| Part I Synthesis, radio-labeling and quality control of 18F-AlNOTA-MAL-Cys39-exendin-4Objective: To synthesize β cell target probe 18F-Al-NOTA-MAL-Cys39-exendin-4 by Al18 F complex strategy and analyze the quality of the product.Methods: Cys39-exendin-4 was conjugated to the MAL-NOTA at first, the fractions containing NOTA-MAL-Cys39-exendin-4 were collected and lyophilized. Then taken the NOTA-MAL-Cys39-exendin-4 as the precursor, use the one-step Al18 F tag to get the product. Compare the HPLC of 18F-Al-NOTA-MAL-Cys39-exendin-4 with the ultraviolet peak of the standard. Test the in vitro stability of 18F-Al-NOTA-MAL-Cys39-exendin-4 through analytical HPLC. Calculate the oil/water partition coefficient of the product. Inject 3.7×107Bq/0.2ml 18F-Al-NOTA-MAL-Cys39-exendin-4 to the mice through the tail vein, observation on 48 h, finally executed and anatomy, observe whether the organs and cells were damaged or not.Results: NOTA-MAL-Cys39-exendin-4 were collected and lyophilized successfully. 18F-Al-NOTA-MAL-Cys39-exendin-4 can obtained in 30 min with one-step Al18 F tag to the precursor. The retention time of 18F-Al-NOTA-MAL-Cys39-exendin-4 was 39.43 min analyzed by analytical HPLC, consistent with the ultraviolet absorption value of NOTAMAL-Cys39-exendin-4. The radiochemical purity was above 99.0% and still above 99% after 6 h. The oil-water partition coefficient is-1.86, tips for hydrophilic. The mice that injected with 18F-Al-NOTA-MAL-Cys39-exendin-4 were alive after 48 h, no organ damaged, no cell difference between the experimental and the control mice.Conclusion: First synthetic precursor NOTA-MAL-Cys39-exendin-4, and then use Al18 F one-step tag synthesis of 18F-Al-NOTA-MAL-Cys39-exendin-4. This strategy have the advantage for simple operation, high yield, high radiochemistry purity and better in vitro stability. The labeling rate, stability and chemical purity are high. In addition, this radiopharmaceutical is low toxicity, meet the requirements of the experiment using in vivo and in vitro, easy to translate to use in clinical diagnose and evaluation. Part II Experiment of β cell imaging with 18F-Al-NOTA-MALCys39-exendin-4Objective:Glucagon-like peptide-1 receptor(GLP-1R) is abundantly expressed on beta cells and may be an ideal target for the pancreas imaging. Monitoring the GLP-1R of pancreas could be benefit for understanding the pathophysiology of diabetes. In the present study, 18F-Al labeled exendin-4 analog, 18F-Al-NOTA-MAL-Cys39-exendin-4, was evaluated for PET imaging GLP-1R in the pancreas.Methods: Measure the cell uptake ratio of 18F-Al-NOTA-MAL-Cys39-exendin-4 by INS-1 cell and PANC-1 cell, and use large dose Cys39-exendin-4 to test if the uptake can be blocked. 50 μCi 18F-Al-NOTA-MAL-Cys39-exendin-4 were injected to 6 mice through tail vein, draw blood at different time and counted by γ counter with standard tube, then calculated according to the software DAS 2.1.1 for pharmacokinetic parameters.The targeting of 18F-Al labeled exendin-4 analog was examined in healthy and streptozotocin induced diabetic rats. Rats were injected with 18F-Al-NOTA-MAL-Cys39-exendin-4 and micro PET imaging was performed at 1h postinjection, followed by ex vivo biodistribution. GLP-1R expression in pancreas was determined through post mortern examinations.Results: The uptake of 18F-NOTA-MAL-Cys39-exendin-4 by Ins-1 cell was reach peak at 60 min and continue to 120 min.unlabled lager dose NOTA-MAL-Cys39-exendin-4 can reduce the uptake level. The uptake of 18F-NOTA-MAL-Cys39-exendin-4 by Panc-1 cell was increased continually to 120 min, but the extent of uptake has been below INS-1 cell all the time. Furthermore, it nearly can’t blocked by Cys39-exendin-4. The blood concentration of normal mice was decreased quickly after injected 18F-Al-NOTA-MAL-Cys39-exendin-4, t1/2α=1.786 min, t1/2β=11.557 min. The pancreas of healthy rats was readily visualized after administration of 18F-Al-NOTA-MAL-Cys39-exendin-4, whereas the pancreas of diabetic rats, as well as those from rats co-injected with excess of unlabeled peptides, was barely visible by micro PET. At 60 min postinjection, the pancreatic uptakes were 1.02±0.15%ID/g and 0.23 ±0.05 %ID/g in healthy and diabetic rats respectively. Under block, the pancreatic uptakes of non-diabetic rats reduced to 0.21±0.07 %ID/g at the same time point. Biodistribution data confirmed the findings of the micro PET imaging. IHC staining clearly revealed that GLP-1R positive cells were significantly higher in the pancreas of healthy rats than diabetic rats.Conclusion: The specific binding of 18F-Al-NOTA-MAL-Cys39-exendin-4 on β cell was confirmed by cell uptake and block experiment. 