Real-Time Impedance-Based Monitoring of the Growth and Inhibition of Osteomyelitis Biofilm Pathogen Staphylococcus aureus Treated with Novel Bisphosphonate-Fluoroquinolone Antimicrobial Conjugates.

Osteomyelitis is a limb- and life-threatening orthopedic infection predominantly caused by Staphylococcus aureus biofilms. Bone infections are extremely challenging to treat clinically. Therefore, we have been designing, synthesizing, and testing novel antibiotic conjugates to target bone infections. This class of conjugates comprises bone-binding bisphosphonates as biochemical vectors for the delivery of antibiotic agents to bone minerals (hydroxyapatite). In the present study, we utilized a real-time impedance-based assay to study the growth of Staphylococcus aureus biofilms over time and to test the antimicrobial efficacy of our novel conjugates on the inhibition of biofilm growth in the presence and absence of hydroxyapatite. We tested early and newer generation quinolone antibiotics (ciprofloxacin, moxifloxacin, sitafloxacin, and nemonoxacin) and several bisphosphonate-conjugated versions of these antibiotics (bisphosphonate-carbamate-sitafloxacin (BCS), bisphosphonate-carbamate-nemonoxacin (BCN), etidronate-carbamate-ciprofloxacin (ECC), and etidronate-carbamate-moxifloxacin (ECX)) and found that they were able to inhibit Staphylococcus aureus biofilms in a dose-dependent manner. Among the conjugates, the greatest antimicrobial efficacy was observed for BCN with an MIC of 1.48 µg/mL. The conjugates demonstrated varying antimicrobial activity depending on the specific antibiotic used for conjugation, the type of bisphosphonate moiety, the chemical conjugation scheme, and the presence or absence of hydroxyapatite. The conjugates designed and tested in this study retained the bone-binding properties of the parent bisphosphonate moiety as confirmed using high-performance liquid chromatography. They also retained the antimicrobial activity of the parent antibiotic in the presence or absence of hydroxyapatite, albeit at lower levels due to the nature of their chemical modification. These findings will aid in the optimization and testing of this novel class of drugs for future applications to pharmacotherapy in osteomyelitis.

Pharmacology of bisphosphonates.

The biological effects of the bisphosphonates (BPs) as inhibitors of calcification and bone resorption were first described in the late 1960s. In the 50 years that have elapsed since then, the BPs have become the leading drugs for the treatment of skeletal disorders characterized by increased bone resorption, including Paget's disease of bone, bone metastases, multiple myeloma, osteoporosis and several childhood inherited disorders. The discovery and development of the BPs as a major class of drugs for the treatment of bone diseases is a paradigm for the successful journey from "bench to bedside and back again". Several of the leading BPs achieved "blockbuster" status as branded drugs. However, these BPs have now come to the end of their patent life, making them highly affordable. The opportunity for new clinical applications for BPs also exists in other areas of medicine such as ageing, cardiovascular disease and radiation protection. Their use as inexpensive generic medicines is therefore likely to continue for many years to come. Fifty years of research into the pharmacology of bisphosphonates have led to a fairly good understanding about how these drugs work and how they can be used safely in patients with metabolic bone diseases. However, while we seemingly know much about these drugs, a number of key aspects related to BP distribution and action remain incompletely understood. This review summarizes the existing knowledge of the (pre)clinical and translational pharmacology of BPs, and highlights areas in which understanding is lacking.

Zoledronate Attenuates Accumulation of DNA Damage in Mesenchymal Stem Cells and Protects Their Function.

Mesenchymal stem cells (MSCs) undergo a decline in function following ex vivo expansion and exposure to irradiation. This has been associated with accumulation of DNA damage and has important implications for tissue engineering approaches or in patients receiving radiotherapy. Therefore, interventions, which limit accumulation of DNA damage in MSC, are of clinical significance. We were intrigued by findings showing that zoledronate (ZOL), an anti-resorptive nitrogen containing bisphosphonate, significantly extended survival in patients affected by osteoporosis. The effect was too large to be simply due to the prevention of fractures. Moreover, in combination with statins, it extended the lifespan in a mouse model of Hutchinson Gilford Progeria Syndrome. Therefore, we asked whether ZOL was able to extend the lifespan of human MSC and whether this was due to reduced accumulation of DNA damage, one of the important mechanisms of aging. Here, we show that this was the case both following expansion and irradiation, preserving their ability to proliferate and differentiate in vitro. In addition, administration of ZOL before irradiation protected the survival of mesenchymal progenitors in mice. Through mechanistic studies, we were able to show that inhibition of mTOR signaling, a pathway involved in longevity and cancer, was responsible for these effects. Our data open up new opportunities to protect MSC from the side effects of radiotherapy in cancer patients and during ex vivo expansion for regenerative medicine approaches. Given that ZOL is already in clinical use with a good safety profile, these opportunities can be readily translated for patient benefit.

Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile Imaging Toolkit for Applications in Bone Biology and Biomedicine.

A bone imaging toolkit of 21 fluorescent probes with variable spectroscopic properties, bone mineral binding affinities, and antiprenylation activities has been created, including a novel linking strategy. The linking chemistry allows attachment of a diverse selection of dyes fluorescent in the visible to near-infrared range to any of the three clinically important heterocyclic bisphosphonate bone drugs (risedronate, zoledronate, and minodronate or their analogues). The resultant suite of conjugates offers multiple options to "mix and match" parent drug structure, fluorescence emission wavelength, relative bone affinity, and presence or absence of antiprenylation activity, for bone-related imaging applications.

In vitro and in vivo effects of zoledronate on senescence and senescence-associated secretory phenotype markers.

In addition to reducing fracture risk, zoledronate has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronate could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronate killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronate or vehicle for 8 weeks, zoledronate significantly reduced circulating SASP factors, including CCL7, IL-1ß, TNFRSF1A, and TGFß1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronate demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronate, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronate significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronate has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo . These data point to the need for additional studies testing zoledronate and/or other bisphosphonate derivatives for senotherapeutic efficacy.

Molecular characterization of a novel geranylgeranyl pyrophosphate synthase from Plasmodium parasites.

We present here a study of a eukaryotic trans-prenylsynthase from the malaria pathogen Plasmodium vivax. Based on the results of biochemical assays and contrary to previous indications, this enzyme catalyzes the production of geranylgeranyl pyrophosphate (GGPP) rather than farnesyl pyrophosphate (FPP). Structural analysis shows that the product length is constrained by a hydrophobic cavity formed primarily by a set of residues from the same subunit as the product as well as at least one other from the dimeric partner. Furthermore, Plasmodium GGPP synthase (GGPPS) can bind nitrogen-containing bisphosphonates (N-BPs) strongly with the energetically favorable cooperation of three Mg(2+), resulting in inhibition by this class of compounds at IC(50) concentrations below 100 nM. In contrast, human and yeast GGPPSs do not accommodate a third magnesium atom in the same manner, resulting in their insusceptibility to N-BPs. This differentiation is in part attributable to a deviation in a conserved motif known as the second aspartate-rich motif: whereas the aspartates at the start and end of the five-residue motif in FFPP synthases and P. vivax GGPPSs both participate in the coordination of the third Mg(2+), an asparagine is featured as the last residue in human and yeast GGPPSs, resulting in a different manner of interaction with nitrogen-containing ligands.

Reduced colon cancer incidence and mortality in postmenopausal women treated with an oral bisphosphonate--Danish National Register Based Cohort Study.

UNLABELLED: In this Danish national register-based cohort study, we examined the effects of alendronate on the development of colon cancers and survival. The incidence of colon cancer and mortality rate, once colon cancer had been diagnosed, were lower in patients treated with alendronate, posing the question whether alendronate acts as chemopreventive. INTRODUCTION: When bisphosphonates are given by mouth, around 99% remains non-absorbed in the intestine. Based on their biochemical actions, we predicted that oral bisphosphonates might prevent colon cancers. METHODS: This is a Danish national register-based cohort study. We identified 30,606 women aged 50+, mean age 71.9 years, who had not previously taken treatments for osteoporosis, who began to take alendronate in 1996-2005, and assigned 124,424 individually age- and gender-matched control subjects. The main outcome measure was colorectal cancers incidence and post-diagnosis survival in patients taking oral alendronate for osteoporosis. RESULTS: Cox proportional hazards analysis of death due to colon cancer showed lower risk in alendronate users, crude hazard ratio (HR) 0.69 (95% CI 0.59-0.81) with an adjusted HR of 0.62 (95% CI 0.52-0.72). The reduction in risk comprised both a lower incidence of colon cancer-adjusted HR 0.69 (95% CI 0.60-0.79) and a lower mortality once colon cancer had been diagnosed, adjusted HR 0.82 (95% CI 0.70-0.97). Weekly alendronate was associated with a greater risk reduction than daily alendronate. The main findings were unaffected by excluding patients from the analysis who had pulmonary disease, a major co-morbid condition in users of alendronate and an important cause of death. CONCLUSIONS: The risk of overall deaths from cancer and in particular death caused by colon cancer was significantly and substantially decreased (40%) in patients treated with alendronate, with survival curves deviating progressively after 2 years. Also, the incidence of colon cancer was lower in those patients.

