High Affinity For Bone Research Articles

Evaluation of antitumor activity and cardiac toxicity of a bone-targeted ph-sensitive liposomal formulation in a bone metastasis tumor model in mice

Chemotherapy for bone tumors is a major challenge because of the inability of therapeutics to penetrate dense bone mineral. We hypothesize that a nanostructured formulation with high affinity for bone could deliver drug to the tumor while minimizing off-target toxicity. Here, we evaluated the efficacy and toxicity of a novel bone-targeted, pH-sensitive liposomal formulation containing doxorubicin in an animal model of bone metastasis. Biodistribution studies with the liposome showed good uptake in tumor, but low accumulation of doxorubicin in the heart. Mice treated with the bone-targeted liposome formulation showed a 70% reduction in tumor volume, compared to 35% reduction for free doxorubicin at the same dose. Both cardiac toxicity and overall mortality were significantly lower for animals treated with the bone-targeted liposomes compared to free drug. Bone-targeted, pH-sensitive, doxorubicin containing liposomes represent a promising approach to selectively delivering doxorubicin to bone tumors while minimizing cardiac toxicity.

A three-dimensional block structure consisting exclusively of carbon nanotubes serving as bone regeneration scaffold and as bone defect filler.

Many recent studies have been conducted to assess the ability of composite materials containing carbon nanotubes (CNTs) with high bone affinity to serve as scaffolds in bone regenerative medicine. These studies have demonstrated that CNTs can effectively induce bone formation. However, no studies have investigated the usefulness of scaffolds consisting exclusively of CNTs in bone regenerative medicine. We built a three-dimensional block entity with maximized mechanical strength from multi-walled CNTs (MWCNT blocks) and evaluated their efficacy as scaffold material for bone repair. When MWCNT blocks containing recombinant human bone morphogenetic protein-2 (rhBMP-2) were implanted in mouse muscle, ectopic bone was formed in direct contact with the blocks. Their bone marrow densities were comparable to those of PET-reinforced collagen sheets with rhBMP-2. On day 1 and day 3, MC3T3-E1 preosteoblasts were attached to the scaffold surface of MWCNT blocks than that of PET-reinforced collagen sheets. They also showed a maximum compression strength comparable to that of cortical bone. Our MWCNT blocks are expected to serve as bone defect filler and scaffold material for bone regeneration.

In vitro potency, in vitro and in vivo efficacy of liposomal alendronate in combination with γδ T cell immunotherapy in mice

Nitrogen-containing bisphosphonates (N-BP), including zoledronic acid (ZOL) and alendronate (ALD), have been proposed as sensitisers in γδ T cell immunotherapy in pre-clinical and clinical studies. Therapeutic efficacy of N-BPs is hampered by their rapid renal excretion and high affinity for bone. Liposomal formulations of N-BP have been proposed to improve accumulation in solid tumours. Liposomal ALD (L-ALD) has been suggested as a suitable alternative to liposomal ZOL (L-ZOL), due to unexpected mice death experienced in pre-clinical studies with the latter. Only one study so far has proven the therapeutic efficacy of L-ALD, in combination with γδ T cell immunotherapy, after intraperitoneal administration of γδ T cell resulting in delayed growth of ovarian cancer in mice. This study aims to assess the in vitro efficacy of L-ALD, in combination with γδ T cell immunotherapy, in a range of cancerous cell lines, using L-ZOL as a comparator. The therapeutic efficacy was tested in a pseudo-metastatic lung mouse model, following intravenous injection of γδ T cell, L-ALD or the combination. In vivo biocompatibility and organ biodistribution studies of L-N-BPs were undertaken simultaneously. Higher concentrations of L-ALD (40–60μM) than L-ZOL (3–10μM) were required to produce a comparative reduction in cell viability in vitro, when used in combination with γδ T cells. Significant inhibition of tumour growth was observed after treatment with both L-ALD and γδ T cells in pseudo-metastatic lung melanoma tumour-bearing mice after tail vein injection of both treatments, suggesting that therapeutically relevant concentrations of L-ALD and γδ T cell could be achieved in the tumour sites, resulting in significant delay in tumour growth.

