99m Tc-Labeled Small-Molecule Inhibitors of Prostate-Specific Membrane Antigen for Molecular Imaging of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer, and small-molecule radiopharmaceuticals targeting PSMA rapidly detect the location and extent of disease. Here we evaluated preclinically 4 novel 99mTc-labeled small-molecule inhibitors of PSMA with the potential for clinical translation for molecular imaging of prostate cancer in humans. Methods: Four PSMA inhibitors derived from the glutamate-urea-glutamate or glutamate-urea-lysine pharmacophores conjugated to CIM or TIM chelators were radiolabeled with 99m Tc and evaluated in vitro and in vivo. Results: High-affinity, saturable binding to PSMA on LNCaP cells was observed with Kd values of 0.64 6 0.46 nM for 99m TcMIP-1427, 1.07 6 0.89 nM for 99m Tc-MIP-1404, 1.75 6 0.32 nM for 99m Tc-MIP-1428, and 4.35 6 0.35 nM for 99m Tc-MIP-1405. 99m Tc-labeled PSMA inhibitors did not bind human prostate cancer PC3 cells, which lack PSMA, demonstrating specificity, and binding was abolished with 2-(phosphonomethyl)pentanedioic acid (PMPA), a structurally unrelated PSMA inhibitor. 99m Tc-labeled PSMA inhibitors were shown to internalize at 37� C. Uptake in LNCaP xenografts ranged from 9.3% to 12.4% injected dose per gram at 1 h after injection and from 7.2% to 11.0% at 4 h, with tumor-to-blood ratios ranging from 29:1 to 550:1 and tumor–to– skeletal muscle ratios ranging from 31:1 to 157:1 at 4 h. 99m TcMIP-1404 exhibited the best combination of high tumor uptake and rapid clearance from kidney and nontarget tissues. 99mTcMIP-1404 specifically bound to PSMA in vivo as demonstrated by the absence of uptake in PC3 xenografts and by competition with PMPA. SPECT/CT imaging corroborated the tissue distribution results, demonstrating uptake only in PSMA-expressing kidney and tumor tissue and clearance through the urinary bladder. Conclusion: These 99mTc-labeled radiopharmaceuticals targeting