Nitrogen-containing Bisphosphonates Research Articles

Fragment-Based Discovery of Non-bisphosphonate Binders of Trypanosoma brucei Farnesyl Pyrophosphate Synthase.

Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). Nitrogen-containing bisphosphonates, a current treatment for bone diseases, have been shown to block the growth of the T. brucei parasites by inhibiting farnesyl pyrophosphate synthase (FPPS); however, due to their poor pharmacokinetic properties, they are not well suited for antiparasitic therapy. Recently, an allosteric binding pocket was discovered on human FPPS, but its existence on trypanosomal FPPS was unclear. We applied NMR and X-ray fragment screening to T. brucei FPPS and report herein on four fragments bound to this previously unknown allosteric site. Surprisingly, non-bisphosphonate active-site binders were also identified. Moreover, fragment screening revealed a number of additional binding sites. In an early structure-activity relationship (SAR) study, an analogue of an active-site binder was unexpectedly shown to bind to the allosteric site. Overlaying identified fragment binders of a parallel T. cruzi FPPS fragment screen with the T. brucei FPPS structure, and medicinal chemistry optimisation based on two binders revealed another example of fragment "pocket hopping". The discovery of binders with new chemotypes sets the framework for developing advanced compounds with pharmacokinetic properties suitable for the treatment of parasitic infections by inhibition of FPPS in T. brucei parasites.

Zoledronate decreases CTLA-4 in vivo and in vitro independently of its action on bone resorption.

Acute phase response (APR) following intravenous zoledronate (ZOL) administration is related to activation and increased proliferation of γδ T cells, attributed to the molecular mechanism of action of nitrogen-containing bisphosphonates (N-BPs). ZOL, however, has also been reported to inhibit the proliferation of regulatory T cells in vitro and to reduce the expression of Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), a negative regulator of T cell activation that is increased in patients with autoimmune diseases. There are, however, no data on the relationship between ZOL treatment and soluble(s)CTLA-4 either in vivo in relevant patient populations or in vitro with the use of assays relevant to the mechanism of action of N-BPs. The objectives of the present study were firstly, to characterize the ZOL-induced APR in patients with inflammatory rheumatic diseases (IRDs) and its relationship with changes in circulating sCTLA-4 and secondly, to investigate the effects of ZOL on CTLA-4 production and expression by peripheral blood mononuclear cells (PBMCs). We studied 10 postmenopausal women with IRDs treated with intravenous ZOL 5mg. Five women experienced APR (APR+) associated with significant decreases in blood lymphocytes and increases in granulocytes and serum CRP. Serum sCTLA-4 values were increased in all patients before ZOL administration and decreased significantly 72h after the ZOL infusion (from 30.0±2.9 to 6.3±1.8ng/ml; p<0.001) with no differences between APR+ and APR- patients. Consistent with the results of the in vivo study, ZOL (1μM) decreased the production of sCTLA-4 by 87% and 57% after 3 and 5days in cultures of peripheral blood mononuclear cells (PBMCs) in vitro, respectively, and inhibited the expression of both cytoplasmic and membrane-bound CTLA-4. Our results reveal a novel immunoregulatory action of ZOL that is not related to its action on bone resorption but might be associated with reported clinically significant extraskeletal outcomes of ZOL treatment.

Molecular mechanisms of action of bisphosphonates and new insights into their effects outside the skeleton.

Bisphosphonates (BP) are a class of calcium-binding drug used to prevent bone resorption in skeletal disorders such as osteoporosis and metastatic bone disease. They act by selectively targeting bone-resorbing osteoclasts and can be grouped into two classes depending on their intracellular mechanisms of action. Simple BPs cause osteoclast apoptosis after cytoplasmic conversion into toxic ATP analogues. In contrast, nitrogen-containing BPs potently inhibit FPP synthase, an enzyme of the mevalonate (cholesterol biosynthesis) pathway. This results in production of a toxic metabolite (ApppI) and the loss of long-chain isoprenoid lipids required for protein prenylation, a process necessary for the function of small GTPase proteins essential for the survival and activity of osteoclasts. In this review we provide a state-of-the-art overview of these mechanisms of action and a historical perspective of how they were discovered. Finally, we challenge the long-held dogma that BPs act only in the skeleton and highlight recent studies that reveal insights into hitherto unknown effects on tumour-associated and tissue-resident macrophages.

