New bone seeking radiopharmaceuticals for the diagnosis & treatment of Osteosarcoma

Background: Osteosarcoma, the most common primary malignancy of bone, occurs predominantly during the pubertal growth spurt. With current multimodal diagnostics and treatment, about 70% of patients with localized disease can achieve long-term remissions. At diagnosis up to 90% of patients have micro-metastases and about 20% have clinically detectable metastases. In patients with metastases, the 5-year overall survival rate is no higher than 30%. Due to these facts, it is obvious, that there is the need for early detection of metastases, and more efficient therapy methods including targeted cancer therapy. All in all, this leads to a demand of new and more effective diagnostic and therapeutic tools for osteosarcoma. Various bone seekers have been used and studied for more than 35 years in nuclear medicine. However, their uptake mechanisms into osseous tissues are still discussed and partly unclear. Meanwhile, although [ 99m Tc]MDP is the so far gold standard for the diagnosis of most osseous malignancies, it is of limited value for the diagnosis of osteosarcoma, since it shows hardly no uptake in osteoid. But, as it is well known, in osteosarcoma, osteoid is mainly and predominantly built by the tumor cells, which underlines the crucial need for a more specific and efficient tracer for this highly malignant disease. Since therapy response and overall survival rates strongly depend on early detection of metastases and skip metastases, it is of special importance. Therefore, we propose that zoledronic acid (ZOL), an N-containing bisphosphonate (N-BP) will serve as such a tracer. Firstly, because it acts as a bisphosphonate (BP) and binds on hydroxyapatite, which leads to inhibition of osteoclastic bone resorption. Secondly, as an N-BP, it inhibits farnesylation and geranyl-geranylation pathways, leading indirectly to cytostatic and proapoptotic effects against human tumor cells. Its anticancer activities are undisputed. In consequence we hypothesize, that it will provide an ideal ligand for a bone-seeking tracer. Aims of the study: The development and evaluation of improved bone seekers, which will allow a more precise and earlier diagnosis of tumors and metastases, resulting in personalized treatment for osteosarcoma patients: 1. radiolabelling of ZOL with three different radionuclides ([99m Tc], [ 68Ga], [ 153 Sm]) 2. the comparative preclinical evaluation of tracers (a) - (c) using (a) in-vivo models (PET, SPECT) (b) pre- vivo bone models (c) in-vitro pathway model 3. formulation of tracers (a) - (c) into liposomal nanoparticles (a) assessment of the formulations (b) evaluation in an in-vivo model (PET, SPECT) Materials and Methods: Radiochemistry: Labelling of ZOL with 3 different radionuclides [ 99m Tc], [ 68Ga] and [ 153 Sm]; assessment of optimum reaction parameters & conditions and quality control. In-vivo model: Evaluation of the three tracers in a murine model displaying human osteosarcoma (tumor and metastases) via SPECT and PET. Pre-vivo bone model: Evaluation of the tracers in-vitro on human bone material and comparison with well known bone seekers. In-vitro pathway model: Exploration of the three tracers and the underlying cellular uptake mechanisms in osteosarcoma cell lines. Nanoparticle formulation: Formulation of the three tracers into a liposomal structure and evaluation in an in-vivo model via SPECT and PET. Conclusion and Outlook: On the basis of these evaluations we will be able to decide, whether radiolabelled ZOL will be applied in clinical studies.

Background: Osteosarcoma, the most common primary bone cancer, occurs mainly in young children and in puberty, as well as in adults. Patients who develop metastases have an especially low survival rate of under 25% in the course of 5 years. To date, no common diagnostic method reliably visualizes all metastases. Hence, there is a strong need for a tool that allows early detection of metastases and more efficient therapy including targeted cancer therapy. Zoledronic acid (ZOL) is the most potent bisphosphonate currently on the market. It shows high affinity towards the bone and leads to direct and indirect anti-tumor activities. Furthermore, preclinical studies proved its significant activity against lung metastases. Hence, we hypothesized that radiolabelled ZOL will be a suitable tracer for Osteosarcoma patients. Aim: The development of a radioactive tracer specifically binding to the tumor and the metastases of osteosarcoma patients. Hence, providing a new tool that would be of special importance to the patients and the clinicians, since therapy response and overall survival rates strongly depend on early detection of metastases. Main findings: High & selective bone uptake of [99mTc]ZOL in vitro (synthetic & human bone compartments) and in vivo (species: mice and dogs) Selective tumor uptake of the tracer in vivo (Veterinary Osteosarcoma patient: dog) Selective uptake on lung metastases in vitro (tissues of lung metastases of human OS patients) Additionally: High and selective uptake in arthritic areas in vivo and ex vivo (rheumatoid arthritis mouse model) Conclusion and Outlook: High selectivity and specificity of [99mTc]ZOL was confirmed in vitro, ex vivo and in vivo. Therefore, on the basis of these findings, [99mTc]ZOL will be further evaluated and applied in a (pre-)clinical study with dog patients from the Veterinary Medicine University. Dogs spontaneously develop osteosarcoma, hence they are an ideal animal model since their OS-tumors and metastases are directly comparable with the human pathology. Hence, the data obtained from the veterinary patients are fully translatable to humans. The outcome ultimately will benefit OS patients in general and the OS patients of the St. Annas Children Hospital.