Optimizing PSMA-617-based inhibitors through charged linker modifications: Insights into structure-activity relationships

<b>Rationale:</b> The introduction of Pluvicto^® ([¹⁷⁷Lu]Lu-vipivotide tetraxetan; [¹⁷⁷Lu]Lu-PSMA-617) marks a milestone in radioligand therapy (RLT) for PSMA-positive metastatic castration-resistant prostate cancer (mCRPC). While dose escalation of [¹⁷⁷Lu]Lu-PSMA-617 and alpha-emitting agents like [²²⁵Ac]Ac-PSMA-617 improves efficacy, it is limited by dose-dependent toxicity in critical organs, including kidneys, bone marrow and salivary glands. Modifications of the linker region in PSMA inhibitors have been proven to highly influence the pharmacokinetic profile. The utilization of charged linker moieties resulted in clinically used PSMA-targeting radiotracers such as [¹⁸F]PSMA-1007. This study explores histidine and/or glutamic acid-modified variants of PSMA-617 to investigate their effects on pharmacokinetic properties. <b>Methods:</b> Based on the core structure of PSMA-617, eleven novel PSMA-targeting inhibitors were synthesized by introducing histidine and/or glutamic acid moieties at three positions within the linker region. Compounds were radiolabeled with [⁶⁸Ga]Ga³⁺ and [¹⁷⁷Lu]Lu³⁺ to assess their chemical and stability properties. Biological activity was evaluated in competitive cell binding and internalization assays with PSMA-expressing LNCaP cells. Dynamic and static small-animal PET imaging studies were conducted with the ⁶⁸Ga-labeled inhibitors in LNCaP bearing BALB/c nu/nu xenografts to investigate their pharmacokinetic profiles. <b>Results:</b> Precursors of linker-modified PSMA inhibitors presented high radiochemical purities (RCPs) for the complexation reactions with both radionuclides (>94%). ⁶⁸Ga-labeled compounds demonstrated significantly lower lipophilicity (ranging from -3.4 to -3.9) compared to the reference compound [⁶⁸Ga]Ga-PSMA-617 (-2.8 ± 0.3). Substantial effects on the affinity to PSMA were observed depending on the position and nature of modification (IC₅₀ ranging from 10.40 ± 2.94 nM to 78.6 ± 44.1 nM). Modification with glutamic acid adjacent to the chelator resulted in a two-fold increase in affinity, while variants containing histidine and glutamic acid led to significant improvements in cell surface binding and internalization (<i>p</i> < 0.05). Dynamic small-animal PET scans with the novel ⁶⁸Ga-labeled variants revealed an improved accumulation in LNCaP xenograft tumors (SUV_{1 h}: 0.21 ± 0.05 to 1.32 ± 0.08 g/ml) accompanied by a fast clearance from the kidneys and background tissue within the initial 60 min. Static PET scans 2 h p.i. confirmed a high tumor uptake and a rapid renal excretion. <b>Conclusion:</b> The introduction of histidine and/or glutamic acid moieties into the linker region of PSMA-617 resulted in measurable changes in pharmacokinetic properties both <i>in vitro</i> and <i>in vivo</i>. While some modifications led to improved tumor-to-kidney ratios and favorable early-stage excretion, it remains challenging to predict clinical off-targeting effects like salivary gland uptake. This study provides important insights into the structure-activity relationships of PSMA-617-related linker modifications and warrant additional investigation, including mechanistic and translational studies, to more accurately evaluate their therapeutic potential.