Borate bioactive glass prevents zoledronate-induced osteonecrosis of the jaw by restoring osteogenesis and angiogenesis.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a severe complication of systemic nitrogen-containing bisphosphonate (N-BP) administration, which leads to osteonecrosis, pain, and infection. Despite much effort, effective remedies are yet to be established. This study aimed to investigate potential recovery effect of borate bioactive glass (BBG) in vitro and in vivo. The effect of BBG on zoledronate-treated bone marrow mesenchymal cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) was explored by cell counting kit-8, EdU assay, flow cytometry, alkaline phosphatase staining, alizarin red staining, angiogenesis experiment, and real-time quantitative polymerase chain reaction. The preventive effect of BBG on zoledronate-induced osteonecrosis of the jaw in rat model was examined by micro-CT, HE staining, and immunohistochemistry. Exposure of BBG to BMSCs and HUVECs increased cell proliferation and restored their osteogenesis and angiogenesis potential in vitro. The BRONJ lesions were satisfactorily repaired and bone mineral density, bone volume/tissue volume, trabecula number, OCN-positive cells, and CD31-positive cells were increased in the BBG-treated groups compared with saline-treated groups. Exposure of BMSCs and HUVECs to BBG restores osteogenesis and angiogenesis inhibited by zoledronate. BBG successfully restores extraction socket healing of BRONJ in rat model.

Zoledronic acid regulates the synthesis and secretion of IL-1\u03b2 through Histone methylation in macrophages

Long-term administration of nitrogen-containing bisphosphonates increases the risk of detrimental side effects, such as bisphosphonate-related osteonecrosis of the jaw (BRONJ). BRONJ development is associated with inflammation, but its pathophysiology remains unknown. Here, we examined whether histone methylation is responsible for zoledronic acid (Zol)-induced inflammatory responses. We found that Kdm6a and Kdm6b markedly increased interleukin 1β expression and Gasdermin D cleavage, which are both activated by Caspase 1, in macrophages. Inhibitors of Kdm6a and Kdm6b robustly abolished Zol-enhanced interleukin 1β synthesis and secretion from macrophages. When Kdm6a and Kdm6b were pharmacologically inhibited in vivo, poor healing of the alveolar socket and inflammatory responses were ameliorated in Zol-treated mice. Taken together, we showed the pathologic role of Kdm6a and Kdm6b in Zol-promoted inflammatory responses and demonstrated that Kdm6a and Kdm6b are potential therapeutic targets for the treatment of BRONJ.

Structure-Dependent Effects of Bisphosphonates on Inflammatory Responses in Cultured Neonatal Mouse Calvaria.

Bisphosphonates (BPs) are classified into two groups, according to their side chain structures, as nitrogen-containing BPs (NBPs) and non-nitrogen-containing BPs (non-NBPs). In this study, we examined the effects of NBPs and non-NBPs on inflammatory responses, by quantifying the inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO), in cultured neonatal mouse calvaria. All examined NBPs (pamidronate, alendronate, incadronate, risedronate, zoledronate) stimulated lipopolysaccharide (LPS)-induced PGE2 and NO production by upregulating COX-2 and iNOS mRNA expression, whereas non-NBPs (etidronate, clodronate, tiludronate) suppressed PGE2 and NO production, by downregulating gene expression. Additionally, [4-(methylthio) phenylthio] methane bisphosphonate (MPMBP), a novel non-NBP with an antioxidant methylthio phenylthio group in its side chain, exhibited the most potent anti-inflammatory activity among non-NBPs. Furthermore, results of immunohistochemistry showed that the nuclear translocation of NF-κB/p65 and tyrosine nitration of cytoplasmic protein were stimulated by zoledronate, while MPMBP inhibited these phenomena, by acting as a superoxide anion (O2−) scavenger. These findings indicate that MPMBP can act as an efficacious agent that causes fewer adverse effects in patients with inflammatory bone diseases, including periodontitis and rheumatoid arthritis.

Nitrogen-Containing Bisphosphonates Are Associated With Reduced Risk of Pneumonia in Patients With Hip Fracture.