Bisphosphonate binding affinity affects drug distribution in both intracortical and trabecular bone of rabbits.

Differences in the binding affinities of bisphosphonates for bone mineral have been proposed to determine their localizations and duration of action within bone. The main objective of this study was to test the hypothesis that mineral binding affinity affects bisphosphonate distribution at the basic multicellular unit (BMU) level within both cortical and cancellous bone. To accomplish this objective, skeletally mature female rabbits (n = 8) were injected simultaneously with both low- and high-affinity bisphosphonate analogs bound to different fluorophores. Skeletal distribution was assessed in the rib, tibia, and vertebra using confocal microscopy. The staining intensity ratio between osteocytes contained within the cement line of newly formed rib osteons or within the reversal line of hemiosteons in vertebral trabeculae compared to osteocytes outside the cement/reversal line was greater for the high-affinity compared to the low-affinity compound. This indicates that the low-affinity compound distributes more equally across the cement/reversal line compared to a high-affinity compound, which concentrates mostly near surfaces. These data, from an animal model that undergoes intracortical remodeling similar to humans, demonstrate that the affinity of bisphosphonates for the bone determines the reach of the drugs in both cortical and cancellous bone.

Bisphosphonates: The role of chemistry in understanding their biological actions and structure-activity relationships, and new directions for their therapeutic use.

The bisphosphonates ((HO)2P(O)CR1R2P(O)(OH)2, BPs) were first shown to inhibit bone resorption in the 1960s, but it was not until 30 years later that a detailed molecular understanding of the relationship between their varied chemical structures and biological activity was elucidated. In the 1990s and 2000s, several potent bisphosphonates containing nitrogen in their R2 side chains (N-BPs) were approved for clinical use including alendronate, risedronate, ibandronate, and zoledronate. These are now mostly generic drugs and remain the leading therapies for several major bone-related diseases, including osteoporosis and skeletal-related events associated with bone metastases. The early development of chemistry in this area was largely empirical and only a few common structural features related to strong binding to calcium phosphate were clear. Attempts to further develop structure-activity relationships to explain more dramatic pharmacological differences in vivo at first appeared inconclusive, and evidence for mechanisms underlying cellular effects on osteoclasts and macrophages only emerged after many years of research. The breakthrough came when the intracellular actions on the osteoclast were first shown for the simpler bisphosphonates, via the in vivo formation of P-C-P derivatives of ATP. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates in the 1980s and 1990s led to the key discovery that the antiresorptive effects of these more complex analogs on osteoclasts result mostly from their potency as inhibitors of the enzyme farnesyl diphosphate synthase (FDPS/FPPS). This key branch-point enzyme in the mevalonate pathway of cholesterol biosynthesis is important for the generation of isoprenoid lipids that are utilized for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Since then, it has become even more clear that the overall pharmacological effects of individual bisphosphonates on bone depend upon two key properties: the affinity for bone mineral and inhibitory effects on biochemical targets within bone cells, in particular FDPS. Detailed enzyme-ligand crystal structure analysis began in the early 2000s and advances in our understanding of the structure-activity relationships, based on interactions with this target within the mevalonate pathway and related enzymes in osteoclasts and other cells have continued to be the focus of research efforts to this day. In addition, while many members of the bisphosphonate drug class share common properties, now it is more clear that chemical modifications to create variations in these properties may allow customization of BPs for different uses. Thus, as the appreciation for new potential opportunities with this drug class grows, new chemistry to allow ready access to an ever-widening variety of bisphosphonates continues to be developed. Potential new uses of the calcium phosphate binding mechanism of bisphosphonates for the targeting of other drugs to the skeleton, and effects discovered on other cellular targets, even at non-skeletal sites, continue to intrigue scientists in this research field.

