Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption.

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.

The Notch pathway regulates the bone gain induced by PTH anabolic signaling.

Parathyroid hormone (PTH) signaling downstream of the PTH 1 receptor (Pth1r) results in both bone anabolic and catabolic actions by mechanisms not yet fully understood. In this study, we show that Pth1r signaling upregulates the expression of several components of the Notch pathway and that Notch signals contribute to the catabolic actions of PTH in bone. We found that constitutive genetic activation of PTH receptor signaling in osteocytes (caPth1rOt ) or treatment with PTH daily increased the expression of several Notch ligands/receptors in bone. In contrast, sustained elevation of endogenous PTH did not change Notch components expression. Deletion of the PTH receptor or sclerostin overexpression in osteocytes abolished Notch increases by PTH. Further, deleting the canonical Notch transcription factor Rbpjk in osteocytes decreased bone mass and increased resorption and Rankl expression in caPth1rOt mice. Moreover, pharmacological bone-targeted Notch inhibition potentiated the bone mass gain induced by intermittent PTH by reducing bone resorption and preserving bone formation. Thus, Notch activation lies downstream of anabolic signaling driven by PTH actions in osteocytes, and Notch pharmacological inhibition maximizes the bone anabolic effects of PTH.

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.

Bisphosphonate use in the horse: what is good and what is not?

BACKGROUND: Bisphosphonates (BPs) are a family of molecules characterized by two key properties: their ability to bind strongly to bone mineral and their inhibitory effects on mature osteoclasts and thus bone resorption. Chemically two groups of BPs are recognized, non-nitrogen-containing and nitrogen-containing BPs. Non-nitrogen-containing BPs incorporate into the energy pathways of the osteoclast, resulting in disrupted cellular energy metabolism leading to cytotoxic effects and osteoclast apoptosis. Nitrogen-containing BPs primarily inhibit cholesterol biosynthesis resulting in the disruption of intracellular signaling, and other cellular processes in the osteoclast. BODY: BPs also exert a wide range of physiologic activities beyond merely the inhibition of bone resorption. Indeed, the breadth of reported activities include inhibition of cancer cell metastases, proliferation and apoptosis in vitro. In addition, the inhibition of angiogenesis, matrix metalloproteinase activity, altered cytokine and growth factor expression, and reductions in pain have been reported. In humans, clinical BP use has transformed the treatment of both post-menopausal osteoporosis and metastatic breast and prostate cancer. However, BP use has also resulted in significant adverse events including acute-phase reactions, esophagitis, gastritis, and an association with very infrequent atypical femoral fractures (AFF) and osteonecrosis of the jaw (ONJ). CONCLUSION: Despite the well-characterized health benefits of BP use in humans, little is known regarding the effects of BPs in the horse. In the equine setting, only non-nitrogen-containing BPs are FDA-approved primarily for the treatment of navicular syndrome. The focus here is to discuss the current understanding of the strengths and weaknesses of BPs in equine veterinary medicine and highlight the future utility of these potentially highly beneficial drugs.

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.

Hydroxychloroquine and a low antiresorptive activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects.

Osteoporosis remains incurable. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation, while the anabolic agent, teriparatide, does not inhibit bone resorption, and thus they have limited efficacy in preventing osteoporotic fractures and cause some side effects. Thus, there is an unmet need to develop dual antiresorptive and anabolic agents to prevent and treat osteoporosis. Hydroxychloroquine (HCQ), which is used to treat rheumatoid arthritis, prevents the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an NF-κB adaptor protein that limits bone resorption and maintains bone formation. We attempted to covalently link HCQ to a hydroxyalklyl BP (HABP) with anticipated low antiresorptive activity, to target delivery of HCQ to bone to test if this targeting increases its efficacy to prevent TRAF3 degradation in the bone microenvironment and thus reduce bone resorption and increase bone formation, while reducing its systemic side effects. Unexpectedly, HABP-HCQ was found to exist as a salt in aqueous solution, composed of a protonated HCQ cation and a deprotonated HABP anion. Nevertheless, it inhibited osteoclastogenesis, stimulated osteoblast differentiation, and increased TRAF3 protein levels in vitro. HABP-HCQ significantly inhibited both osteoclast formation and bone marrow fibrosis in mice given multiple daily PTH injections. In contrast, HCQ inhibited marrow fibrosis, but not osteoclast formation, while the HABP alone inhibited osteoclast formation, but not fibrosis, in the mice. HABP-HCQ, but not HCQ, prevented trabecular bone loss following ovariectomy in mice and, importantly, increased bone volume in ovariectomized mice with established bone loss because HABP-HCQ increased bone formation and decreased bone resorption parameters simultaneously. In contrast, HCQ increased bone formation, but did not decrease bone resorption parameters, while HABP also restored the bone lost in ovariectomized mice, but it inhibited parameters of both bone resorption and formation. Our findings suggest that the combination of HABP and HCQ could have dual antiresorptive and anabolic effects to prevent and treat osteoporosis.