The objective of this work was to study the risk of pneumonia and pneumonia mortality among patients receiving nitrogen-containing bisphosphonates (N-BPs), non-N-BP anti-osteoporosis medications, and no anti-osteoporosis medications after hip fracture. We studied a historical cohort using a population-wide database. Patients with first hip fracture during 2005-2015 were identified and matched by time-dependent propensity score. The cohort was followed until December 31, 2016, to capture any pneumonia and pneumonia mortality. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using Cox-proportional hazards regression. Absolute risk difference (ARD) and number needed to treat (NNT) were calculated. We identified 54,047 patients with hip fracture. Of these, 4041 patients who received N-BPs and 11,802 without anti-osteoporosis medication were propensity score-matched. N-BPs were associated with a significantly lower risk of pneumonia compared with no treatment (6.9 versus 9.0 per 100 person-years; HR 0.76; 95% CI, 0.70 to 0.83), resulting in an ARD of 0.02 and NNT of 46. A similar association was observed with pneumonia mortality (HR 0.65; 95% CI, 0.56 to 0.75). When N-BPs were compared with non-N-BP anti-osteoporosis medications, the association remained significant. N-BPs were associated with lower risks of pneumonia and pneumonia mortality. Randomized controlled trials are now required to determine whether N-BPs, non-vaccine-based medications, can reduce pneumonia incidence in high risk groups. © 2020 American Society for Bone and Mineral Research.

Screening for side-effect susceptibility

Bone Osteoporosis is typically treated with nitrogen-containing bisphosphonates (N-BPs) to inhibit bone resorption; however, N-BPs can cause serious side effects, including osteonecrosis and fracture. To understand susceptibility to these side effects, Surface et al. investigated the role of the gene ATRAID in mediating response to N-BPs. Mice deficient in this gene had weaker bone and did not respond to N-BP treatment in models of osteoporosis. Patients with coding variants in ATRAID taking N-BPs presented with fractures and osteonecrosis of the jaw. The authors determined that ATRAID is necessary for inhibition of osteoclast function by N-BPs. It may thus be prudent to screen patients for variants in ATRAID to avoid N-BP side effects. Sci. Transl. Med. 12 , eaav9166 (2020).

ATRAID regulates the action of nitrogen-containing bisphosphonates on bone.

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFF) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase, resulting in defects in protein prenylation. Yet, it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Last, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare nonsynonymous coding variants in patients with ONJ or an AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.

Alendronate alleviates the symptoms of experimental autoimmune encephalomyelitis

Nitrogen-containing bisphosphonates, such as alendronate, have been widely used to treat osteoporosis because they may target multiple signals in the mevalonate cascade. The present study evaluated the therapeutic effects of alendronate on experimental autoimmune encephalomyelitis (EAE), which is a prototypical autoimmune disease model. EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. The mice were checked daily for clinical symptoms, such as paralysis, and the levels of inflammatory cytokines were analyzed using ELISA, western blot analyses, and immunohistochemistry. The daily oral administration of alendronate to EAE-induced mice significantly reduced the severity of paralysis and lowered T cell proliferation. Additionally, histopathological examinations confirmed that alendronate mitigated inflammation in the spinal cord after EAE induction, suppressed the infiltration of CD68-positive inflammatory cells, and reduced the production of various pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, as well as inducible nitric oxide synthase (iNOS). Furthermore, the alendronate-treated group exhibited a decrease in the number of iNOS-positive inflammatory cells compared to the vehicle-treated group. Taken together, the present results suggest that alendronate alleviated neuro-inflammation in the spinal cords of EAE-induced mice, which is an animal model of multiple sclerosis, possibly by inhibiting the downstream effects of the mevalonate cascade.

Calcium Phosphate Ceramics Can Prevent Bisphosphonate-Related Osteonecrosis of the Jaw.

Bisphosphonate-associated osteonecrosis of the jaw (BRONJ), a post-surgical non-healing wound condition, is one of the most common side effects in patients treated with nitrogen-containing bisphosphonates. Its physiopathology has been related with suppression of bone turnover, of soft tissue healing and infection. Biphasic calcium phosphates (BCP) are used as a drug delivery vehicle and as a bone substitute in surgical wounds. Due to their capacity to adsorb zoledronate, it was hypothesized these compounds might have a protective effect on the soft tissues in BRONJ wounds. To address this hypothesis, a reproducible in vivo model of BRONJ in Wistar rats was used. This model directly relates chronic bisphosphonate administration with the development of osteonecrosis of the jaw after tooth extraction. BCP granules were placed in the alveolus immediately after tooth extraction in the test group. The animals were evaluated through nuclear medicine, radiology, macroscopic observation, and histologic analysis. Encouragingly, calcium phosphate ceramics were able to limit zoledronate toxicity in vivo and to favor healing, which was evidenced by medical imaging (nuclear medicine and radiology), macroscopically, and through histology. The studied therapeutic option presented itself as a potential solution to prevent the development of maxillary osteonecrosis.

