Reduction of Negative Charge in Mercaptoacetyl-Based Chelators Influences the Biodistribution of Prostate-Specific Membrane Antigen-Targeting Pseudopeptides Labeled with Technetium-99m

Prostate cancer (PCa) is the most common cancer and the second leading cause of death among men worldwide. Significant progress has been made in managing PCa by targeting the prostate-specific membrane antigen (PSMA), which holds great promise for improving the accuracy and effectiveness of diagnosis. Previously, we reported a high-affinity glutamate–urea–lysine (EuK)-based PSMA-targeting tracer, BQ0413, containing the maE₃ chelator for labeling with technetium-99m for single-photon emission tomography diagnostic imaging. BQ0413 demonstrated efficient tumor targeting in PCa patients with concomitant elevated activity retention in the kidneys, which is typical for EuK-based PSMA-targeting tracers. We hypothesized that a decrease in the tracer’s total negative charge, by substituting negatively charged glutamate residues in the maE₃ chelator with polar neutral serine, could decrease activity retention in the kidneys. The present study aimed to evaluate the tumor targeting and biodistribution profile of two new PSMA-targeting tracers, BQ0500 (maESE) and BQ0501 (maS₃), in comparison to BQ0413 (maE₃). Conjugates were successfully radiolabeled with technetium-99m and characterized in vitro and in vivo. [^99mTc]Tc-BQ0500 and [^99mTc]Tc-BQ0501 demonstrated PSMA-specific binding to PC3-pip cells with picomolar affinity; however, the affinity was 3–5-fold compromised in comparison with the reference [^99mTc]Tc-BQ0413. Full replacement of glutamate residues by serines in [^99mTc]Tc-BQ0501 resulted in an improved overall clearance from normal organs with a moderately increased accumulation of activity in the gastrointestinal tract. [^99mTc]Tc-BQ0501 demonstrated efficient tumor targeting and improved tumor-to-background ratios. These results suggest that chelator modifications, such as charge alteration, play a critical role in improving tumor targeting and pharmacokinetics for EuK-based PSMA-targeting tracers.