Synthesis And Evaluation Of New ^99mTc-Labeled Small-Molecule Inhibitors Of Prostate-specific Membrane Antigen

Objective:Here we evaluated preclinically a novel ^99mTc-labeled small-molecule inhibitors of PSMA,which derived from the glutamate-urea-R pharmacophores conjugated to G?Gly?GGC?Cys?,and quality control was performed.we also was evaluated it's value both in vitro and in vivo,using prostate cancer cells-22Rv1?PSMA⁺?and PC-3?PSMA^-?and PCa models?PC-3 and 22Rv1?,as a diagnostic imaging agent for prostate cancer.Methods:PSMA inhibitor GGGC-PSMA was synthesized using solid-phase synthesis of Fmoc/tBu polypeptide and was radiolabeled by ^99mTc at room temperature.The labeling rate and radiochemical purity of the molecular probe were analyzed and determined by high liquid chromatography.To study the stability of the probe in vitro,after incubation for 1,2,4,6,8,and12 hours with normal saline and fresh human serum at 37?,respectively,the radiochemical purity at different time points was detected by RT-HPLC.The human prostate cancer 22Rv1 cells?PSMA⁺?and PC-3 cells?PSMA^-?were incubated by standing adherent.In logarithmic growth phase,male BALB/c-nu/nu mice were subcutaneously inoculated in the right trunk with1·10⁷ 22Rv1 cells in normal saline or 1·10⁷ PC-3 cells in phosphate-buffered saline,the studies were performed When the tumors reachedapproximately1cmindiameter.Thestabilityof^99mTc-GGGC-PSMA in vivo was tested by RP-HPLC,the urine of the mice bearing 22Rv1 tumors was collected be injected via the tail vein with^99mTc-GGGC-PSMA.Cellular uptake,retention kinetics,internalization,and blocking studies of the probe were investigated in the human prostate cancer cell lines 22Rv1?PSMA-positive?and PC-3?PSMA-negative?,to assesse pharmacokinetics and specificity of the probe in vitro.The affinity constant?K_d?of ^99mTc-labeled PSMA inhibitors was determined by saturation binding analysis in the human prostate cancer cell lines 22Rv1?PSMA-positive?.Organ distribution experiments were performed with 4 mice models bearing an 22Rv1 tumor and 3 mice models bearing a PC-3 tumor.The mice were administered via tail vein injection of the tracer.At 4 h after injection,the animals were sacrificed,and organs of interest were dissected,Including Blood,heart,liver,spleen,lung,kidney,stomach,small intestine,brain,bone,muscle and tumor,and the tissue were weighed and radioactivities were measured with a?-counter and calculated as the percentage injected dose per gram?%ID/g?.For the SPECT studies,^99mTc-GGGC-PSMA were injected via tail vein into a mouse bearing an 22Rv1 tumor xenograft with or without excess nonradiolabeled compound blockade,and a mouse bearing an PC-3 tumor xenograft.At 1,2,4,6 and 8h after injection,the mice were anesthetized and imaged.The ratio of radioactive counts in the tumor to that in the contralateral equivalent region?tumor to nontumor[T/NT]?were calculated by drawing regions of interest?ROI?at each time point.All experimental variables were expressed as mean±SD and were performed with Two-sample t?or t??test or Wilcoxon rank sum test using SPSS?version 22.0?and GraphPad Prism?version 5.0?.P values of less than 0.05 were considered significant.Results:The ^99mTc-complex was assessed by HPLC,the retention time of^99mTc-GGGC-PSMA was 11.66 min and the radiochemical purity provided yields of?99.29±0.43?%after 20 min?n=3?without purification,at room temperature.The stability of ^99mTc-GGGC-PSMA both in vitro and in vivo was tested by incubation of ^99mTc-GGGC-PSMA in fresh human serum or normal saline for 1?2?4?6?8?12h at 37?.The radiochemical purity of the molecular probe was above 97%within 12 h by the RP-HPLC analysis,and the position of the radiation peak was stable.The stability of radiolabeled compound in vivo,this date showed 2 peaks and the main peak is similar to the retention time of ^99mTc-GGGC-PSMA.The small peak before the main peak is irrelevant with the retention time of ⁹⁹Tc^mO^4-,which may be the metabolite of the peptide,but not free ^99mTc.The results of cell uptake experiments showed that 22Rv1 and PC3,which were incubated with^99mTc-labeled PSMA inhibitors,cells uptake were?2.21±0.24?