Insights about the structure of farnesyl diphosphate synthase (FPPS) and the activity of bisphosphonates on the proliferation and ultrastructure of Leishmania and Giardia

BackgroundThe enzyme farnesyl diphosphate synthase (FPPS) is positioned in the intersection of different sterol biosynthesis pathways such as those producing isoprenoids, dolichols and ergosterol. FPPS is ubiquitous in eukaryotes and is inhibited by nitrogen-containing bisphosphonates (N-BP). N-BP activity and the mechanisms of cell death as well as damage to the ultrastructure due to N-BP has not yet been investigated in Leishmania infantum and Giardia. Thus, we evaluated the effect of N-BP on cell viability and ultrastructure and then performed structural modelling and phylogenetic analysis on the FPPS enzymes of Leishmania and Giardia.MethodsWe performed multiple sequence alignment with MAFFT, phylogenetic analysis with MEGA7, and 3D structural modelling for FPPS with Modeller 9.18 and on I-Tasser server. We performed concentration curves with N-BP in Leishmania promastigotes and Giardia trophozoites to estimate the IC50via the MTS/PMS viability method. The ultrastructure was evaluated by transmission electron microscopy, and the mechanism of cell death by flow cytometry.ResultsThe nitrogen-containing bisphosphonate risedronate had stronger anti-proliferative activity in Leishmania compared to other N-BPs with an IC50 of 13.8 µM, followed by ibandronate and alendronate with IC50 values of 85.1 µM and 112.2 µM, respectively. The effect of N-BPs was much lower on trophozoites of Giardia than Leishmania (IC50 of 311 µM for risedronate). Giardia treated with N-BP displayed concentric membranes around the nucleus and nuclear pyknosis. Leishmania had mitochondrial swelling, myelin figures, double membranes, and plasma membrane blebbing. The same population labelled with annexin-V and 7-AAD had a loss of membrane potential (TMRE), indicative of apoptosis. Multiple sequence alignments and structural alignments of FPPS proteins showed that Giardia and Leishmania FPPS display low amino acid identity but possess the conserved aspartate-rich motifs.ConclusionsGiardia and Leishmania FPPS enzymes are phylogenetically distant but display conserved protein signatures. The N-BPs effect on FPPS was more pronounced in Leishmania than Giardia. This might be due to general differences in metabolism and differences in the FPPS catalytic site.

Indigenous Microbiota Protects against Inflammation-Induced Osteonecrosis

Medication-related osteonecrosis of the jaw (MRONJ) is a rare intraoral lesion that occurs in patients undergoing long-term and/or high-dose therapy with nitrogen-containing bisphosphonates, a RANKL inhibitor, antiangiogenic agents, or mTOR inhibitors. The presence of pathogenic bacteria is highly associated with advanced stages of MRONJ lesions; however, the exact role of indigenous microbes in MRONJ development is unknown. Here, we report that the normal oral flora in mice protects against inflammation-induced osteonecrosis. In mice that developed osteonecrosis following tooth extraction, there was increased bacterial infiltration when compared with healed controls. Antibiotic-mediated oral dysbiosis led to a local inhibition of bone resorption in the presence of ligature-induced periodontitis (LIP). There was no significant difference in empty lacunae, necrotic bone formation, osteoclast number, and surface area in antibiotic-treated as compared with conventionally colonized mice following extraction of healthy teeth after zoledronic acid infusions. However, extraction of LIP teeth led to increased empty lacunae, necrotic bone, and osteoclast surface area in antibiotic- and zoledronic acid–treated mice as compared with conventionally colonized mice. Our findings suggest that the presence of the indigenous microbiota protects against LIP-induced osteonecrosis.

Use of alendronate in the therapy of osteoporosis

Treatment of osteoporosis is a difficult task due to the need for long-term medication and maximum safety. The main goal of osteoporosis treatment is to prevent bone fractures. According to the national clinical guidelines for the treatment of osteoporosis, nitrogen-containing bisphosphonates are among the first-line drugs for the prevention and treatment of this disease. The diversity in the side chain structure determines the strength with which bisphosphonates bind to hydroxyapatite, are distributed, and remain in bone tissue for a certain time after treatment has been completed. They are the drugs of choice as they have proven efficacy in reducing the risk of fractures, and at the same time an acceptable safety profile. Bisphosphonates are the most well-known and studied drugs, successfully used in all countries for the treatment of various forms of osteoporosis. When using oral forms of bisphosphonates, the most expected undesirable phenomenon is esophagitis. The development of new forms of well-known and proven medicines that reduce adverse events and increase adherence to treatment is extremely important and promising. Of the latest achievements in this regard, it should be noted a new form of alendronate-Binosto-effervescent soluble tablet, the use of which reduces the risk of irritating effects on the upper gastrointestinal tract.