Development of a bone-targeted pH-sensitive liposomal formulation containing doxorubicin: physicochemical characterization, cytotoxicity, and biodistribution evaluation in a mouse model of bone metastasis.

BackgroundDespite recent advances in cancer therapy, the treatment of bone tumors remains a major challenge. A possible underlying hypothesis, limitation, and unmet need may be the inability of therapeutics to penetrate into dense bone mineral, which can lead to poor efficacy and high toxicity, due to drug uptake in healthy organs. The development of nanostructured formulations with high affinity for bone could be an interesting approach to overcome these challenges.PurposeTo develop a liposomal formulation with high affinity for hydroxyapatite and the ability to release doxorubicin (DOX) in an acidic environment for future application as a tool for treatment of bone metastases.Materials and methodsLiposomes were prepared by thin-film lipid hydration, followed by extrusion and the sulfate gradient-encapsulation method. Liposomes were characterized by average diameter, ζ-potential, encapsulation percentage, X-ray diffraction, and differential scanning calorimetry. Release studies in buffer (pH 7.4 or 5), plasma, and serum, as well as hydroxyapatite-affinity in vitro analysis were performed. Cytotoxicity was evaluated by MTT assay against the MDA-MB-231 cell line, and biodistribution was assessed in bone metastasis-bearing animals.ResultsLiposomes presented suitable diameter (~170 nm), DOX encapsulation (~2 mg/mL), controlled release, and good plasma and serum stability. The existence of interactions between DOX and the lipid bilayer was proved through differential scanning calorimetry and small-angle X-ray scattering. DOX release was faster when the pH was in the range of a tumor than at physiological pH. The bone-targeted formulation showed a strong affinity for hydroxyapatite. The encapsulation of DOX did not interfere in its intrinsic cytotoxicity against the MDA-MB-231 cell line. Biodistribution studies demonstrated high affinity of this formulation for tumors and reduction of uptake in the heart.ConclusionThese results suggest that bone-targeted pH-sensitive liposomes containing DOX can be an interesting strategy for selectively delivering this drug into bone-tumor sites, increasing its activity, and reducing DOX-related toxicity.

An indirect evaluation of bone saturation with zoledronic acid after long-term Q4 week dosing using plasma and urine pharmacokinetics.

537 Background: Zoledronic acid (ZA) has a high binding affinity for human bone. This post-hoc analysis from the OPTIMIZE-2 trial evaluated whether there is evidence for bone saturation of ZA follo...

PTH(1-34) Treatment Increases Bisphosphonate Turnover in Fracture Repair in Rats.

Bisphosphonates (BP) are antiresorptive drugs with a high affinity for bone. Despite the therapeutic success in treating osteoporosis and metabolic bone diseases, chronic BP usage has been associated with reduced repair of microdamage and atypical femoral fracture (AFF). The latter has a poor prognosis, and although anabolic interventions such as teriparatide (PTH(1-34) ) have been suggested as treatment options, there is a limited evidence base in support of their efficacy. Because PTH(1-34) acts to increase bone turnover, we hypothesized that it may be able to increase BP in turnover in the skeleton, which, in turn, may improve bone healing. To test this, we employed a mixture of fluorescent Alexa647-labelled pamidronate (Pam) and radiolabeled (14) C-ZA (zoledronic acid). These traceable BPs were dosed to Wistar rats in models of normal growth and closed fracture repair. Rats were cotreated with saline or 25 μg/kg/d PTH(1-34) , and the effects on BP liberation and bone healing were examined by X-ray, micro-CT, autoradiography, and fluorescent confocal microscopy. Consistent with increased BP remobilization with PTH(1-34) , there was a significant decrease in fluorescence in both the long bones and in the fracture callus in treated animals compared with controls. This was further confirmed by autoradiography for (14) C-ZA. In this model of acute BP treatment, callus bone volume (BV) was significantly increased in fractured limbs, and although we noted significant decreases in callus-bound BP with PTH(1-34) , these were not sufficient to alter this BV. However, increased intracellular BP was noted in resorbing osteoclasts, confirming that, in principle, PTH(1-34) increases bone turnover as well as BP turnover.

