation and the matrix-maturation stages. We found that the two different BPs have opposing effects on specific alkaline phosphatase (ALP) activity, on stromal-cell proliferation, and on cell-mediated mineralization. These BPs differentially interact with cell-associated phosphohydrolysis, particularly at a concentration of 10⁻² of ALP K_m, in which alendronate inhibits whereas VS-6 did not inhibit phosphatase activity. VS-6 treatment resulted in similar and significantly increased mineralization at 10 and 1 μM drug concentrations, respectively. In contrast, mineralization was similar to control, and significantly decreased at 10 and 1 μM drug concentrations, respectively, under alendronate treatment. J. Cell. Biochem. 68:186–194, 1998. © 1998 Wiley-Liss, Inc.     

Bisphosphonates, specific inhibitors of osteoclast function and a class of drugs for osteoporosis therapy

Osteoporosis is a result of the disruption of bone homeostasis that is carried out by bone-forming osteoblasts and bone-degrading osteoclasts. The most common treatment of osteoporosis is N-containing bisphosphonates, a class of non-hydrolyzable pyrophosphate analogs. They have strong affinity to Ca(2+) of hydroxyapatite with high specificity and can only be liberated from the bone in an acidic environment. These properties bestow them unique pharmacokinetic features including specific and strong retention at bone resorption surface, uptaken specifically by osteoclasts, quick excretion of non-retained free bisphosphonates, long half-life, and recyclability. Such properties underlie the drugs' high efficacy, minor side effects, and intermittent dosing regimens. Further studies show that bisphosphonates inhibit farnesyl pyrophosphate synthase, a critical enzyme required for synthesis of isoprenyl and geranylgeranyl, and inhibit prenylation and geranylgeranylation of small G-proteins such as Rac and Rho. This leads to defective actin ring formation at the sealed zone, a subcellular structure essential for bone resorption, and a decrease in bone resorption. Bisphosphonates are also used to treat Paget's disease of bone, osteolytic bone metastases, and hypercalcemia. Moreover, these properties also make N-BPs a good candidate as a bone-seeking agent. Here we update our understanding of this remarkable class of anti-resorption drugs.     

Indirect osteoblast differentiation by liposomal clodronate

Bisphosphonates impair function of osteoclasts and prevent bone resorption, the mechanism of which has been studied extensively. However, the possible effects of bisphosphonates on chondroblast differentiation and calcium deposition by osteoblasts have only been demonstrated recently. Moreover, cells from monocytic lineage are capable of stimulating osteoblast proliferation. Hence, susceptibility of osteoblasts to various factors requires further investigation. A primary culture of bone marrow‐derived stromal cells was treated with liposomal clodronate (0.1, 0.5, or 1.0 mg/ml) or conditioned medium from liposomal clodronate. Liposomal clodronate (0.25 mg) was injected into mouse femur for in vivo experiments. The effects of liposomal clodronate were examined by alkaline phosphatase staining and/or activity assay, and real‐time RT‐PCR was used for studying the effect on osteogenic gene expression. Administration of liposomal clodronate to bone marrow‐derived mesenchymal stromal cell culture enhanced alkaline phosphatase activity and mRNA levels of Runx2 and Dlx5. In addition, conditioned medium from liposomal clodronate also stimulated osteogenic characteristics similar to those of observed in vitro, and the number of exosomes in the conditioned medium was highest when pre‐treated with liposomal clodronate. Western blot analysis revealed the presence of RANK proteins in exosomes collected from conditioned medium of liposomal clodronate. Identical observations were obtained in vivo, as liposomal clodronate‐injected mouse femur showed increased alkaline phosphatase activity and Runx2 and Dlx5 mRNA expressions, even though the numbers of monocytes and macrophages were reduced. In conclusion, osteoblast differentiation was promoted via soluble RANK‐containing exosomes in response to clodronates.     

The effects of bisphosphonates on ectopic soft tissue mineralization caused by mutations in the ABCC6 gene

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable ectopic mineralization disorders. Most cases of PXE and many cases of GACI harbor mutations in the ABCC6 gene. There is no effective treatment for these disorders. We explored the potential efficacy of bisphosphonates to prevent ectopic calcification caused by ABCC6 mutations by feeding Abcc6^−/− mice with diet containing etidronate disodium (ETD) or alendronate sodium trihydrate (AST) in quantities corresponding to 1x, 5x, or 12x of the doses used to treat osteoporosis in humans. The mice were placed on diet at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks by quantitation of the calcium deposits in the dermal sheath of vibrissae, a progressive biomarker of the mineralization, by computerized morphometry of histopathologic sections and by direct chemical assay of calcium. We found that ETD, but not AST, at the 12x dosage, significantly reduced mineralization, suggesting that selected bisphosphonates may be helpful for prevention of mineral deposits in PXE and GACI caused by mutations in the ABCC6 gene, when combined with careful monitoring of efficacy and potential side-effects.     

