What a difference a methylene makes: replacing Glu with Asp or Aad in the Lys-urea-Glu pharmacophore of PSMA-targeting radioligands to reduce kidney and salivary gland uptake.

The aim of this study was to investigate the effect of replacing Glu in the Lys-urea-Glu PSMA-targeting pharmacophore of [Ga]Ga-HTK03041 with a close analog on the uptake of kidneys, salivary glands and PSMA-expressing tumor xenografts. HTK03161, HTK03149 and HTK03189A/B were obtained by replacing Glu in HTK03041 with Asp, Aad (L-2-aminoadipic acid) and Api (2-aminopimelic acid), respectively. PSMA binding affinities were measured by competition binding assays. PET imaging and biodistribution studies of Ga-labeled ligands were performed in LNCaP tumor-bearing mice. The best candidate HTK03149 was selected and radiolabeled with Lu, and SPECT imaging and biodistribution studies were performed in LNCaP tumor-bearing mice. Radiation dosimetry calculation was conducted using the OLINDA software. Radioligand therapy study was performed in LNCaP tumor-bearing mice treated with [Lu]Lu-HTK03149 (9.3-148 MBq), [Lu]Lu-PSMA-617 (37 MBq) or Lu-HTK03149 (500 pmol). PSMA binding affinities (K) of Ga-HTK03161, Ga-HTK03149, Ga-HTK03189A and Lu-HTK03149 were 3.88±0.66, 6.99±0.80, 550±35.7 and 1.57±0.42 nM, respectively. PET imaging showed that all Ga-labeled HTK03161, HTK03149 and HTK03189A/B were excreted mainly via the renal pathway and had minimal uptake in all organs/tissues including kidneys and salivary glands. Tumor xenografts were clearly visualized in PET images of [Ga]Ga-HTK03161 and [Ga]Ga-HTK03149 but were barely visualized using [Ga]Ga-HTK03189A/B. Tumor uptake values for [Ga]Ga-HTK03161, [Ga]Ga-HTK03149, [Ga]Ga-HTK0189A and [Ga]Ga-HTK03189B were 12.7±1.91, 19.1±6.37, 2.10±0.28 and 0.67±0.15 %IA/g, respectively at 1h post-injection, and their average kidney and salivary gland uptake values were 2.13-4.41 and 0.20-0.23 %IA/g, respectively. Longitudinal SPECT imaging studies showed that [Lu]Lu-HTK03149 was excreted mainly through the renal pathway with high uptake in LNCaP tumors and minimal uptake in all normal organs/tissues. The tumor uptake of [Lu]Lu-HTK03149 peaked at 4h post-injection (20.9±2.99 %IA/g) and the uptake was sustained over time. Compared to [Lu]Lu-PSMA-617, [Lu]Lu-HTK03149 had 145% increase in tumor absorbed dose but 70% less in kidney absorbed dose, leading to an 7.1-fold increase in tumor-to-kidney absorbed dose ratio. Radioligand therapy studies showed that only half of the [Lu]Lu-PSMA-617 injected dosage was needed for [Lu]Lu-HTK03149 to achieve the same median survival. Replacing Glu in the PSMA-targeting Lys-urea-Glu pharmacophore of [Ga]Ga-HTK03041 with Asp and Aad generates [Ga]Ga-HTK03161 and [Ga]Ga-HTK03149, respectively, and the new derivatives retain high uptake in LNCaP tumors and have minimal uptake in normal organs/tissues including kidneys and salivary glands. [Lu]Lu-HTK03149 also retain high uptake in LNCaP tumors and has minimal uptake in normal organs/tissues, and is, therefore, promising for clinical translation to treat prostate cancer.