Targeted delivery of mesenchymal stem cells to the bone

Osteoporosis is a disease of excess skeletal fragility that results from estrogen loss and aging. Age related bone loss has been attributed to both elevated bone resorption and insufficient bone formation. We developed a hybrid compound, LLP2A–Ale in which LLP2A has high affinity for the α4β1 integrin on mesenchymal stem cells (MSCs) and alendronate has high affinity for bone. When LLP2A–Ale was injected into mice, the compound directed MSCs to both trabecular and cortical bone surfaces and increased bone mass and bone strength. Additional studies are underway to further characterize this hybrid compound, LLP2A–Ale, and how it can be utilized for the treatment of bone loss resulting from hormone deficiency, aging, and inflammation and to augment bone fracture healing. This article is part of a Special Issue entitled “Stem Cells and Bone”.

Immunosuppressed Microenvironment – An Emerging Target in Prostate Cancer Management

Although the prognosis of metastatic castrate-resistant prostate cancer (mCRPC) has dramatically changed in the last decade, with median survivals improving from about a year to almost 3 years, current hormonal and chemotherapeutic approaches ultimately result in resistance. An enhanced understanding of the microenvironment of prostate cancer may explain the mechanisms underlying this resistance and provide novel therapeutic targets. The tumour microenvironment promotes the growth and spread of prostate cancer through suppression of immune responses. Many cellular and molecular components of the immunosuppressed tumour microenvironment have been identified as potential targets for therapeutic intervention, including myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAMs), toll-like receptors (TLRs) and the pro-inflammatory protein S100A9. Several agents have demonstrated an ability to modulate the tumour cell microenvironment, including immunotherapies such as sipuleucel T and ipilimumab. In preclinical models, tasquinimod has been shown to bind to S100A9 and therefore has the potential to affect accumulation and function of MDSCs as well as enhancing anti-tumour immune responses. It is now in phase III development. The bone microenvironment also represents a valuable therapeutic target: in clinical studies, denosumab, a rank-L inhibitor, delays time to first skeletal event, despite showing no improvement in overall survival (OS). Radium-223, an alpha-emitter with high bone affinity, delays bone metastasis as well as significantly improving OS.

Bilateral atypical femoral fractures in a patient with multiple myeloma treated with intravenous bisphosphonate therapy.

Bisphosphonates are currently the standard approach to managing bone disease in multiple myeloma. Bisphosphonates have high bone affinity that inhibits osteoclastic activity and additionally reduces the growth factors released from malignant or osteoblastic cells, thereby impairing abnormal bone remodeling which leads to osteolysis. However, patients of multiple myeloma may be at a higher risk of atypical femoral fractures because the treatment for malignant myeloma requires notably higher cumulative doses of bisphosphonates. Here we present a patient with bilateral atypical femoral fractures and multiple myeloma treated with intravenous bisphosphonate therapy.

Reversing bone loss by directing mesenchymal stem cells to bone

Bone regeneration by systemic transplantation of mesenchymal stem cells (MSCs) is problematic due to the inability to control the MSCs' commitment, growth, and differentiation into functional osteoblasts on the bone surface. Our research group has developed a method to direct the MSCs to the bone surface by conjugating a synthetic peptidomimetic ligand (LLP2A) that has high affinity for activated α4β1 integrin on the MSC surface, with a bisphosphonates (alendronate) that has high affinity for bone (LLP2A-Ale), to direct the transplanted MSCs to bone. Our in vitro experiments demonstrated that mobilization of LLP2A-Ale to hydroxyapatite accelerated MSC migration that was associated with an increase in the phosphorylation of Akt kinase and osteoblastogenesis. LLP2A-Ale increased the homing of the transplanted MSCs to bone as well as the osteoblast surface, significantly increased the rate of bone formation and restored both trabecular and cortical bone loss induced by estrogen deficiency or advanced age in mice. These results support LLP2A-Ale as a novel therapeutic option to direct the transplanted MSCs to bone for the treatment of established bone loss related to hormone deficiency and aging.

