Preparation, Optimisation, and In Vitro Evaluation of [18F]AlF-NOTA-Pamidronic Acid for Bone Imaging PET.

[18F]sodium fluoride ([18F]NaF) is recognised to be superior to [99mTc]-methyl diphosphate ([99mTc]Tc-MDP) and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) in bone imaging. However, there is concern that [18F]NaF uptake is not cancer-specific, leading to a higher number of false-positive interpretations. Therefore, in this work, [18F]AlF-NOTA-pamidronic acid was prepared, optimised, and tested for its in vitro uptake. NOTA-pamidronic acid was prepared by an N-Hydroxysuccinimide (NHS) ester strategy and validated by liquid chromatography-mass spectrometry analysis (LC-MS/MS). Radiolabeling of [18F]AlF-NOTA-pamidronic acid was optimised, and it was ensured that all quality control analysis requirements for the radiopharmaceuticals were met prior to the in vitro cell uptake studies. NOTA-pamidronic acid was successfully prepared and radiolabeled with 18F. The radiolabel was prepared in a 1:1 molar ratio of aluminium chloride (AlCl3) to NOTA-pamidronic acid and heated at 100 °C for 15 min in the presence of 50% ethanol (v/v), which proved to be optimal. The preliminary in vitro results of the binding of the hydroxyapatite showed that [18F]AlF-NOTA-pamidronic acid was as sensitive as [18F]sodium fluoride ([18F]NaF). Normal human osteoblast cell lines (hFOB 1.19) and human osteosarcoma cell lines (Saos-2) were used for the in vitro cellular uptake studies. It was found that [18F]NaF was higher in both cell lines, but [18F]AlF-NOTA-pamidronic acid showed promising cellular uptake in Saos-2. The preliminary results suggest that further preclinical studies of [18F]AlF-NOTA-pamidronic acid are needed before it is transferred to clinical research.

Preparation, Characterization, and Radiolabeling of [68Ga]Ga-NODAGA-Pamidronic Acid: A Potential PET Bone Imaging Agent.

Early diagnosis of bone metastases is crucial to prevent skeletal-related events, and for that, the non-invasive techniques to diagnose bone metastases that make use of image-guided radiopharmaceuticals are being employed as an alternative to traditional biopsies. Hence, in the present work, we tested the efficacy of a gallium-68 (68Ga)-based compound as a radiopharmaceutical agent towards the bone imaging in positron emitting tomography (PET). For that, we prepared, thoroughly characterized, and radiolabeled [68Ga]Ga-NODAGA-pamidronic acid radiopharmaceutical, a 68Ga precursor for PET bone cancer imaging applications. The preparation of NODAGA-pamidronic acid was performed via the N-Hydroxysuccinimide (NHS) ester strategy and was characterized using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MSn). The unreacted NODAGA chelator was separated using the ion-suppression reverse phase-high performance liquid chromatography (RP-HPLC) method, and the freeze-dried NODAGA-pamidronic acid was radiolabeled with 68Ga. The radiolabeling condition was found to be most optimum at a pH ranging from 4 to 4.5 and a temperature of above 60 °C. From previous work, we found that the pamidronic acid itself has a good bone binding affinity. Moreover, from the analysis of the results, the ionic structure of radiolabeled [68Ga]Ga-NODAGA-pamidronic acid has the ability to improve the blood clearance and may exert good renal excretion, enhance the bone-to-background ratio, and consequently the final image quality. This was reflected by both the in vitro bone binding assay and in vivo animal biodistribution presented in this research.

Bisphosphonates in breast cancer.

Bisphosphonates are osteoclast inhibitors, currently being used in oncology to prevent or delay bone morbidity in cancer. Oral and intravenous formulations of bisphosphonates have been found to be efficacious in preventing skeletal-related events such as bone pain, pathologic fractures, spinal cord compression and hypercalcemia of malignancy, in patients with bone metastatic breast cancer. Bisphosphonates are also used to prevent bone loss associated with anti-estrogen therapy using aromatase inhibitors. In addition to its role in preventing bone resorption, several pre-clinical studies have noted an anti-tumor role as well. Recent research effort has particularly focused on investigating an adjuvant role for bisphosphonates in early breast cancer. Recently, few randomized trials have found a beneficial effect for adjuvant use of the aminobisphosphonate, zoledronate, in older patients who are post-menopausal. This review article will summarize the various clinical studies investigating the role of bisphosphonates in breast cancer.

In vitro Effect of Geranylgeraniol (GGOH) on Bisphosphonate-Induced Cytotoxicity of Oral Mucosa Cells.