Pamidronate: A model compound of the pharmacology of nitrogen-containing bisphosphonates; A Leiden historical perspective

Pamidronate [3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD)] was the first nitrogen-containing bisphosphonate (N-BP) investigated in clinical studies. In contrast to other clinically used bisphosphonates, pamidronate was discovered and its properties were initially studied in an Academic Institution. On the occasion of the 50th Anniversary of the first publications on the biological effects of bisphosphonates, I review in this article the contribution of Leiden investigators to the development of pamidronate that led to the recognition of the significance of the Nitrogen atom in the side chain of bisphosphonates for their action on bone resorption and to the formulation of principles for the use of N-BPs in the management of patients with different skeletal disorders.

A nitrogen-containing bisphosphonate inhibits osteoblast attachment and impairs bone healing in bone-compatible scaffold

Compromised osteoblast attachment on hydroxyapatite could be involved in the development of bone healing failure. We developed a bone-compatible scaffold that mimics bone structure with sub-micron hydroxyapatite (HA) surfaces, so that we could evaluate the effects of nitrogen-containing bisphosphonate (N-BP) on osteoblast behavior and bone healing. Human osteoblasts were seeded onto the bone-compatible scaffold with or without N-BP, and cell attachment and spreading behavior were evaluated 4 and 24 h after seeding. Then, mineralization was evaluated at 7 and 14 days. The osteoconductive activity of the scaffold was evaluated by implantation for 3 and 6 weeks into a rat cranial bone defect. The numbers of osteoblasts and their diameters were significantly less in N-BP-binding scaffolds than in untreated scaffolds at 4 and 24 h. Mineralization were also significantly less in the N-BP-binding scaffolds than in controls at 7 and 14 days. In vivo study revealed bone formation in N-BP-binding scaffolds was significantly less than in untreated scaffolds at 3 and 6 weeks. These results suggest that N-BP-binding to HA inhibited osteoblast attachment and spreading, thereby compromising bone healing process in the injured bone defect site.

Basic Studies on the Mechanism, Prevention, and Treatment of Osteonecrosis of the Jaw Induced by Bisphosphonates

Since the first report in 2003, bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been increasing, without effective clinical strategies. Osteoporosis is common in elderly women, and bisphosphonates (BPs) are typical and widely used anti-osteoporotic or anti-bone-resorptive drugs. BRONJ is now a serious concern in dentistry. As BPs are pyrophosphate analogues and bind strongly to bone hydroxyapatite, and the P-C-P structure of BPs is non-hydrolysable, they accumulate in bones upon repeated administration. During bone-resorption, BPs are taken into osteoclasts and exhibit cytotoxicity, producing a long-lasting anti-bone-resorptive effect. BPs are divided into nitrogen-containing BPs (N-BPs) and non-nitrogen-containing BPs (non-N-BPs). N-BPs have far stronger anti-bone-resorptive effects than non-N-BPs, and BRONJ is caused by N-BPs. Our murine experiments have revealed the following. N-BPs, but not non-N-BPs, exhibit direct and potent inflammatory/necrotic effects on soft-tissues. These effects are augmented by lipopolysaccharide (the inflammatory component of bacterial cell-walls) and the accumulation of N-BPs in jawbones is augmented by inflammation. N-BPs are taken into soft-tissue cells via phosphate-transporters, while the non-N-BPs etidronate and clodronate inhibit this transportation. Etidronate, but not clodronate, has the effect of expelling N-BPs that have accumulated in bones. Moreover, etidronate and clodronate each have an analgesic effect, while clodronate has an anti-inflammatory effect via inhibition of phosphate-transporters. These findings suggest that BRONJ may be induced by phosphate-transporter-mediated and infection-promoted mechanisms, and that etidronate and clodronate may be useful for preventing and treating BRONJ. Our clinical trials support etidronate being useful for treating BRONJ, although additional clinical trials of etidronate and clodronate are needed.