18F-Al-NOTA-MAL-Cys39-exendin-4 in the pancreas is mediated by GLP-1R-specific mechanisms in rodents. The favorable preclinical data indicated that the tracer may be suitable for non-invasive monitoring functional pancreatic beta cells. Part III Synthesis, radio-labeling and quality control of 18F-PC-10Objective: To synthesize 18F-PC-10 and analyze the quality of the product.Methods:PC-7 was taken as the precursor, and prepare the tag intermediates 18F-SFB. CFN-MPS-100 fluorine multifunction radiopharmaceutical chemical synthesis module was adopted to complete the radioactive fluorination reaction, and the crude product was purified by semi-preparative HPLC, the solvent was removed by rotary evaporation. Radio-HPLC were applied for quality control. Inject 3.7×107Bq/0.2ml 18F-PC-10 to the mice through the tail vein, observation on 48 h, finally executed and anatomy, observe whether the organs and cells were damaged or not.Results: 18F-PC-10 was obtained in 100-120 min with the radiochemical yield of(15±3)%(no decay corrected, n=3). The radiochemical purity was above 99.0% and still above 99% after 6 h. The product was colorless solution, with p H value of 6-8. The retention time of 18F-PC-10 was 20 min analyzed by analytical HPLC. The retention time of 18F-PC-10 was consistent with the ultraviolet absorption value of 19F-PC-10. The plasma protein binding rate of 18F-PC-10 was 25.54%. The mice that injected with 18F-PC-10 were alive after 48 h, no organ damaged, no cell difference between the experimental and the control mice.Conclusion:18F-PC-10 could be synthesized with semi-automatic synthesis method based on the CFN-MPS-100 fluorine multifunction module. The labeling rate, stability and chemical purity are high.This radiopharmaceutical is low toxicity, meet the requirements of the experiment using in vivo and in vitro. Part IV Experiment of β cell imaging with 18F-PC-10Objective:PROK1R is expressed on beta cells and may be an ideal target for the pancreas imaging. Measure the cell uptake ratio of 18F-PC-10 by INS-1 cell. Study the pharmacokinetics of 18F-PC-10 in mice, and perform micro PET imaging for normal mice to show the bio-distribution and characteristics of image, to verify the feasibility of islet beta cell imaging.Methods: Measure the cell uptake ratio of 18F-PC-10 by INS-1 cell and PANC-1 cell, and use large dose PC-7 to test if the uptake can be blocked.50 μCi 18F-PC-10 were injected to 6 mice through tail vein, draw blood at different time and counted by γ counter with standard tube, then calculated according to the software DAS 2.1.1 for pharmacokinetic parameters. Mice were injected with 18F-PC-10 and 4 time point’s micro PET imaging was performed postinjection, followed by in vivo biodistribution and image analysis.Results: The uptake of 18F-PC-10 by Ins-1 cell was reach peak at 60 min and continue to 120 min. The uptake of 18F-PC-10 by Panc-1 cell was similar. The block effect of PC-7 was not significant. The blood concentration of normal mice was decreased slowly after injected 18F-PC-10, t1/2α=1.419 min, t1/2β=75.338 min. The pancreas of healthy mice was readily visualized at 5min PET image postinjection of 18F-PC-10. The uptake of pancreas was decreased gradually, and the uptake of intestines was markedly increased. It is hard to visualize pancreas and draw the ROI. 18F-PC-10 is metabolized by liver, and excreted mainly by intestines, secondary by urinary system.Conclusion: The specific binding of 18F-PC-10 on β cell was confirmed by cell uptake. The in-vivo elimination of 18F-PC-10 is slow. Although the pancreas uptake is high, the image is influenced by radioactive excretion of intestines. The favorable image may be acquired by increase the hydrophilic of 18F-PC-10. |
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