Medication-related osteonecrosis of the jaw (MRONJ) is an often-severe complication found in patients receiving bisphosphonates in the management of Paget's, osteoporosis and metastatic bone cancer. Mucosal breakdown with bone exposure is a primary clinical presentation of MRONJ linked to the inhibitory effect of nitrogen-containing bisphosphonates (N-BP) on the mevalonate pathway. Geranylgeraniol (GGOH) has demonstrated a rescue effect on N-BP-treated osteoclasts but the biological effects on oral soft tissues and cells remain unclear. This study aimed to determine whether GGOH could prevent bisphosphonate induced toxicity to oral mucosa cells in vitro. Primary oral fibroblasts and keratinocytes were exposed to different GGOH concentrations or GGOH in combination with two nitrogen-containing bisphosphonates, zoledronic acid (ZA) or pamidronic acid (PA), for 72 h. The metabolic activity of each cell type was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GGOH without bisphosphonates significantly reduced the metabolic activity of oral mucosa cells. Fibroblasts treated with GGOH and ZA in combination showed a slight increase in metabolic status compared to fibroblasts treated with ZA alone, however this positive effect was not observed in keratinocytes. In the presence of PA, GGOH was unable to increase the metabolic activity of either cell type. These findings demonstrate that GGOH is toxic to oral mucosa cells and that GGOH was not able to prevent bisphosphonate induced toxicity. These data show that GGOH does not have therapeutic potential for bisphosphonate-induced soft tissue toxicity in MRONJ and the use of GGOH as an MRONJ treatment should be strongly reconsidered.

Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro.

Medication-related osteonecrosis of the jaw (MRONJ) is a side effect of bisphosphonate therapy, characterised by exposed necrotic bone. The soft tissues of the oral mucosa no longer provide a protective barrier and MRONJ patients experience pain, infections and difficulties eating. We hypothesised that hydroxyapatite (Ca5(PO4)3(OH)) could reduce bisphosphonate concentrations and protect the oral mucosa by exploiting bisphosphonate's calcium binding affinity. The effect of zoledronic acid (ZA) and pamidronic acid (PA) on the metabolism of oral fibroblasts, oral keratinocytes and three-dimensional oral mucosa models was investigated and then repeated in the presence of hydroxyapatite granules. Without hydroxyapatite, ZA and PA significantly reduced the metabolic activity of oral cells in a dose-dependent manner. Both drugs reduced epithelial thickness and 30 µM ZA resulted in loss of the epithelium. Hydroxyapatite granules had a protective effect on oral cells, with metabolic activity retained. Oral mucosa models retained their multi-layered epithelium when treated with ZA in the presence of hydroxyapatite granules and metabolic activity was comparable to controls. These results demonstrate hydroxyapatite granules protected oral soft tissues from damage caused by bisphosphonate exposure. Porous hydroxyapatite granules are currently used for socket preservation and this data suggests their potential to prevent MRONJ in at-risk patients.

Preparation and evaluation of rhenium-188-pamidronate as a palliative treatment in bone metastasis.

OBJECTIVE: Rhenium-188-hydroxyethylidene diphosphonate (188Re-HEDP) as a first generation bisphosphonate has been widely used for bone seeking radiopharmaceutical in cases of metastatic bone disease. No study has been yet reported on preparing a complex of 188Re with pamidronate (3-aminohydroxypropylidene-1,1-bisphosphonic acid) (PMA) as a second generation bisphosphonate. Based on this fact, it was hypothesized that a bone-seeking 188Re-PMA radiopharmaceutical could be developed as an agent for palliative radiotherapy of bone pain due to skeletal metastases. METHODS: Pamidronate was labeled with 188ReO4- eluted from the alumina based 188W/188Re generator. Labeling was optimized, and radiochemical analysis was performed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Biodistribution of this radioconjugate was evaluated and verified further in mice. RESULTS: 188Re-PMA was prepared successfully in a high labeling yield (˃95%) corresponding to a specific activity of 124MBq/µmol and good in vitro stability, but it is likely to consist of multiple species. In biodistribution studies selective uptake and retention of activity in the skeletal system (0.81±0.25% ID/g and 0.57±0.16 at 4 and 48h in bone post injection respectively) followed by clearance in the soft tissues were observed. CONCLUSION: These results show that due to its biological capabilities it would be advantageous to use 188Re-PMA for bone pain palliation therapy.

A preliminary study of pamidronic acid downregulation of angiogenic factors IGF-1/PECAM-1 expression in circulating level in bone metastatic breast cancer patients.

OBJECTIVE: To evaluate the expressions of circulating angiogenic factors affected by pamidronic acid (PA) intravenous infusion in bone metastatic breast cancer patients and the impact on their prognosis. METHODS: Peripheral blood of ten bone metastatic breast cancer patients was collected for serum insulin-like growth factor-1 (IGF-1) and platelet endothelial cell adhesion molecule-1 expression detection just before and 2 days after PA infusion. RESULTS: Both IGF-1 and platelet endothelial cell adhesion molecule-1 concentrations decreased after PA treatment for 48 hours (P<0.05). Modification was defined as >20% decrease recorded 2 days after PA administration. The decrease of IGF-1 was more significant in breast cancer patients who had received previous hormonotherapy. Moreover, the progression-free survival of first-line chemotherapy treatment of IGF-1 modified patients was longer than that of IGF-1 unmodified patients (P=0.009). CONCLUSION: PA treatment could suppress circulating serum IGF-1 and platelet endothelial cell adhesion molecule-1 concentrations; moreover, the prognosis of patients in IGF-1 unmodified group was relatively poor.