Bisphosphonates in dentistry: Historical perspectives, adverse effects, and novel applications.

Studies of the potential role of bisphosphonates in dentistry date back to physical chemical research in the 1960s, and the genesis of the discovery of bisphosphonate pharmacology in part can be linked to some of this work. Since that time, parallel research on the effects of bisphosphonates on bone metabolism continued, while efforts in the dental field included studies of bisphosphonate effects on dental calculus, caries, and alveolar bone loss. While some utility of this drug class in the dental field was identified, leading to their experimental use in various dentrifice formulations and in some dental applications clinically, adverse effects of bisphosphonates in the jaws have also received attention. Most recently, certain bisphosphonates, particularly those with strong bone targeting properties, but limited biochemical effects (low potency bisphosphonates), are being studied as a local remedy for the concerns of adverse effects associated with other more potent members of this drug class. Additionally, low potency bisphosphonate analogs are under study as vectors to target active drugs to the mineral surfaces of the jawbones. These latter efforts have been devised for the prevention and treatment of oral problems, such as infections associated with oral surgery and implants. Advances in the utility and mechanistic understanding of the bisphosphonate class may enable additional oral therapeutic options for the management of multiple aspects of dental health.

Bisphosphonates for delivering drugs to bone.

Advances in the design of potential bone-selective drugs for the treatment of various bone-related diseases are creating exciting new directions for multiple unmet medical needs. For bone-related cancers, off-target/non-bone toxicities with current drugs represent a significant barrier to the quality of life of affected patients. For bone infections and osteomyelitis, bacterial biofilms on infected bones limit the efficacy of antibiotics because it is hard to access the bacteria with current approaches. Promising new experimental approaches to therapy, based on bone-targeting of drugs, have been used in animal models of these conditions and demonstrate improved efficacy and safety. The success of these drug-design strategies bodes well for the development of therapies with improved efficacy for the treatment of diseases affecting the skeleton. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

2009 Santa Fe Bone symposium.

Osteoporosis is a common skeletal disease with serious clinical consequences because of fractures. Despite the availability of clinical tools to diagnose osteoporosis and assess fracture risk, and drugs proven to reduce fracture risk, it remains a disease that is underdiagnosed and undertreated. When treatment is started, it is commonly not taken correctly or long enough to be effective. Recent advances in understanding of the regulators and mediators of bone remodeling have led to new therapeutic targets and the development of drugs that may offer advantages over current agents in reducing the burden of osteoporotic fractures. Many genetic factors that play a role in the pathogenesis of osteoporosis and metabolic bone disease have now been identified. At the 2009 Santa Fe Bone Symposium, held in Santa Fe, New Mexico, USA, the links between advances in genetics, basic bone science, recent clinical trials, and new and emerging therapeutic agents were presented and explored. Socioeconomic challenges and opportunities in the care of osteoporosis were discussed. This is a collection of medical essays based on key presentations at the 2009 Santa Fe Bone Symposium.

History of alendronate.

Alendronate was synthesized in 1970s in a search for inhibitors of calcification. Istituto Gentili investigators identified it as a potent inhibitor of bone resorption and obtained a patent covering its use in the treatment of osteoporosis and other disorders of excessive bone resorption in the 1980s. Merck licensed alendronate in 1988 and its pharmaceutical chemists reformulated it as a sodium salt with good solubility in a tablet that reduced its potential for esophageal irritation. Clinical trials proved that it reduced bone turnover, increased BMD and reduced the risk of vertebral fractures in postmenopausal osteoporotic women. Merck sponsored a large clinical trials that won FDA approval for treatment of osteoporosis in postmenopausal women and showed that it reduced the risk of spine and hip fractures. Its approval in the US in 1995 spurred sales of bone densitometers and BMD testing to screen for low bone mineral density and identify osteoporosis. Bone mass measurement was supported by medical society guidelines and reimbursement by Medicare and other insurers in the USA. A 70 mg weekly instead of 10 mg daily dose of alendronate produced the same effect on BMD and biochemical markers of bone remodelling with greater convenience and reduced potential for upper GI adverse events. Consequently, by 2006, about 30 million prescriptions for alendronate were written annually in the U.S. for about 15% of postmenopausal women in the U.S. Thereafter, publicity about rare but concerning atypical femoral fractures (AFF) and osteonecrosis of the jaw (ONJ) along with the expiry of Merck's patent (in 2008) and cessation of their promotion of alendronate, and a decline in use of densitometry led to a steady slide in its use even among patients for whom the benefits of alendronate far outweigh its potential risks. Nevertheless, in 25 years since its regulatory approval, alendronate has undoubtedly prevented millions of fractures world-wide.

Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia.

ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.

Zoledronate.

Zoledronate is the most potent and most long-acting bisphosphonate in clinical use, and is administered as an intravenous infusion. Its major uses are in osteoporosis, Paget's disease, and in myeloma and cancers to reduce adverse skeletal related events (SREs). In benign disease, it is a first- or second-line treatment for osteoporosis, achieving anti-fracture efficacy comparable to that of the RANKL blocker, denosumab, over 3 years, and it reduces fracture risk in osteopenic older women. It is the preferred treatment for Paget's disease, achieving higher rates of remissions which are much more prolonged than with any other agent. Some trials have suggested that it reduces mortality, cardiovascular disease and cancer, but these findings are not consistent across all studies. It is nephrotoxic, so should not be given to those with significant renal impairment, and, like other potent anti-resorptive agents, can cause hypocalcemia in patients with severe vitamin D deficiency, which should be corrected before administration. Its most common adverse effect is the acute phase response, seen in 30-40% of patients after their first dose, and much less commonly subsequently. Clinical trials in osteoporosis have not demonstrated increases in osteonecrosis of the jaw or in atypical femoral fractures. Observational databases are currently inadequate to determine whether these problems are increased in zoledronate users. Now available as a generic, zoledronate is a cost-effective agent for fracture prevention and for management of Paget's disease, but wider provision of infusion facilities is important to increase patient access. There is a need to further explore its potential for reducing cancer, cardiovascular disease and mortality, since these effects could be substantially more important than its skeletal actions.

Diagnosis and Management of Paget's Disease of Bone in Adults: A Clinical Guideline.

An evidence-based clinical guideline for the diagnosis and management of Paget's disease of bone (PDB) was developed using GRADE methodology, by a Guideline Development Group (GDG) led by the Paget's Association (UK). A systematic review of diagnostic tests and pharmacological and nonpharmacological treatment options was conducted that sought to address several key questions of clinical relevance. Twelve recommendations and five conditional recommendations were made, but there was insufficient evidence to address eight of the questions posed. The following recommendations were identified as the most important: 1) Radionuclide bone scans, in addition to targeted radiographs, are recommended as a means of fully and accurately defining the extent of metabolically active disease in patients with PDB. 2) Serum total alkaline phosphatase (ALP) is recommended as a first-line biochemical screening test in combination with liver function tests in screening for the presence of metabolically active PDB. 3) Bisphosphonates are recommended for the treatment of bone pain associated with PDB. Zoledronic acid is recommended as the bisphosphonate most likely to give a favorable pain response. 4) Treatment aimed at improving symptoms is recommended over a treat-to-target strategy aimed at normalizing total ALP in PDB. 5) Total hip or knee replacements are recommended for patients with PDB who develop osteoarthritis in whom medical treatment is inadequate. There is insufficient information to recommend one type of surgical approach over another. The guideline was endorsed by the European Calcified Tissues Society, the International Osteoporosis Foundation, the American Society of Bone and Mineral Research, the Bone Research Society (UK), and the British Geriatric Society. The GDG noted that there had been a lack of research on patient-focused clinical outcomes in PDB and identified several areas where further research was needed. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Zoledronate in the prevention of Paget's (ZiPP): protocol for a randomised trial of genetic testing and targeted zoledronic acid therapy to prevent SQSTM1-mediated Paget's disease of bone.

