| The epidermal growth factor receptor(EGFR)was abnormally expressed or mutated in many types of tumors.The discovery of targeted epidermal growth factor receptor small molecule tyrosine kinase inhibitor(EGFR-TKIs)has provided new treatment options for many patients.And the quality of life and survival time for patients was significantly improved.However,these drugs are not broad-spectrum and only sensitive and effective in patients with positive EGFR gene mutation.Therefore,how to screen sensitive groups for these targeted drugs to formulate personalized treatment plans is a difficult problem to be solved in clinical work.At present,the DNA direct sequencing method is still the "gold standard" for the EGFR gene mutation detection.But it was high cost,time-consuming and requires high quality and content of tumor tissue samples.So,the direct sequencing method is not easy to be widely used in clinical practice.The development of positron emission computed tomography(PET/CT)is a revolution in medical imaging.PET was used to provide detailed functional and metabolic molecular information of lesions through specific positron probes and CT was used to accurately anatomize lesions.Targeted EGFR PET imaging based on EGFR-TKIs is expected to enable non-invasive screening of sensitive individuals for EGFR-TKIs,to clarify the distribution of tumor lesions,and to perform early diagnosis,preoperative localization,postoperative contrast,and efficacy monitoring of solid tumors.Among EGFR-TKIs imaging agents targeting EGFR,11C-Erlotinib has the greatest application prospects and there have been dozens of clinical imaging reports.However,due to the short half-life of 11C(20.39 min),11C-Erlotinib can only be used in PET/CT centers with accelerators,and the number of patients that can be satisfied in a single production was limited(for a PET/CT scanner,the maximum amount of drug each production only can be satisfied 3 patients).It is not easy to make a large specimen study.The development of positron nuclide with long half-life(e.g.18F,T1/2-109.8 min)to label Erlotinib for clinical imaging is urgently required.The purpose of this study was to label erlotinib with 18F and perform PET imaging studies targeting EGFR to evaluate its feasibility as a targetting EGFR PET probe and to screen sensitive individual for EGFR-TKIs.The main methods of this study are as follows:"Click chemistry" method was used to label Erlotinib with 18F.18F-FEA-Erlotinib was synthesized by two kinds of labeling pathway:the "two-step method" and "one-step method".And its automatic synthesis is carried out in the automated synthesis module.The reference compound 19F-FEA-Erlotinib was synthesized and confirmed by 1H-NMR and MS.According to the quality control of radioactive drugs of Chinese Pharmacopoeia,the quality control(QC)method of probe 18F-FEA-Erlotinib was established and the QC was performed with the method.Then the stability of the probe 18F-FEA-Erlotinib in PBS and FBS was evaluated for 2 h,and the lipid-water partition coefficient log P value was determined.The IC50 value of the affinity between the probe and EGFR protein was measured by the Kinase-Glo Plus luminescence kinase assay.After that,the cellular uptake and efflux of the probe was tested in three tumor cell lines HepG2,HCC827,and A431,and Erlotinib inhibition of cell uptake and efflux experiments were performed in HCC827 cells.Then the stability,acute toxicity,in vivo biodistribution and pharmacokinetics based on quantitative analysis of PET imaging in mice was evaluated,followed by Micro-PET/CT imaging of 18F-FEA-Erlotinib in three HepG2,HCC827,A431 tumor-bearing mouse model,and dynamic imaging in HCC827 model.Finally,comparative PET imaging studies of 18F-FEA-Erlotinib,11C-Erlotinib and 18F-FDG were carried out in HCC827 tumor-bearing mouse model.The following results were obtained:The yields of 18F-FEA-Erlotinib by "two-step"and "one-step" synthesis methods were 25 ± 20%and 55 ± 2%respectively,and the time was 75 min and 33 min,respectively.The quality of 18F-FEA-Erlotinib injection was in line with the