Targeting integrins to promote bone formation and repair

The ageing skeleton experiences a progressive decline in the rate of bone formation, which can eventually result in osteoporosis--a common disease characterized by reduced bone mass and altered bone microarchitecture which can result in fractures. One emerging therapy involves the identification of molecules that target bone-marrow mesenchymal stromal cells (MSCs) and promote their differentiation into osteoblasts, thereby counteracting bone loss. This Review highlights the discovery that some integrins, a family of heterodimeric transmembrane proteins that can interact with matrix proteins and generate intracellular signals, can be targeted to promote homing of MSCs to bone, osteogenic differentiation and bone formation. Specifically, priming of the α(5)β(1) integrin, which is required for osteoblastic differentiation of MSCs, leads to increased bone formation and improved bone repair in mice. Additionally, treatment with a peptidomimetic ligand of the α(4)β(1) integrin coupled to an agent with a high affinity for bone improves the homing of MSCs to bone and promotes osteoblast differentiation and bone formation, leading to increased bone mass in osteopenic mice. Strategies that target key integrins expressed by MSCs might, therefore, translate into improved therapies for age-related bone loss and possibly other disorders.

Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass

Aging reduces the number of mesenchymal stem cells (MSCs) in the bone marrow which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct the MSCs to the bone surface by attaching a synthetic high affinity and specific peptidomimetic ligand (LLP2A) against integrin α4β1 on the MSC surface, to a bisphosphonate (alendronate, Ale) that has high affinity for bone. LLP2A-Ale increased MSCs migration and osteogenic differentiation in vitro. A single intravenous injection of LLP2A-Ale increased trabecular bone formation and bone mass in both xenotransplantation and immune competent mice. Additionally, LLP2A-Ale prevented trabecular bone loss after peak bone acquisition was achieved or following estrogen deficiency. These results provide a proof of principle that LLP2A-Ale can direct MSCs to the bone to form new bone and increase bone strength.

Synthesis and evaluation of a novel 68Ga-chelate-conjugated bisphosphonate as a bone-seeking agent for PET imaging

(68)Ga is a positron-emitting nuclide that has significant imaging potential given that, unlike cyclotron-produced (18)F, the isotope can be produced on-site utilizing a (68)Ge/(68)Ga generator. We recently synthesized a novel bone-seeking agent by coupling a bisphosphonate with the (68)Ga chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). This study presents a first report on the potential of this (68)Ga bone-seeking radiopharmaceutical in the detection of bone metastases. 4-Amino-1-hydroxybutylidene-1,1-bisphosphonate was conjugated with 2-[4,7-di(carboxymethyl)-1,4,7-triazonan-1-yl]pentanedioic acid, yielding 2-[4,7-di(carboxymethyl)-1,4,7-triazonan-1-yl]-5-[(4-hydroxy-4,4-diphosphonobutyl)amino]-5-oxopentanoic acid (NOTA-BP). (68)Ga-labeled NOTA-BP ([(68)Ga]NOTA-BP) was prepared by complexation of NOTA-BP with [(68)Ga] gallium chloride and evaluated in in vitro experiments, biodistribution experiments and micro-positron emission tomography (PET) imaging experiments. The labeling of NOTA-BP with (68)Ga was completed by heating for 10 min. [(68)Ga]NOTA-BP was determined to have a radiochemical purity of over 95%, a high affinity for hydroxyapatite and a high stability in plasma. In in vivo biodistribution experiments, [(68)Ga]NOTA-BP demonstrated high bone uptake potential. Compared with (99m)Tc-labeled methylene diphosphonate ([(99m)Tc]MDP) and [(18)F]fluoride, [(68)Ga]NOTA-BP exhibited faster blood clearance and a higher bone-to-blood ratio. In addition, mouse model bone metastasis was detected by micro-PET imaging at 1 h postinjection of [(68)Ga]NOTA-BP. We have developed a novel (68)Ga-radiolabeled bone-seeking agent. This [(68)Ga]NOTA-BP complex was found to have a high bone affinity and rapid blood clearance, and may thus prove to be useful as a bone-seeking agent for clinical PET.

