Study On The Mechanism Of Cell Uptake And Preclinical Biology Evaluation About ¹⁸F-FDGly-NH-Phe

Background:Glucose analogue [¹⁸F]-fluorodeoxyglucose is the most widely used and irreplaceable tumor PET imaging agent.However,there are still some limitations in the clinical application of¹⁸F-FDG.For instance,the brain tumor imaging,the differentiation of tumor and inflammation,and so on.In order to improve the sensitivity and specificity of imaging,we have successfully synthesized a new glucosamine molecule probe—¹⁸F-fluoro-deoxyglucose p-aminophenylalanine amine(¹⁸F-FDGly-NH-Phe)as a new imaging agent for tumors.It contains both glucose and amino acid functional groups,and can interact with glucose transporter and amino acid transporters at the same time.It is used as a multiple metabolic PET imaging agent of tumor.In this study,the uptake mechanism of¹⁸F-FDGly-NH-Phe in tumor cells was studied from cell level,and the imaging results of new imaging agents in Kunming mice were studied.Objective:1.In vitro: Uptake studies of cell molecular probes¹⁸F-FDGly-NH-Phe and preliminary comparison with¹⁸F-FDG.Study on the mechanism of cell uptake level¹⁸F-FDGly-NH-Phe molecular probes,to make clear whether the molecular probe is double targeting,verification of amino acid transport channel and¹⁸F-FDGly-NH-Phe is involved in protein synthesis or not.2.In vivo : Study on¹⁸F-FDGly-NH-Phe molecular probe in vivo biodistribution of Kunming rats and results of PET/CT imaging,and comparison with¹⁸F-FDG PET/CT image results.Method:This study was divided into the cell experiment part and the preclinical animal model experiment part.The cell experiment part includes three contents.Firstly,We measured the uptake and elution rate of¹⁸F-FDG and¹⁸F-FDGly-NH-Phe in laryngeal carcinoma Hep-2 cells at different culture times to evaluate the difference and similarity between the new molecular probe and¹⁸F-FDG.Secondly,we investigated whether there is a dual target of¹⁸F-FDGly-NH-Phe and which transport mechanism is dominant.Thirdly,to study the in vitro stability and fat solubility of¹⁸F-FDGly-NH-Phe.The preclinical experiment part includes tumor and inflammation models made of Kunming mice,PET / CT imaging,the evaluation of image features,and the detection of biological distribution.Result:1.The maximal uptake rate of¹⁸F-FDGly-NH-Phe was 9.33 ± 0.19% at 80 min in laryngeal carcinoma Hep-2 cells.2.Laryngeal carcinoma Hep-2 cells have dual targeting.When the concentration of 2-DG was 100 ?M,the inhibition rate of¹⁸F-FDGly-NH-Phe was 45.66 ± 1.92%.With the increase of 2-DG concentration?200?M to 300?M?,the inhibition rate did not increase significantly.That means 100 ?M of 2-DG has reached the inhibitory balance.The inhibition rate of phenylalanine to ¹⁸F-FDGly-NH-Phe increases with the increase of phenylalanine concentration.When the phenylalanine concentration was 300 ?M,the inhibition rate of¹⁸F-FDGly-NH-Phe was 76.77 ± 5.49%.When 2-DG and phenylalanine were present in the culture environment of laryngeal carcinoma Hep-2cells at the same time,with the increase of 2-DG and phenylalanine concentration?100?M,200?M and 300?M?,the inhibition rate?69.61 ± 5.67%,73.43 ± 2.14%,79.25± 2.33%?of¹⁸F-FDGly-NH-Phe was increased.3.The addition of three different amino acid transporter inhibitors?BCH,Me AIB,serine?in the Na +-containing PBS system with 100?M 2-DG blocking glucose transport pathway were studied.The inhibitory rates of¹⁸F-FDGly-NH-Phe were 76.95 ± 7.70%,69.53 ± 2.66% and 76.19 ± 0.72% respectively.In the choline system without Na+,the uptake of¹⁸F-FDGly-NH-Phe was not significantly inhibited.That means the uptake of¹⁸F-FDGly-NH-Phe mainly through Na+dependent B0,+system,A system,ASC system.¹⁸F-FDGly-NH-Phe is hardly involved in the synthesis of proteins.4.In vivo tumor inflammation model mice biodistribution results:The ratio of tumor/muscle:¹⁸F-FDGly-NH-Phe?6.97 ± 1.32?>¹⁸F-FDG?4.40 ±0.67?;The ratio of inflammation/muscle:¹⁸F-FDGly-NH-Phe?1.56 ± 0.19?<¹⁸F-FDG?3.61 ± 0.82?;The ratio of tumor/ inflammation:¹⁸F-FDGly-NH-Phe?4.56 ± 1.31?>¹⁸F-FDG?1.24± 0.16?;P<0.05?Conclusion1.Laryngeal carcinoma Hep-2 cells have dual targeting to¹⁸F-FDGly-NH-Phe and can enter cells both through glucose and amino acid transporters.2.That uptake of¹⁸F-FDGly-NH-Phe mainly through Na+dependent B0,+system,A system,ASC system.¹⁸F-FDGly-NH-Phe is hardly involved in the synthesis of proteins.3.¹⁸F-FDGly-NH-Phe is well-stabled and water-soluble.4.S¹⁸0 tumor-bearing mice had confirmed that¹⁸F-FDGly-NH-Phe was significantly lower in inflammation and brain uptake than¹⁸F-FDG.And the uptake in tumors were high.¹⁸F-FDGly-NH-Phe appears to be a potential PET imaging agent for multi-target tumor metabolic imaging.