Study On The Preparation And Metabolism Of Prostate Cancer Targeting Probe ⁶⁸Ga-FSH4

Objective:Prostate cancer?PCa?is a malignant tumor in older men.In Occident,the incidence rate is the highest and the fatality rate is 16.56%.In recent years,the average annual growth rate of prostate cancer has reached 12.07%in China.Therefore,it is important to make early diagnosis of prostate tumor.In this study,we designed a new type of radioactive polypeptide probe⁶⁸Ga-FSH4 to provide a new method for early diagnosis of prostate cancer.Methods:After chelating reaction,NOTA-MAL-FSH4 were synthesized.New probe⁶⁸Ga-FSH4 were made and the markers were quality control analysed and in vitro stability studied.Cultured human prostate cancer PC3 cells to study the uptake and block of and competition binding experiment ⁶⁸Ga-FSH4.Constructed the prostate cancer-bearing nude mouse model and performed the Micro-PET imaging.The distribution of the probe in vivo was observed through biodistribution experiments and verified the reliability with PET imaging.The recovery rate of tissue samples in vivo was detected by Radio-HPLC,and the degradation of the probe in vivo was observed.In vitro single-factor studies were performed using Radio-TLC to observe the degradation of peptides by trypsin and the inhibitory effect of aprotinin on probe hydrolysis.The homogenate of external organs was studied by Radio-HPLC and Radio-TLC,and verified by in vivo experiments.Result:The labeling yield of ⁶⁸Ga-FSH4 is 90%,radiochemical purities?RCPs?were more than 95%,and the stability was excellent at 37°C in PBS buffer for 2 h.In vitro cell uptake and blocking experiment showed that the uptake values at 30 min and 60 min were?1.04±0.08?and?1.21±0.35?%AD/10⁵,respectively,and maintained at a high level;added the aprotinin,the uptake values at 30 min and 60min were?0.49±0.08?and?0.51±0.06?%AD/10⁵,respectively.It is indicated that FSH4 can significantly reduce the tumor uptake of radioactive probes.Analysised competitive binding experiment:the IC50 value of?127.5±1.21?nM in PC3 cells,indicating that the probe has excellent specificity for FSHR and high affinity.MicroPET imaging showed that the tumor of PC3 was clearly visible,and the values of the probe at 30 min and 60 min were?4.33±0.27?and?2.36±0.11?%ID/g,respectively.And this probe was removed rapidly in the tumor and normal tissue organs.The biodistribution of nude mice coincided with the results of Micro-PET imaging.Using the Radio-HPLC to detect the recovery rates of⁶⁸Ga-FSH4 in plasma,tumor,liver and kidney.And the sample recovery rates were 66.32%,51.92%and40.33%,respectively.And the sample recovery rate in the kidneys is almost 0%,indicating that the probe is rapidly metabolized in the vivo and mainly metabolized by the liver in the kidney.Added 4 mg of Aprotinin,MicroPET imaging showed the uptake values of the probe at 30 min and 60 min were?5.64±0.21?and?9.21±0.15?%ID/g,respectively.The biodistribution of nude mice coincided with the results of Micro-PET imaging.In the stability of in vitro trypsin solution studies showed the degradation rate of the probe in 200?L 1.6×10^-3?g/?L trypsin was19.80%.After added 4 mg aprotinin,the hydrolysis of the probe was effectively inhibited.It was indicated that trypsin can cause the hydrolysis of polypeptides and the aprotinin can inhibit this hydrolysis.In vitro organ homogenate experiments found that Radio-TLC can detect the⁶⁸Ga-FSH4 with different levels of metabolism in different organ tissues;Radio-HPLC can detect the degradation rate of ⁶⁸Ga-FSH4in plasma,tumor and liver was 70.28%,72.88%,and 30.96%,respectively.And it is mainly metabolized by the kidney.After added aprotinin,the hydrolysis of the probe was effectively inhibited.The vivo experiment consistent wih the results of the vitro experiment.Conclusion:The probe⁶⁸Ga-FSH4 was successfully prepared.It was able to specifically target FSHR and the addition of aprotinin can optimize its metabolism in vivo.It is expected to provide a new diagnostic method for the prostate patients.