%,?2.88±0.30?%,?17.72±0.68?%,?16.06±0.78?%,?11.19±1.68?%;?0.55±0.01?%,?0.58±0.06?%,?0.66±0.02?%,?0.64±0.08?%,?0.54±0.00?%,respectively for 1?2?4?6?8h,indicating that the uptake rate increased firstly and then gradually decreased with the prolongation of time,and the uptake rate reached a peak at 4h;while the labeled compound showed almost no accumulation in PC-3 cells?PSMA^-?with time.The results of cell retention of^99mTc-GGGC-PSMA in human prostate 22Rv1 cells showed that the molecular probe retention rate was relatively high at 4h,reaching 68.66%.As a result of the internalization experiment,the internalization rate showed a downward trend as a whole.The inhibition experiment with 500-fold and 1000-fold excess of unlabeled GGGC-PSMA in 22Rv1 cells showed the same results as those in PC-3 cells.The cell uptake rate after incubation for 4 hours was reduced from?17.72±0.68?%to?0.55±0.02?%,?0.43±0.02?%.Saturation binding analysis was conducted to determine the affinity of^99mTc-GGGC-PSMA for PSMA expressed on 22Rv1 cells.The cells were incubated with 0–200 nM ^99mTc-GGGC-PSMA inhibitor to determine the K_d.Consistent with the order of potency of ^99mTc-compounds in a competitive inhibition assay?22?,the affinity for PSMA?K_d?was 1.316.The results of the organ distribution experiments are summarized that,^99mTc-GGGC-PSMA was injected in 22Rv1?PSMA-positive?tumor–bearing mice and PC-3?PSMA-negative?tumor–bearing mice via tail vein,blood?0.60±0.20?%ID/g,heart?3.37±1.18?%ID/g,liver?0.89±0.9?%ID/g,spleen?7.78±1.46?%ID/g,lung?3.04±0.78?%ID/g,kidney?325.97±58.27?%ID/g,stomach?1.50±0.22?%ID/g,small intestine?0.93±0.20?%ID/g,muscle?0.81±0.21?%ID/g,bone?1.54±0.50?%ID/g,brain?0.06±0.02?%ID/g,tumor?5.74±2.14?%ID/g;blood?0.41±0.10?%ID/g,heart?0.91±0.48?%ID/g,liver?1.06±0.15?%ID/g,spleen?8.83±0.87?%ID/g,lung?1.76±0.54?%ID/g,kidney?328.72±86.33?%ID/g,stomach?0.57±0.33?%ID/g,smallintestine?0.55±0.03?%ID/g,muscle?0.76±0.05?%ID/g,bones?0.44±0.04?%ID/g,brain?0.02±0.01?%ID/g,tumor?0.34±0.07?%ID/g,respectively.The%ID/g values of 22Rv1 tumor-bearing mice and PC-3 tumor-bearing mice were?5.74±2.14versus 0.34±0.07,respectively?,indicating that the molecular probe can be taken by tumor tissues?PSMA⁺?.SPECT imaging experiments showed that after injection of^99mTc-GGGC-PSMA for 1h in 22Rv1 tumor-bearing mice,the tumor tissue showed obvious concentration of radionuclides,and the degree of concentration increased with time;on the contrary,the tumor site of PC-3tumor-bearing mice no significant radionuclide concentration was observed in the experiment.The T/NT ratios of the groups at different time points were compared.The 22Rv1 tumor-bearing mice was significantly higher than the PC-3 tumor-bearing mice?t=3.547⁴.878,P=0.001⁰.008?.For the blockade experiment,it showed that the radionuclide uptake at the tumor site was blocked by unlabeled GGGC-PSMA,and the T/NT ratio at each time point was statistically different.Conclusion:^99mTc labeled PSMA small molecule inhibitor,labeling method is simple,high labeling rate,no purification,can be directly applied in vivo imaging experiments,good stability in vitro and in vivo,can be specifically taken up by cells with PSMA-positive.It can be used for tumor?PSMA⁺?specific imaging in vivo,and has high targeting to tumor tissues.Therefore,we clearly demonstrated the feasibility of SPECT imaging of prostate cancer with ^99mTc-GGGC-PSMA as a new radiotracer.