Hydroxychloroquine and a low activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects.

Osteoporosis is incurable because there are no dual antiresorptive and anabolic therapeutic agents that can be administered long-term. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation and thus have limited effect in preventing osteoporotic fracture. Hydroxychloroquine (HCQ), which is used to treat rheumatoid arthritis, prevents the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an NF-κB adaptor protein that limits bone resorption and maintains bone formation. We attempted to covalently link HCQ to a hydroxyalklyl BP (HABP) with anticipated low antiresorptive activity, to target delivery of HCQ to bone to test if this targeting increases its efficacy to prevent TRAF3 degradation in the bone microenvironment and thus reduce bone resorption and increase bone formation, while reducing its systemic side effects. Unexpectedly, HABP-HCQ was found to exist as a salt in aqueous solution, composed of a protonated HCQ cation and a deprotonated HABP anion. Nevertheless, it inhibited osteoclastogenesis, stimulated osteoblast differentiation, and increased TRAF3 protein levels in vitro. HABP-HCQ significantly inhibited both osteoclast formation and bone marrow fibrosis in mice given multiple daily PTH injections. In contrast, HCQ inhibited fibrosis, but not osteoclast formation, while the HABP alone inhibited osteoclast formation, but not fibrosis, in the mice. HABP-HCQ, but not HCQ, prevented trabecular bone loss following ovariectomy in mice and, importantly, increased bone volume in ovariectomized mice with established bone loss because HABP-HCQ increased bone formation and decreased bone resorption parameters simultaneously. In contrast, HCQ increased bone formation, but did not decrease bone resorption parameters, while HABP also restored the bone lost in ovariectomized mice, but it inhibited parameters of both bone resorption and formation. Our findings suggest that the combination of HABP and HCQ could have dual antiresorptive and anabolic effects to prevent and treat osteoporosis.

Evidence of bisphosphonate-conjugated sitafloxacin eradication of established methicillin-resistant S. aureus infection with osseointegration in murine models of implant-associated osteomyelitis.

Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve "target-and-release" drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (hydroxybisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.

Evidence of Bisphosphonate-Conjugated Sitafloxacin Eradication of Established Methicillin-Resistant S. aureus Infection with Osseointegration in Murine Models of Implant-Associated Osteomyelitis.

Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve "target-and-release" drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (bisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy, while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.

Fluorescent risedronate analogue 800CW-pRIS improves tooth extraction-associated abnormal wound healing in zoledronate-treated mice.

Background: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a rare but serious side effect of nitrogen-containing bisphosphonate drugs (N-BPs) frequently prescribed to reduce skeletal-related events in bone malignancies and osteoporosis. BRONJ is associated with abnormal oral wound healing after dentoalveolar surgery and tooth extraction. We previously found that N-BP chemisorbed to bone mineral hydroxyapatite was dissociated by secondary applied N-BP. This study investigated the effect of the surface equilibrium-based removal of N-BP from jawbone on tooth extraction wound healing of zoledronate (ZOL)-treated mice. Methods: A pharmacologically inactive N-BP derivative (the 4-pyridyl isomer of risedronate equipped with a near-infrared 800CW fluorescent imaging dye, 800CW-pRIS) was designed and synthesized. 800CW-pRIS was intra-orally injected or topically applied in a deformable nano-scale vesicle formulation (DNV) to the palatal tissue of mice pretreated with ZOL, a potent N-BP. The female C56BL6/J mice were subjected to maxillary molar extraction and oral wound healing was compared for 800CW-pRIS/ZOL, ZOL and untreated control groups. Results: 800CW-pRIS is confirmed to be inactive in inhibiting prenylation in cultured osteoclasts while retaining high affinity for hydroxyapatite. ZOL-injected mice exhibit delayed tooth extraction wound healing with osteonecrosis relative to the untreated controls. 800CW-pRIS applied topically to the jaw one week before tooth extraction significantly reduces gingival oral barrier inflammation, improves extraction socket bone regeneration, and prevents development of osteonecrosis in ZOL-injected mice. Conclusions: Topical pre-treatment with 800CW-RIS in DNV is a promising approach to prevent the complication of abnormal oral wound healing associated with BRONJ while retaining the anti-resorptive benefit of legacy N-BP in appendicular or vertebrate bones.

