Sarcosine-Based Pharmacokinetic Optimization and Fluorescent Dye Library Evaluation of Dual-Labeled PSMA Inhibitors...

Objectives: Fluorescence-guided surgery (FGS) targeting prostate-specific membrane antigen (PSMA) holds promise for improving surgical precision in prostate cancer. Since conjugation of fluorescent dyes to targeting vectors can substantially alter pharmacokinetic properties, we systematically evaluated a library of fluorescent dyes conjugated to a PSMA-617-derived scaffold incorporating sarcosine-based spacers to identify candidates with favorable biodistribution and optical profiles for clinical translation. Methods: Nineteen fluorescent dyes spanning NIR, large Stokes shift, and STED-compatible categories were conjugated to a dual-labeled PSMA-617-derived precursor (Glu-urea-Lys-2Nal-TXA-Sar₁₀-Lys(DOTA)-Sar₅-βAla; hereafter DP). Compounds were radiolabeled with ⁶⁸Ga or ¹⁷⁷Lu and characterized for serum stability, lipophilicity, binding affinity, and internalization in LNCaP^PSMA+ cells. In vivo pharmacokinetics were assessed in LNCaP xenograft-bearing BALB/c nu/nu mice by µPET/MRI (1 and 2 h p.i., 500 pmol ⁶⁸Ga), organ distribution (0.5, 1, and 2 h p.i., 60 pmol ¹⁷⁷Lu), and clinical-grade endoscopic fluorescence imaging. Results: All conjugates retained hydrophilic character (logD: -3.72 to -1.79), low nanomolar binding affinity (K_i: 18-87 nM), and high serum stability (94-100% intact at 24 h). Despite comparable in vitro properties, dye conjugation markedly influenced in vivo pharmacokinetics: tumor uptake at 2 h p.i. ranged from 1 to 23%ID/g and kidney accumulation from 3 to 82%ID/g. Visible-range dyes exhibited faster renal washout within the imaging window and higher tumor-to-background contrast than NIR fluorophores. Fluorescence signal intensity did not correlate with radiotracer-derived uptake, underscoring the importance of dye-specific photophysical properties. Conclusions: DP-12 (SulfoCy5), DP-15 (Alexa Fluor 647), and DP-18 (Tide Fluor 5WS) were identified as lead candidates combining favorable pharmacokinetics with strong fluorescence contrast, warranting further evaluation toward fluorescence-guided prostate cancer surgery.