Preparation and evaluation of a radiogallium complex-conjugated bisphosphonate as a bone scintigraphy agent

(68)Ga is a radionuclide of great interest as a positron emitter for positron emission tomography (PET). To develop a new bone-imaging agent with radiogallium, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as a chelating site and Ga-DOTA complex-conjugated bisphosphonate, which has a high affinity for bone, was prepared and evaluated. Although we are interested in developing (68)Ga-labeled bone imaging agents for PET, in these initial studies (67)Ga was used because of its longer half-life. DOTA-conjugated bisphosphonate (DOTA-Bn-SCN-HBP) was synthesized by conjugation of 2-(4-isothiocyanatebenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). (67)Ga-DOTA-Bn-SCN-HBP was prepared by coordination with (67)Ga, and its in vitro and in vivo evaluations were performed. (67)Ga-DOTA-Bn-SCN-HBP was prepared with a radiochemical purity of over 95% without purification. (67)Ga-DOTA-Bn-SCN-HBP had great affinity for hydroxyapatite in binding assay. In biodistribution experiments, (67)Ga-DOTA-Bn-SCN-HBP accumulated in bone rapidly but was hardly observed in tissues other than bone. Pretreatment of an excess amount of alendronate inhibited the bone accumulation of (67)Ga-DOTA-Bn-SCN-HBP. (67)Ga-DOTA-Bn-SCN-HBP showed ideal biodistribution characteristics as a bone-imaging agent. These findings should provide useful information on the drug design of bone imaging agents for PET with (68)Ga.

Cancer‐induced bone loss and associated pain‐related behavior is reduced by risedronate but not its phosphonocarboxylate analog NE‐10790

Prostate, breast and lung cancers readily develop bone metastases which lead to fractures, hypercalcemia and pain. Malignant growth in the bones depends on osteoclast-mediated bone resorption and in this regard bisphosphonate compounds, which have high-bone affinity and inhibit osteoclast activity, have been found to alleviate bone cancer symptoms. In this study, the bisphosphonate risedronate and its phosphonocarboxylate derivative NE-10790 was tested in a murine bone cancer pain model. Risedronate decreased bone cancer-related bone destruction and pain-related behavior and decreased the spinal expression of glial fibrillary acidic protein, whereas NE-10790 had no effect on these parameters. Furthermore, risedronate but not NE-10790 induced dose-dependent toxicity in NCTC-2472 cells in vitro. Furthermore, the direct toxic effect of risedronate on tumor cells observed in vitro opens the possibility that a direct toxic effect on tumor cells may also be present in vivo and be related to the efficacy of bisphosphonate compounds. In conclusion, these results suggest that risedronate treatment may lead to an increased life quality, in patient suffering from bone cancer, in terms of decreased osteolysis and pain, and merits further study.

Phase II Trial of Consolidation Docetaxel and Samarium-153 in Patients With Bone Metastases From Castration-Resistant Prostate Cancer

To assess docetaxel combined with samarium-153-ethylene diamine tetramethylene phosphonic acid (EDTMP), a radiopharmaceutical with a high affinity for bone, in patients with castration-resistant prostate cancer (CRPC). Patients with bone metastases from CRPC who achieved a response or stabilization after four cycles of docetaxel and estramustine were given consolidation docetaxel 20 mg/m(2)/wk for 6 weeks and samarium-153-EDTMP (37 MBq/kg) during week 1. Prostate-specific antigen (PSA) response was assessed by using consensus criteria, and pain was assessed by using a visual analog scale (VAS). This study used a Simon two-step design with PSA-progression-free survival (PFS) as the primary end point. Forty-three patients were included in the trial. A PSA response was obtained in 77% (95% CI, 61% to 82%). The pain response rate was 69% (95% CI, 49% to 85%). At least five of the six planned weekly injections of docetaxel were administered to 34 patients (81%). The consolidation docetaxel-samarium-153-EDTMP regimen was well tolerated; there was no febrile neutropenia, and only two episodes (5%) of rapidly reversible grade 3 thrombocytopenia occurred. Although a serum PSA relapse eventually occurred in all patient cases, this regimen resulted in pain control in the long-term. The median PSA-PFS was 6.4 months (95% CI, 6 to 7 months). The median survival was 29 months (95% CI, 22 to 31); the 1-year survival rate was 77% (62% to 87%); and the 2-year survival rate was 56% (41% to 70%). Combining docetaxel and samarium-153-EDTMP in patients with bone metastases from CRPC is well tolerated, and it yields major pain relief that persists long after treatment. Overall survival compares favorably with that expected in this population of patients, most of whom exhibit symptoms.