Bone-Targeted Bortezomib Inhibits Bortezomib-Resistant Multiple Myeloma in Mice by Providing Higher Levels of Bortezomib in Bone.

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic and blood cancers. The difficulty of eliminating bone-residing cancer, especially drug-resistant cancer, necessitates novel, alternative treatments to manipulate tumor cells and their microenvironment, with minimal off-target effects. To this end, bone-targeted conjugate (BP-Btz) was generated by linking bortezomib (Btz, an anticancer, bone-stimulatory drug) to a bisphosphonate (BP, a targeting ligand) through a cleavable linker that enables spatiotemporally controlled delivery of Btz to bone under acidic conditions for treating multiple myeloma (MM). Three conjugates with different linkers were developed and screened for best efficacy in mouse model of MM. Results demonstrated that the lead candidate BP-Btz with optimal linker could overcome Btz resistance, reduced tumor burden, bone destruction, or tumor metastasis more effectively than BP or free Btz without thrombocytopenia and neurotoxicity in mice bearing myeloma. Furthermore, pharmacokinetic and pharmacodynamic studies showed that BP-Btz bound to bone matrix, released Btz in acidic conditions, and had a higher local concentration and longer half-life than Btz in bone. Our findings suggest the potential of bone-targeted Btz conjugate as an efficacious Btz-resistant MM treatment mechanism. © 2021 American Society for Bone and Mineral Research (ASBMR).

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.

Mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) revealed by targeted removal of legacy bisphosphonate from jawbone using competing inert hydroxymethylene diphosphonate.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) presents as a morbid jawbone lesion in patients exposed to a nitrogen-containing bisphosphonate (N-BP). Although it is rare, BRONJ has caused apprehension among patients and healthcare providers and decreased acceptance of this antiresorptive drug class to treat osteoporosis and metastatic osteolysis. We report here a novel method to elucidate the pathological mechanism of BRONJ by the selective removal of legacy N-BP from the jawbone using an intra-oral application of hydroxymethylene diphosphonate (HMDP) formulated in liposome-based deformable nanoscale vesicles (DNV). After maxillary tooth extraction, zoledronate-treated mice developed delayed gingival wound closure, delayed tooth extraction socket healing and increased jawbone osteonecrosis consistent with human BRONJ lesions. Single cell RNA sequencing of mouse gingival cells revealed oral barrier immune dysregulation and unresolved proinflammatory reaction. HMDP-DNV topical applications to nascent mouse BRONJ lesions resulted in accelerated gingival wound closure and bone socket healing as well as attenuation of osteonecrosis development. The gingival single cell RNA sequencing demonstrated resolution of chronic inflammation by increased anti-inflammatory signature gene expression of lymphocytes and myeloid-derived suppressor cells. This study suggests that BRONJ pathology is related to N-BP levels in jawbones and demonstrates the potential of HMDP-DNV as an effective BRONJ therapy.

Efficacy of Bisphosphonate-Conjugated Sitafloxacin in a Murine Model of S. aureus Osteomyelitis: Evidence of "Target & Release" Kinetics and Killing of Bacteria Within Canaliculi.