Dental implications of bisphophonate-related osteonecrosis

doi: 10.1111/j.1741‐2358.2012.00622.x
Dental implications of bisphophonate‐related osteonecrosis Objectives: The aim is to explore the current theories about clinical , pathological and dental management of bisphosphonate related osteonecrosis of the jaws. Also discussed are the actions of bisphosphonates, pathogenesis related to the susceptibility of jaws, the predisposing risk factors for the development of bisphosphonate‐related osteonecrosis of the jaws (BRONJ) and diagnostic criteria based on the literature review. Discussion: Osteoporosis is a disease that generally affects the mineral status of both cortical and trabecular bone in post menopausal women. Bisphosphonates are a group of drugs that preserve and increase bone mass. Bisphosphonate drugs are classified according to use and method of delivery. The bisphosphonates used for the treatment of osteoporosis are taken orally. Little is known about the side effects and dangers of the long‐term use of therapeutic doses of Bisphosphonates. A recent complication reported is osteonecrosis of jaws. The use of IV bisphosphonates for multiple myeloma and metastatic bone diseases suggests that dosage, length of treatment, and route of administration, as well as cofactors such as use of glucocorticoids and immunosuppressive agents, and dental surgery, could all be related to the incidence of BRONJ. This review provides an update on current knowledge about clinical, pathological and management aspects of BRONJ. Conclusions: Little evidence exists to direct the prosthodontic management of patients with a history of bisphosphonate use. Patients with active osteonecrosis related to bisphosphonate use have reduced tissue tolerance to function with removable prostheses and decreased potential for osseointegration of dental implants. Decisions should be based on clinical judgment tempered by the presenting conditions, medical profile, and patient needs. A better understanding would help in a dental setting to prevent any complication and help to improve the prognosis for those being treated for osteoradionecrosis.Until further evidence emerges regarding management of patients with active bisphosphonate‐ related osteonecrosis, conservative prosthodontic treatment is reasonable and prudent.     

The effect of two bisphosphonates on human neutrophil chemiluminescence and myeloperoxidase activity

In order to assess the importance of chlorine in a drug molecule as an influence on myeloperoxidase-mediated inflammatory cell functions, the effect of the chlorinated bisphosphonate, clodronate, on human neutrophil chemiluminescence and myeloperoxidase (MPO) activity was compared to the non-chlorinated structural analogue, etidronate. The results suggested that the presence of chlorine may be important to the enhancement of MPO activity. In addition both drugs manifested low toxicity and both of these observations may have relevance to host defence.     

Inhibition of osteoblast function in vitro by aminobisphosphonates

Bisphosphonates are analogues of pyrophosphate, a key physicochemical inhibitor of mineralisation. We examined the direct actions of bisphosphonates on the function of cultured osteoblasts derived from rat calvariae. Treatment with zoledronate, the most potent bisphosphonate studied, reduced osteoblast number at concentrations ≥100 nM and was strongly toxic at 10 µM, causing a threefold decrease in osteoblast viability after 2 days and a 90% decrease in cell numbers after 14 days. In control osteoblast cultures on plastic, abundant formation of ‘trabecular’ mineralised bone matrix nodules began after 10 days. Continuous exposure to zoledronate inhibited bone mineralisation at concentrations as low as 10 nM. Pamidronate and clodronate exerted similar effects but at higher doses (≥1 and ≥10 µM, respectively). Short‐term or intermittent exposure of osteoblasts to zoledronate and pamidronate (1–10 µM) was sufficient to inhibit bone mineralisation by ≥85%. Zoledronate but not pamidronate or clodronate also strongly inhibited osteoblast alkaline phosphatase activity at concentrations ≥100 nM and soluble collagen production at concentrations ≥1 µM. We additionally studied the effects of zoledronate on osteoblasts cultured on dentine, a bone‐like mineralised substrate, observing similar inhibitory effects, although at concentrations 10–100‐fold higher; this shift presumably reflected adsorption of zoledronate to dentine mineral. Thus, zoledronate blocked bone formation in two ways: first, a relatively non‐toxic, selective inhibition of mineralisation at concentrations in the low nanomolar range and second, a cytotoxic inhibition of osteoblast growth and function at concentrations ≥1 µM. Although no data are available on the bisphosphonate concentrations that osteoblasts could be exposed to in vivo, our results are consistent with earlier observations that bisphosphonates may inhibit bone formation. J. Cell. Biochem. 106: 109–118, 2009. © 2008 Wiley‐Liss, Inc.     

Modifications to the dNTP triphosphate moiety: From mechanistic probes for DNA polymerases to antiviral and anti-cancer drug design

Abnormal replication of DNA is associated with many important human diseases, most notably viral infections and neoplasms. Existing approaches to chemotherapeutics for diseases associated with dysfunctional DNA replication classically involve nucleoside analogues that inhibit polymerase activity due to modification in the nucleobase and/or ribose moieties. These compounds must undergo multiple phosphorylation steps in vivo, converting them into triphosphosphates, in order to inhibit their targeted DNA polymerase. Nucleotide monophosphonates enable bypassing the initial phosphorylation step at the cost of decreased bioavailability. Relatively little attention has been paid to higher nucleotides (corresponding to the natural di- and triphosphate DNA polymerase substrates) as drug platforms due to their expected poor deliverability. However, a better understanding of DNA polymerase mechanism and fidelity dependence on the triphosphate moiety is beginning to emerge, aided by systematic incorporation into this group of substituted methylenebisphosphonate probes. Meanwhile, other bridging, as well as non-bridging, modifications have revealed intriguing possibilities for new drug design. We briefly survey some of this recent work, and argue that the potential of nucleotide-based drugs, and intriguing preliminary progress in this area, warrant acceptance of the challenges that they present with respect to bioavailability and metabolic stability.