BACKGROUND-AIM Bisphosphonates such as zoledronic, alendronic and risedronic acids are a class of drugs clinically used to prevent bone density loss and osteoporosis. Based on their structure a new set of P-C P bisphosphonates (BPs) was projected to be then transformed in radiotracers, as the anion of Ra 223, in place of the two chlorides of 223Radium chloride transformed into 223Ra-bisphosphate to be used in the treatment of bone metastases derived from several types of tumors, i.e., prostate cancer. METHODS Suitable methodologies to prepare C-P-C BPs were developed. Human farnesyl pyrophosphate synthase (hFPPS) and geranylgeranyl pyrophosphate synthase (hGGPPS) were obtained by gene expression and protein purification: expression vectors containing cDNA sequences encoding for hFPPS or hGGPPS, pET-6xHis/hFPPS and pET-6xHis/ hGGPPS, and were individually used to transform E. coli BL21 competent cells. The expression and the purification methods were the same for both enzymes. IC50 values were determined using a colorimetric assay. Cytotoxic effect was evaluated by crystal violet assay and CCK-8 intracellular dehydrogenase assay [the activity of the intracellular dehydrogenases was assessed by using the Cell Counting Kit-8]. Hydroxyapatite affinity assay of BPs expressing affinity for bone mineral represents was accomplished to predict the in vivo interaction with bone tissue and eventually the possible clinical efficacy. Effects of novel BPs on mineralization of pre-osteblastic cell line: in the mineralization assay performed on pre-osteoblastic like cell line MC3T3-E1, the compounds under investigation were capable to induce osteoblastogenesis and mineralization in the nanomolar concentration range (30e500 nM) after 10-15 days of incubation of the cells with the mineralization medium (MM). Ex vivo investigation on the effects of BPs on murine bone marrow cells: in the mineralization assay, performed on bone marrow cells extracted from tibia and femur of the mice, the compounds under investigation were capable to induce osteoblasto genesis and mineralization in the nanomolar concentration range (30e50 nM), after 10-15 days of incubation of the cells with the MM. RESULTS Novel P-C-P bisphosphonates were synthesized for targeting hFPPS and hGGPPS, key enzymes of the mevalonate pathway, and capable of anti-proliferative action on a number of cell lines (PC3, MG63, MC3T3, RAW 264.7, J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone formation and death. Among sixteen compounds, [1-hydroxy-2-(pyrimidin-2 ylamino)ethane-1,1-diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in crystal-violet and cell-dehydrogenase activity assays at 100 mM concentration. 10 reduced differentiated osteoclasts number similarly with zoledronic acid in osteoclastogenesis assay. At nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in MC3T3 and murine bone marrow cells. 10 significantly inhibited the activity of hFPPS showing an IC50 of 0.31 mM and a remarkable hydroxyapatite binding of 90%. Docking calculations were performed identifying putative interactions between some novel bisphosphonates and both hFPPS and hGGPPS CONCLUSIONS The novel [1-hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-diyl]bis(phosphonic acid was found to behave similarly or even better than zoledronic acid as a antiresorptive agent. Attempts of its functionalization to subsequently complexation with stable divalent ions, barium or strontium simulating Ra 223, were accomplished.

NOVEL BISPHOSPHONATES, WITH ANTIRESORPTIVE EFFECT IN BONE MINERALIZATION AND OSTEOCLASTOGENESIS, AS MOIETIES OF RA 223-BASED RADIOTRACERS

A. Scilimati;M. G. Perrone;S. Ferorelli;
2019

Abstract

BACKGROUND-AIM Bisphosphonates such as zoledronic, alendronic and risedronic acids are a class of drugs clinically used to prevent bone density loss and osteoporosis. Based on their structure a new set of P-C P bisphosphonates (BPs) was projected to be then transformed in radiotracers, as the anion of Ra 223, in place of the two chlorides of 223Radium chloride transformed into 223Ra-bisphosphate to be used in the treatment of bone metastases derived from several types of tumors, i.e., prostate cancer. METHODS Suitable methodologies to prepare C-P-C BPs were developed. Human farnesyl pyrophosphate synthase (hFPPS) and geranylgeranyl pyrophosphate synthase (hGGPPS) were obtained by gene expression and protein purification: expression vectors containing cDNA sequences encoding for hFPPS or hGGPPS, pET-6xHis/hFPPS and pET-6xHis/ hGGPPS, and were individually used to transform E. coli BL21 competent cells. The expression and the purification methods were the same for both enzymes. IC50 values were determined using a colorimetric assay. Cytotoxic effect was evaluated by crystal violet assay and CCK-8 intracellular dehydrogenase assay [the activity of the intracellular dehydrogenases was assessed by using the Cell Counting Kit-8]. Hydroxyapatite affinity assay of BPs expressing affinity for bone mineral represents was accomplished to predict the in vivo interaction with bone tissue and eventually the possible clinical efficacy. Effects of novel BPs on mineralization of pre-osteblastic cell line: in the mineralization assay performed on pre-osteoblastic like cell line MC3T3-E1, the compounds under investigation were capable to induce osteoblastogenesis and mineralization in the nanomolar concentration range (30e500 nM) after 10-15 days of incubation of the cells with the mineralization medium (MM). Ex vivo investigation on the effects of BPs on murine bone marrow cells: in the mineralization assay, performed on bone marrow cells extracted from tibia and femur of the mice, the compounds under investigation were capable to induce osteoblasto genesis and mineralization in the nanomolar concentration range (30e50 nM), after 10-15 days of incubation of the cells with the MM. RESULTS Novel P-C-P bisphosphonates were synthesized for targeting hFPPS and hGGPPS, key enzymes of the mevalonate pathway, and capable of anti-proliferative action on a number of cell lines (PC3, MG63, MC3T3, RAW 264.7, J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone formation and death. Among sixteen compounds, [1-hydroxy-2-(pyrimidin-2 ylamino)ethane-1,1-diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in crystal-violet and cell-dehydrogenase activity assays at 100 mM concentration. 10 reduced differentiated osteoclasts number similarly with zoledronic acid in osteoclastogenesis assay. At nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in MC3T3 and murine bone marrow cells. 10 significantly inhibited the activity of hFPPS showing an IC50 of 0.31 mM and a remarkable hydroxyapatite binding of 90%. Docking calculations were performed identifying putative interactions between some novel bisphosphonates and both hFPPS and hGGPPS CONCLUSIONS The novel [1-hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-diyl]bis(phosphonic acid was found to behave similarly or even better than zoledronic acid as a antiresorptive agent. Attempts of its functionalization to subsequently complexation with stable divalent ions, barium or strontium simulating Ra 223, were accomplished.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11586/231606
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