S. aureus infection of bone is difficult to eradicate due to its ability to colonize the osteocyte-lacuno-canalicular network (OLCN), rendering it resistant to standard-of-care (SOC) antibiotics. To overcome this, we proposed two bone-targeted bisphosphonate-conjugated antibiotics (BCA): bisphosphonate-conjugated sitafloxacin (BCS) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS). Initial studies demonstrated that the BCA kills S. aureus in vitro. Here we demonstrate the in vivo efficacy of BCS and HBCS versus bisphosphonate, sitafloxacin, and vancomycin in mice with implant-associated osteomyelitis. Longitudinal bioluminescent imaging (BLI) confirmed the hypothesized "target and release"-type kinetics of BCS and HBCS. Micro-CT of the infected tibiae demonstrated that HBCS significantly inhibited peri-implant osteolysis versus placebo and free sitafloxacin (p < 0.05), which was not seen with the corresponding non-antibiotic-conjugated bisphosphonate control. TRAP-stained histology confirmed that HBCS significantly reduced peri-implant osteoclast numbers versus placebo and free sitafloxacin controls (p < 0.05). To confirm S. aureus killing, we compared the morphology of S. aureus autolysis within in vitro biofilm and infected tibiae via transmission electron microscopy (TEM). Live bacteria in vitro and in vivo presented as dense cocci ~1 µm in diameter. In vitro evidence of autolysis presented remnant cell walls of dead bacteria or "ghosts" and degenerating (non-dense) bacteria. These features of autolyzed bacteria were also present among the colonizing S. aureus within OLCN of infected tibiae from placebo-, vancomycin-, and sitafloxacin-treated mice, similar to placebo. However, most of the bacteria within OLCN of infected tibiae from BCA-treated mice were less dense and contained small vacuoles and holes >100 nm. Histomorphometry of the bacteria within the OLCN demonstrated that BCA significantly increased their diameter versus placebo and free antibiotic controls (p < 0.05). As these abnormal features are consistent with antibiotic-induced vacuolization, bacterial swelling, and necrotic phenotype, we interpret these findings to be the initial evidence of BCA-induced killing of S. aureus within the OLCN of infected bone. Collectively, these results support the bone targeting strategy of BCA to overcome the biodistribution limits of SOC antibiotics and warrant future studies to confirm the novel TEM phenotypes of bacteria within OLCN of S. aureus-infected bone of animals treated with BCS and HBCS.

Bisphosphonates in veterinary medicine: The new horizon for use.

Bisphosphonates (BPs) are characterized by their ability to bind strongly to bone mineral and inhibit bone resorption. However, BPs exert a wide range of pharmacological activities beyond the inhibition of bone resorption, including the inhibition of cancer cell metastases and angiogenesis and the inhibition of proliferation and apoptosis in vitro. Additionally, the inhibition of matrix metalloproteinase activity, altered cytokine and growth factor expression, as well as reductions in parameters of pain have also been reported. In humans, clinical BP use has transformed the treatment of post-menopausal osteoporosis, rare bone diseases such as osteogenesis imperfecta, as well as multiple myeloma and metastatic breast and prostate cancer, albeit not without infrequent but significant adverse events. Despite the well-characterized health benefits of BP use in humans, the evidence-base for the therapeutic efficacy of BPs in veterinary medicine is, by comparison, limited. Notwithstanding, BPs are used widely in small animal veterinary practice for the medical management of hyperparathyroidism, idiopathic hypercalcemia in cats, as well as for the palliative care of bone tumors which are common in dogs, and in particular, primary bone tumors such as osteosarcoma. Palliative BP treatment has also recently increased in veterinary oncology to alleviate tumor-associated bone pain. In equine veterinary practice, non-nitrogen-containing BPs are FDA-approved to control clinical signs associated with navicular syndrome in adult horses. However, there are growing concerns regarding the off-label use of BPs in juvenile horses. Here we discuss the current understanding of the strengths, weaknesses and current controversies surrounding BP use in veterinary medicine to highlight the future utility of these potentially beneficial drugs.

A Bisphosphonate With a Low Hydroxyapatite Binding Affinity Prevents Bone Loss in Mice After Ovariectomy and Reverses Rapidly With Treatment Cessation.