INTRODUCTION: Paget's disease of bone (PDB) is characterised by increased and disorganised bone remodelling affecting one or more skeletal sites. Complications include bone pain, deformity, deafness and pathological fractures. Mutations in sequestosome-1 (SQSTM1) are strongly associated with the development of PDB. Bisphosphonate therapy can improve bone pain in PDB, but there is no evidence that treatment alters the natural history of PDB or prevents complications. The Zoledronate in the Prevention of Paget's disease trial (ZiPP) will determine if prophylactic therapy with the bisphosphonate zoledronic acid (ZA) can delay or prevent the development of PDB in people who carry SQSTM1 mutations. METHODS AND ANALYSIS: People with a family history of PDB aged >30 years who test positive for SQSTM1 mutations are eligible to take part. At the baseline visit, participants will be screened for the presence of bone lesions by radionuclide bone scan. Biochemical markers of bone turnover will be measured and questionnaires completed to assess pain, health-related quality of life (HRQoL), anxiety and depression. Participants will be randomised to receive a single intravenous infusion of 5 mg ZA or placebo and followed up annually for between 4 and 8 years at which point baseline assessments will be repeated. The primary endpoint will be new bone lesions assessed by radionuclide bone scan. Secondary endpoints will include changes in biochemical markers of bone turnover, pain, HRQoL, anxiety, depression and PDB-related skeletal events. ETHICS AND DISSEMINATION: The study was approved by the Fife and Forth Valley Research Ethics Committee on 22 December 2008 (08/S0501/84). Following completion of the trial, a manuscript will be submitted to a peer-reviewed journal. The results of this trial will inform clinical practice by determining if early intervention with ZA in presymptomatic individuals with SQSTM1 mutations can prevent or slow the development of bone lesions with an adverse event profile that is acceptable. TRIAL REGISTRATION NUMBER: ISRCTN11616770.

Structure-activity relationships among the nitrogen containing bisphosphonates in clinical use and other analogues: time-dependent inhibition of human farnesyl pyrophosphate synthase.

The nitrogen-containing bisphosphonates (N-BPs) are the main drugs currently used to treat diseases characterized by excessive bone resorption. The major molecular target of N-BPs is farnesylpyrophosphate synthase. N-BPs inhibit the enzyme by a mechanism that involves time dependent isomerization of the enzyme. We investigated features of N-BPs that confer maximal slow and tight-binding by quantifying the initial and final K(i)s and calculating the isomerization constant K(isom) for many N-BPs. Disruption of the phosphonate-carbon-phosphonate backbone resulted in loss of potency and reduced K(isom). The lack of a hydroxyl group on the geminal carbon also reduced K(isom). The position of the nitrogen in the side chain was crucial to both K(i) and K(isom). A correlation of K(isom) and also final K(i) with previously published in vivo potency reveals that the isomerization constant ( R = -0.77, p < 0.0001) and the final inhibition of FPPS by N-BPs ( R = 0.74, p < 0.0001) are closely linked to antiresorptive efficacy.

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.

Selective targeting of death receptor 5 circumvents resistance of MG-63 osteosarcoma cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a tumor necrosis factor superfamily member, targets death receptors and selectively kills malignant cells while leaving normal cells unaffected. However, unlike most cancers, many osteosarcomas are resistant to TRAIL. To investigate this resistance, we characterized the response of MG-63 osteosarcoma cells and hPOB-tert osteoblast-like cells to TRAIL and agonist antibodies to death receptor 4 (DR4) and death receptor 5 (DR5). We found that MG-63 osteosarcoma cells and hPOB-tert osteoblast-like cells show no or very little response to TRAIL or a DR4 agonist, but MG-63 cells undergo apoptosis in response to a DR5 agonist. Analysis of TRAIL receptor expression showed that normal osteoblastic and osteosarcoma cells express a variety of TRAIL receptors but this does not correlate to TRAIL responsiveness. Production of the soluble decoy receptor osteoprotegerin also could not explain TRAIL resistance. We show that TRAIL activates the canonical caspase-dependent pathway, whereas treatment with cycloheximide increases the sensitivity of MG-63 cells to TRAIL and anti-DR5 and can also sensitize hPOB-tert cells to both agents. Proapoptotic and antiapoptotic protein expression does not significantly differ between MG-63 and hPOB-tert cells or change following treatment with TRAIL or anti-DR5. However, sequencing the death domain of DR4 in several osteoblast-like cells showed that MG-63 osteosarcoma cells are heterozygous for a dominant-negative mutation, which can confer TRAIL resistance. These results suggest that although the dominant-negative form of the receptor may block TRAIL-induced death, an agonist antibody to the active death receptor can override cellular defenses and thus provide a tailored approach to treat resistant osteosarcomas.