The objective of this research proposal is to improve the early detection and characterization of various tumors, in particular, breast cancers by developing sigma receptor-specific radiopharmaceuticals that would bind to breast tumor cells and breast biopsied tumor membrane preparations with very high affinity and possess a low non-specific binding component and be used in conjunction with SPECT technology for non-invasive imaging. The applicants reported having recently characterized the expression of a high density of sigma receptors on MCF-7 (adenocarcionma), T47D (ductal), and MDA MB231 (adenocarcinoma) breast tumor cells and human prostate tumor cells (LN CaP) using tritiated sigma ligands, [H-3]1,3-di-tolylguanidine (DTG). Furthermore, the applicants reported having synthesized several radioiodinated radiopharmaceuticals that posess high affinity at both sigma-1 and simga-2 receptors. The applicants also recently reported the binding characteristics of these novel radiopharmaceuticals to sigma receptor on these tumor cells. The in vitro binding of radio-iodinated IBP's and IDEMP in biopsied breast tumor membrane preparations (from four patients) showed a high affinity, saturable, dose dependent binding with sigma ligands such as haloperidol and IBP's. The Scatchard plot for the binding of [3H]DTG to membrane preparations of breast tumor from a patient showed saturable, high affinity binding (Kd=57.3 nM, Bmax = 9070 fmol/mg protein). The study of membrane preparations obtained from normal breast tissue (two patients) revealed no detectable specific binding. The novel Tc-99m labeled sigma receptor-binding chelate [Tc-99m]EN6 was shown to exhibit specific binding to human ductal breast cancer cell (T47D) membranes. Scatard's analysis of [Tc-99m]EN6 (spiked with [Tc-99g]EN6 binding in T47D breast cancer cells, showed a saturable binding, with a Kd = 43 +/-14.7 nM and a Bmax = 3121+/-130 fmol/mg protein. The applicants proposed to synthesize several classes of Tc-99m labeled chelates possessing sigma pharmacophores. The radiolabeled compounds will be purified using HPLC methods and characterized by the comparison of retention time of authentic Tc-99g compounds under identical conditions. The sigma-1 and sigma-2 binding affinities of Tc-99m and Tc-99g chelates will be determined in guinea pig brain membranes and rat liver membranes respectively. For the radiolabeled ligands possessing high in vitro affinity and low non-specific binding, the in vivo clearance and pharmacokinetics will be studied in Sprague-Dawley rats. Based upon these results of in vitro binding studies in different pharmacological assays, and the results of biodistribution studies in rats, nude mice will be inoculated with breast tumors and the uptake and retention of the selected radiopharmaceutical probes will be studied. These studies were projected to enable the applicants to develop optimal radiopharmaceutical for future Phase I clinical trials.