СУМІСНА ДІЯ НОВИХ ПІРАЗОЛВМІСНИХ БІСФОСФОНАТІВ І ВІТАМІНУ D3 ЗА ЕКСПЕРИМЕНТАЛЬНОГО ОСТЕОПОРОЗУ

Bisphosphonates remain one of the most effective drugs to treat diseases of the musculoskeletal system. Among a wide range of possible options, the methods of bioinformatic analysis were developed, selected and synthesized analogs of bisphosphonates, the radical of which were selected pyrazole derivatives. The development of new advanced bisphosphonate analogues has broad prospects for medical application. The study is devoted to the investigation of the combined action of synthesized nitrogen-containing bisphosphonates (pyrazole-containing analogues) and vitamin D3 in the correction of mineral metabolism disturbances in osteoporosis. The work was performed on a rat model of alimentary osteoporosis, which was characterized by the development of hypocalcemia, hypophosphatemia, decreased serum 25OHD concentration and severe bone tissue demineralization. New pyrazole-containing bisphosphonates inhibited the process of bone demineralization (resorption) and improved mineral metabolism in rats with alimentary osteoporosis with varying efficiency. The action of the investigated substances resulted in an increase in the concentration of calcium, inorganic phosphate and a decrease in the activity of alkaline phosphatase and its isoenzymes in the serum. The ash content, calcium and phosphorus level in the ash of the tibia was increased as well, along with improving osteometric characteristics of the tibia (length and thickness of the proximal epimetaphysis). The combination of pyrazole-containing bisphosphonates and vitamin D3, which normalizes the concentration of 25OHD in the serum and provides the synthesis of biologically active, hydroxylated form of cholecalciferol, has been more effective in the correction of mineral metabolism impairments in rats with alimentary osteoporosis. According to the experimental results, the combination of vitamin D3 with bisphosphonate ISP-30, as a substance with the highest biological efficiency in regulating bone remodeling, enhancing osteogenesis and inhibition of resorption, is the most promising for further research.

Biological Effects of Anti-RANKL Antibody and Zoledronic Acid on Growth and Tooth Eruption in Growing Mice

The anti-bone resorptive drugs denosumab, an anti-human-RANKL antibody, and zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, have recently been applied for treatment of pediatric patients with bone diseases, though details regarding their effects in growing children have yet to be fully elucidated. In the present study, we administered these anti-resorptive drugs to mice from the age of 1 week and continued once-weekly injections for a total of 7 times. Mice that received the anti-RANKL antibody displayed normal growth and tooth eruption, though osteopetrotic bone volume gain in long and alveolar bones was noted, while there were nearly no osteoclasts and a normal of number osteoblasts observed. In contrast, ZOL significantly delayed body growth, tooth root formation, and tooth eruption, with increased osteoclast and decreased osteoblast numbers. These findings suggest regulation of tooth eruption via osteoblast differentiation by some types of anti-resorptive drugs.

NMR for screening and a biochemical assay: Identification of new FPPS inhibitors exerting anticancer activity.

Farnesyl pyrophosphate synthase (FPPS) is a crucial enzyme for the synthesis of isoprenoids and the key target of nitrogen-containing bisphosphonates (N-BPs). N-BPs are potent and selective FPPS inhibitors that are used in the treatment of bone-related diseases, but have poor pharmacokinetic properties. Given the key role played by FPPS in many cancer-related pathways and the pharmacokinetic limits of N-BPs, hundreds of molecules have been screened to identify new FPPS inhibitors characterized by improved drug-like properties that are useful for broader therapeutic applications in solid, non-skeletal tumours. We have previously shown that N6-isopentenyladenosine (i6A) and its related compound N6-benzyladenosine (2) exert anti-glioma activity by interfering with the mevalonate pathway and inhibiting FPPS. Here, we report the design and synthesis of a panel of N6-benzyladenosine derivatives (compounds 2a-m) incorporating different chemical moieties on the benzyl ring. Compounds 2a-m show in vitro antiproliferative activity in U87MG glioma cells and, analogous to the bisphosphonate FPPS inhibitors, exhibit immunogenic properties in ex vivo γδ T cells from stimulated peripheral blood mononuclear cells (PBMCs). Using saturation transfer difference (STD) and quantitative 1H nuclear magnetic resonance (NMR) experiments, we found that 2f, the N6-benzyladenosine analogue that includes a tertbutyl moiety in the para position of the benzyl ring, is endowed with increased FPPS binding and inhibition compared to the parent compounds i6A and 2. N6-benzyladenosine derivatives, characterized by structural features that are significantly different from those of N-BPs, have been confirmed to be promising chemical scaffolds for the development of non N-BP FPPS inhibitors, exerting combined cytotoxic and immunostimulatory activities.