Is Thiourea the Weak Link? An Investigation of the In Vitro and In Vivo Destabilization of [²⁰³Pb]- and [²¹²Pb]Pb²⁺...

Fibroblast activation protein (FAP) is overexpressed in a variety of cancers, making it an attractive target for bifunctional chelator-based radiopharmaceuticals. This study initially aimed to assess the effect of chelator structure on the biodistribution of ²⁰³Pb/²¹²Pb-labeled FAP inhibitor (FAPI) bioconjugates. However, suboptimal in vivo biodistribution and imaging results suggested the bioconjugate was unstable. RadioHPLC analysis of urine samples suggest the thiourea bond, formed during conjugation between an amine on the biomolecule, and an isothiocyanate-functionalized chelator, is unstable in vivo, resulting in detachment of the radiometal-chelator complex from the targeting vector, resulting in poor tumor accumulation. To determine whether this instability was specific to the FAPI system, a peptide-based (Cyclic melanocyte stimulating hormone, CycMSH) bioconjugate targeting the melanocortin-1 receptor was synthesized using the same thiourea linkage. Identical metabolites were observed, supporting the hypothesis that thiourea bonds are unstable in vivo with this theranostic isotope pair. Subsequently, the effect of bioconjugation chemistry, specifically thiourea and amide bonds, on the stability and biodistribution of ²⁰³Pb/²¹²Pb-labeled bioconjugates was assessed. Modifying the bioconjugation linker to be an amide bond, formed by utilizing a chelate containing an active ester instead of an isothiocyanate, led to significantly improved in vitro and in vivo stability, as demonstrated by radioHPLC and biodistribution and imaging studies in both models. These findings highlight the importance of the choice of bioconjugation chemistry in the development of lead-based radiopharmaceuticals and emphasize the importance of selecting stable linkages to ensure optimal radiometal retention and tumor targeting.