Hybrid Tracers Based on Cyanine Backbones Targeting Prostate-Specific Membrane Antigen: Tuning Pharmacokinetic Properties and Exploring Dye–Protein Interaction

Prostate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)–targeted radiotracers, for example, ^99mTc-labeled PSMA tracer analogs for radioguided surgery. The purpose of this study was to develop a second-generation ^99mTc-labeled PSMA-targeted tracer incorporating a fluorescent dye. Methods: Several PSMA-targeted hybrid tracers were synthesized: glutamic acid-urea-lysine (EuK)-Cy5-mas₃, EuK-(SO₃)Cy5-mas₃, EuK-Cy5(SO₃)-mas₃, EuK-(Ar)Cy5-mas₃, and EuK-Cy5(Ar)-mas₃; the Cy5 dye acts as a functional backbone between the EuK targeting vector and the 2-mercaptoacetyl-seryl-seryl-seryl (mas₃) chelate to study the dye's interaction with PSMA's amphipathic entrance funnel. The compounds were evaluated for their photophysical and chemical properties and PSMA affinity. After radiolabeling with ^99mTc, we performed in vivo SPECT imaging, biodistribution, and fluorescence imaging on BALB/c nude mice with orthotopically transplanted PC346C tumors. Results: The dye composition influenced the photophysical properties (brightness range 0.3–1.5 × 10⁴ M⁻¹ × cm⁻¹), plasma protein interactions (range 85.0% ± 2.3%–90.7% ± 1.3% bound to serum, range 76% ± 0%–89% ± 6% stability in serum), PSMA affinity (half-maximal inhibitory concentration [IC₅₀] range 19.2 ± 5.8–175.3 ± 61.1 nM) and in vivo characteristics (tumor-to-prostate and tumor-to-muscle ratios range 0.02 ± 0.00–154.73 ± 28.48 and 0.46 ± 0.28–5,157.50 ± 949.17, respectively; renal, splenic, and salivary retention). Even though all tracer analogs allowed tumor identification with SPECT and fluorescence imaging, ^99mTc-EuK-(SO₃)Cy5-mas₃ had the most promising properties (e.g., half-maximal inhibitory concentration, 19.2 ± 5.8, tumor-to-muscle ratio, 5,157.50 ± 949.17). Conclusion: Our findings demonstrate the intrinsic integration of a fluorophore in the pharmacophore in PSMA-targeted small-molecule tracers. In this design, having 1 sulfonate on the indole moiety adjacent to EuK (^99mTc-EuK-(SO₃)Cy5-mas₃) yielded the most promising tracer candidate for imaging of PSMA.