requirements radioactive drug.The probe was stable in PBS and FBS,radiochemical purity was still greater than 97%after 2 h,the log P of 18F-FEA-Erlotinib was 2.36 ± 0.01,and IC50 value of probe binding to EGFR protein was 11.3 ?M.Weston blot assay showed that the EGFR expression of HepG2,HCC827,and A431 cells with a tendency of increasing trend.HCC827 uptake of 18F-FEA-Erlotinib was significantly higher in the cell uptake experiments.The percentage of added dose(%AD)of radiation at 60 min was 5.82 ± 0.62 and the%AD values of HepG2 and A431 cells at 60 min were 0.98 ± 0.32 and 1.01 ± 0.46 respectively.From the efflux experiment,we found that the retention of probes in HCC827 cells was 2.90 ± 0.43 after 1 h.Then the inhibited uptake and efflux experiment by Erlotinib for HCC827 cells were performed.The%AD decreased to 0.86 ± 0.25 after 1 h and the inhibitory effect was obvious.After 1 hour of metabolism of 18F-FEA-Erlotinib in mice,the HPLC analysis of plasma showed that there were two small radioactive peaks at 3.0 and 7.5 min,with peak areas of 6.2%and 9.3%respectively,and the prototype peak accounted for 84.5%at 9 min.In the acute toxicity test,there was no abnormal death in mice,and no major pathological changes were observed in the main organs.Pharmacokinetic analysis of the probe metabolized in mice confirmed to the two-compartment model.In vivo biological distribution,the liver,intestinal and kidney radioactive distribution was higher than muscle,bone and other organs.The quantitative analysis of dynamic Micro PET imaging in HCC827 tumor-bearing mice found that the liver,intestine,and kidneys had high radioactive doses,the tumor uptake was significant and the values of tumor/muscle,tumor/brain,tumor/heart,tumor/lung were 3.19 ± 0.50,15.00 ± 4.20,1.15 ± 0.32,3.85 ± 0.62 respectively.The tumor/muscle values of HepG2,HCC827,and A431 tumor-bearing mice at 1 h static imaging were 1.16 ± 0.32,3.16 ± 0.63,1.02 ± 0.29 respectively.After blocked by 100 mg/kg Erlotinib for HCC827 tumor-bearing mice,the tumor/muscle value was reduced to 1.21 ±0.42.Finally,1 h static PET imaging of 18F-FEA-Erlotinib,11C-Erlotinib,and 18F-FDG was performed in the HCC827 tumor-bearing mouse model,the probes showed tumor/muscle values of 3.41 ± 0.72,2.10 ± 0.63,and 1.27 ± 0.34 respectively.The conclusions are as follows:It was found that the "one-step method" for the synthesis of 18F-FEA-Erlotinib had high yield,short time,and the quality conforms to the requirements.The probe was a lipid-soluble molecule and was stable,safe and reliable in vivo and in vitro.18F-FEA-Erlotinib was mainly metabolised by the liver and intestine and small part of probe was metabolised by the kidneys,and the pharmacokinetics accord with the two-compartment model and suitable as an imaging probe.In vitro cell uptake experiments and micro-PET imaging of tumor-bearing mice showed that the uptake of the probes in HCC827 cells and its tumors was high,and the uptake of the other two models was low.While inhibition of Erlotinib in HCC827 cells and tumor-bearing mice models were obvious.Combined with the cell and tumor tissue EGFR expression,it was shown that there was no significant correlation between the uptake of the probe and the expression level of EGFR,and the high uptake of HCC827 was due to the EGFR exon 19 deletion mutation.PET imaging of 18F-FEA-Erlotinib can be used to screen Erlotinib-sensitive individuals.Finally,the comparison of the 18F-FEA-Erlotinib,11C-Erlotinib,and 18F-FDG probes in the HCC827 tumor-bearing mice indicates that 18F-FEA-Erlotinib tumor imaging has better contrast.In a word,PET imaging of probe 18F-FEA-Erlotinib can be used to screen Erlotinib-sensitive individuals.In this paper,a new PET probe,18F-FEA-Erlotinib,was prepared.Through quality control,in vivo and in vitro biological evaluation,and targeting EGFR Micro PET imaging studies,it was found that the probe could be used as a PET imaging probe targeting EGFR.The potential application of relevant tumor diagnostic evaluations requires further study. |
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