The Synthesis,Radiolabeling,in Vitro And In Vivo Study Of Novel PSMA-targeted Radiotracers

Objective:Prostate-specific membrane antigen（PSMA）is over-expressed in the membrane of prostate cancer cells than in normal cells.Having these intriguing characteristics,many small-molecule PSMA inhibitors have been developed,with particular interest in those using Lys-urea-Glu scaffold,due to their high affinity for PSMA.However,there is limited research on the relationship between terminal amino group modification on the lysine and its biological properties,and structure-activity relationship is still unclear.In addition,PSMA drugs with excellent in vivo stability,high specificity,and good in vivo metabolic properties are barely developed.Therefore,to improve drug efficacy and develop new PSMA inhibitors,it is important to further investigate the structure-activity relationship and optimize its chemical structure.In this study,we have developed five ⁶⁸Ga-labeled PSMA-targeted tracers,namely⁶⁸Ga-Flu-1,⁶⁸Ga-Flu-2,⁶⁸Ga-9-Ant,⁶⁸Ga-1-Nal,and ⁶⁸Ga-1-Noi.Our aim is to investigate the effect of lipophilic bulky groups on the affinity and pharmacokinetic of PSMA inhibitors and evaluate their clinical potential by comparing their in vitro and in vivo properties with those of ⁶⁸Ga-PSMA-11,which is widely used in clinical settings.Methods:（1）The target products were synthesized in multiple steps and subsequently characterized by High Performance Liquid Chromatography-Mass Spectroscopy（HPLC-MS）and Nuclear Magnetic Resonance Spectroscopy（NMR）;（2）The target products were then labeled with radionuclide ⁶⁸Ga for quality control;（3）The stability in PBS buffer solution and serum was determined and hydrophilicity was evaluated by measuring their Log P values;（4）Study the uptake,internalization,and specific binding ability of⁶⁸Ga-labeled inhibitors in human prostate cancer LNCa P cells;（5）Using SPF NOD/SCID mice to establish LNCa P xenograft model and then study micro-PET/CT imaging and biodistribution.The uptake values of tumor,kidney,blood,and major organs were calculated and compared.（6）Auto Dock-GPU was used for molecular docking of small molecules with PSMA to obtain docking scoring and possible conformations and orientations of ligands at binding sites.Results:（1）The target molecules were successfully synthesized via multiple-step reactions,and the chemical structure was confirmed by HPLC-MS and NMR.The yield of the final product was 12.50%for Flu-117.84%for Flu-2,15.07%for 9-Ant,15.64%for 1-Nal,and 17.65%for 1-Noi;（2）⁶⁸Ga-labeled PSMA inhibitors were obtained in 88.53%-99.98%radiochemical purity and up to 20 MBq/μg of the highest specific activity;（3）These radioligands have good stability in vitro which remained stable after being placed in PBS buffer solution or serum for 2 h.The hydrophilicity of⁶⁸Ga-Flu-1,⁶⁸Ga-Flu-2,⁶⁸Ga-9-Ant,⁶⁸Ga-1-Nal,and ⁶⁸Ga-1-Noi increased gradually from left to right;（4）In vitro binding experiments showed that the specific binding of radioligands to LNCa P cells increased over incubation time.⁶⁸Ga-Flu-1 revealed a much higher uptake and internalization percentage than the other four radioligands under the same condition.⁶⁸Ga-Flu-1 showed34.57±4.14%ID/10⁶cells uptake and 21.30±0.83%ID/10⁶cells internalization at 120 min.Cell competitive inhibition assay showed that the affinity of nat Ga-Flu-1 was significantly higher than that of ^natGa-PSMA-11.The calculated IC₅₀values for ^natGa-Flu-1 was 9.62±1.70 n M and that for ^natGa-PSMA-11 was 16.60±1.69 n M;（5）Micro PET/CT imaging showed high tumor uptake and enhanced tumor to background ratio in LNCa P tumor-bearing mice.Five radioligands demonstrated favorable biodistribution characteristics and were mainly excreted via the renal pathway.The LNCa P tumor uptake of⁶⁸Ga-Flu-1 reached a peak at 60 min p.i.（52.07±14.83%ID/g）and remained at a high level of uptake within 120 min after administration;（6）The binding affinities of Flu-1,9-Ant,1-Nal,Flu-2,and 1-Noi for PSMA increased gradually.Among them,Flu-1 formed a suitable steric complementation with the binding pocket of PSMA protein,and there were more hydrogen bonds between Flu-1 and PSMA amino acid residues.Conclusion:In conclusion,introducing bulky and lipophilic substituents into Lys-urea-Glu-based PSMA-targeting inhibitors promoted PSMA⁺LNCa P tumor uptake and optimized their pharmacokinetic profile.This resulted significant changes in both their overall hydrophilicity and interaction with PSMA.These ligands showed moderate to excellent binding and internalization properties in vitro and highly specific uptake and favorable retention times in vivo compared to the gold standard ⁶⁸Ga-PSMA-11.With considerably high uptake in tumor and fast kidney pharmacokinetics,⁶⁸Ga-Flu-1 warrants further development as a potential PSMA-targeted PET tracer.