Preclinical evaluation of 99mTc-EC20 for imaging folate receptor-positive tumors

Abstract

Our group previously reported on the synthesis and characterization of a novel 99mTc-based folate-peptide chelator called EC20. This agent was found to bind folate receptor (FR)-positive cells and tissues with high affinity and was deemed useful for radiodiagnostic applications. In this study, we investigated the effect of D-amino acid substitution within EC20 on its tissue biodistribution. Expanded in vivo studies were also performed with unmodified 99mTc-EC20 to determine the effect of tumor FR expression, tumor size, tumor location, route of dose administration, and rodent diet on the agent's tissue biodistribution pattern. Methods: EC20 and EC53, the all-D-isomer of EC20, were synthesized and radiolabeled with 99mTc. The relative affinity of EC53 to the FR with respect to EC20 was then determined in cultured tumor cells. The ability of 99mTc-EC20 and 99mTc-EC53 to target tumors in vivo was examined using BALB/c mice with subcutaneously inoculated M109 or 4T1 cells, yielding 0.1- to 0.5-g tumors in 20 d. Results: The D-amino acid substitutions of EC20 were found to reduce the uptake of the agent into tumor and major organs. Subsequent studies using the original 99MTc-EC20 agent confirmed that its net tumor uptake was specific and proportional to FR expression levels in tumor cells as well as linear with respect to the overall tumor size. Further, 99mTc-EC20 uptake was found to be independent of both solid tumor location (intraperitoneal vs. subcutaneous) and the route of administration (intraperitoneal vs. intravenous). Interestingly, leucovorin supplementation of a commonly used folate-deficient laboratory chow had no effect on the agent's overall tissue biodistribution pattern. But, tumor-to-nontumor ratios could be increased up to 2.7-fold when 1 equivalent of free folic acid was coinjected with 99mTc-EC20. Conclusion: Taken together, these results confirm that 99mTc-EC20 has the potential to be a clinically useful noninvasive radiodiagnostic agent for detecting the locus of FR-positive cancers.