Bisphosphonates (BPs) are a mainstay of osteoporosis treatment; however, concerns about bone health based on oversuppression of remodeling remain. Long-term bone remodeling suppression adversely affects bone material properties with microdamage accumulation and reduced fracture toughness in animals and increases in matrix mineralization and atypical femur fractures in patients. Although a "drug holiday" from BPs to restore remodeling and improve bone quality seems reasonable, clinical BPs have long functional half-lives because of their high hydroxyapatite (HAP) binding affinities. This places a practical limit on the reversibility and effectiveness of a drug holiday. BPs with low HAP affinity and strong osteoclast inhibition potentially offer an alternative approach; their antiresorptive effect should reverse rapidly when dosing is discontinued. This study tested this concept using NE-58025, a BP with low HAP affinity and moderate osteoclast inhibition potential. Young adult female C57Bl/6 mice were ovariectomized (OVX) and treated with NE-58025, risedronate, or PBS vehicle for 3 months to test effectiveness in preventing long-term bone loss. Bone microarchitecture, histomorphometry, and whole-bone mechanical properties were assessed. To test reversibility, OVX mice were similarly treated for 3 months, treatment was stopped, and bone was assessed up to 3 months post-treatment. NE-58025 and RIS inhibited long-term OVX-induced bone loss, but NE-58025 antiresorptive effects were more pronounced. Withdrawing NE-58025 treatment led to the rapid onset of trabecular resorption with a 200% increase in osteoclast surface and bone loss within 1 month. Cessation of risedronate treatment did not lead to increases in resorption indices or bone loss. These results show that NE-58025 prevents OVX-induced bone loss, and its effects reverse quickly following cessation treatment in vivo. Low-HAP affinity BPs may have use as reversible, antiresorptive agents with a rapid on/off profile, which may be useful for maintaining bone health with long-term BP treatment. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Bisphosphonate drugs have actions in the lung and inhibit the mevalonate pathway in alveolar macrophages.

Bisphosphonates drugs target the skeleton and are used globally for the treatment of common bone disorders. Nitrogen-containing bisphosphonates act by inhibiting the mevalonate pathway in bone-resorbing osteoclasts but, surprisingly, also appear to reduce the risk of death from pneumonia. We overturn the long-held belief that these drugs act only in the skeleton and show that a fluorescently labelled bisphosphonate is internalised by alveolar macrophages and large peritoneal macrophages in vivo. Furthermore, a single dose of a nitrogen-containing bisphosphonate (zoledronic acid) in mice was sufficient to inhibit the mevalonate pathway in tissue-resident macrophages, causing the build-up of a mevalonate metabolite and preventing protein prenylation. Importantly, one dose of bisphosphonate enhanced the immune response to bacterial endotoxin in the lung and increased the level of cytokines and chemokines in bronchoalveolar fluid. These studies suggest that bisphosphonates, as well as preventing bone loss, may boost immune responses to infection in the lung and provide a mechanistic basis to fully examine the potential of bisphosphonates to help combat respiratory infections that cause pneumonia.

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.

Development of Bisphosphonate-Conjugated Antibiotics to Overcome Pharmacodynamic Limitations of Local Therapy: Initial Results with Carbamate Linked Sitafloxacin and Tedizolid.

The use of local antibiotics to treat bone infections has been questioned due to a lack of clinical efficacy and emerging information about Staphylococcus aureus colonization of the osteocyte-lacuno canalicular network (OLCN). Here we propose bisphosphonate-conjugated antibiotics (BCA) using a "target and release" approach to deliver antibiotics to bone infection sites. A fluorescent bisphosphonate probe was used to demonstrate bone surface labeling adjacent to bacteria in a S. aureus infected mouse tibiae model. Bisphosphonate and hydroxybisphosphonate conjugates of sitafloxacin and tedizolid (BCA) were synthesized using hydroxyphenyl and aminophenyl carbamate linkers, respectively. The conjugates were adequately stable in serum. Their cytolytic activity versus parent drug on MSSA and MRSA static biofilms grown on hydroxyapatite discs was established by scanning electron microscopy. Sitafloxacin O-phenyl carbamate BCA was effective in eradicating static biofilm: no colony formation units (CFU) were recovered following treatment with 800 mg/L of either the bisphosphonate or α-hydroxybisphosphonate conjugated drug (p < 0.001). In contrast, the less labile tedizolid N-phenyl carbamate linked BCA had limited efficacy against MSSA, and MRSA. CFU were recovered from all tedizolid BCA treatments. These results demonstrate the feasibility of BCA eradication of S. aureus biofilm on OLCN bone surfaces and support in vivo drug development of a sitafloxacin BCA.