Synthesis of tetracycline analogs and their bone affinities

Tetracycline analogs were designed and synthesized and their bone affinities were tested on hydroxyapatite. The results showed that the carbonyl-amide-enol structure in A ring and phenol–ketone structure in BCD ring may be responsible for tetracycline's high bone affinity and either A ring or BCD ring has a planar conformation is essential.

Synthesis of Conjugatable Bisphosphonates for Molecular Imaging of Large Animals

Synthesis of Conjugatable Bisphosphonates for Molecular Imaging of Large Animals

Recombinant Expression, Isolation, and Proteolysis of Extracellular Matrix-Secreted Phosphoprotein-24 kDa

Secreted phosphoprotein-24 kDa (spp24) is an extracellular matrix protein first cloned from bone. Bovine spp24 is transcribed as a 203 amino acid residue protein that undergoes cleavage of a secretory peptide to form the mature protein (spp24, residues 24 to 203). While not osteogenic itself, spp24 is degraded to a pro-osteogenic protein, spp18.5, in bone. Both spp18.5 and spp24 contain a cyclic TRH1 (TGF-beta receptor II homology-1) domain similar to that found in the receptor itself and in fetuin. A synthetic peptide corresponding to the TRH1 domain of spp18.5 and spp24 specifically binds BMP-2 and enhances the rate and magnitude of BMP-2-induced ectopic bone formation in vivo. The parental protein, spp24, exhibits a high affinity for bone and mineral complexes, but its abundance there is low, suggesting that it is rapidly degraded. The availability of recombinant spp24 and its degradation products would facilitate the elucidation of their structure:function relationships. We describe here the expression of His6-tagged bovine spp24 (residues 24 to 203) in E. coli, its purification by high-resolution IMAC (immobilized metal affinity chromatography), and the characterization of the full-length recombinant 21.5 kDa protein and its two major 16 kDa and 14.5 kDa degradation products (spp24, residues 24 to 157, and spp24, residues 24 to 143) by mass spectroscopy. The recombinant spp24 protein was resistant to proteolysis by MC3T3-E1 osteoblastic cell extracts in the absence of calcium; however, in the presence of 4 mM Ca, it can undergo essentially complete proteolysis to small peptides, bypassing the 16 kDa and 14.5 kDa intermediates. This confirms the proteolytic susceptibility of spp24. It also suggests that the levels of spp24 in bone may be regulated, in part, by calcium-dependent proteolysis mediated by osteoblastic cells.

Organ Regeneration in Porous Hydoroxyapatite

HA has a high affinity for bone as well as various tissues. In the present study, we investigated an affinity for abdominal organs. Coralline hydroxyapatite ceramic (HA, cubic structure 4x4x4mm, Interpore 500) was used in this experiment. We made two incisions in the lower back of a 5-week-old male nude mouse, and implanted HA blocks. One was placed around the liver at the right side and another one was placed around the kidney at the left side. The organ fibrous capsule was not removed. At 6 weeks after implantation, mice were sacrificed under overanesthesia and HA blocks were retrieved and prepared for histological analysis. In the HE stain of HA blocks around liver, liver tissue is invaded into the HA pore areas. Hepatocyte proliferation in trabecular pattern was seen in contact with the surfaces of many HA pores. Within some pores, hepatic lobular pattern, Glisson sheath or central vein could be detected. In the HA around kidney, renal tissue was observed in many pores. The pore areas of HA were fullfilled with grumerulus and urinary tube tissues. In contact with the surfaces of some HA blocks, the tissue invasion of pancreas and spleen tissue were recognized. These results indicate that porous HA has a high affinity for the celiac organs, and has a stimulatory effect on celiac organ regeneration. Especially, concerning the regeneration of kidney, it has not been reported yet, so this report is very interesting. HA is also very useful as a scaffold of the organ regeneration.