| Objective:In this study, the preparation of 99mTc-HYNIC-PEG4-E[PEG4-c (RGDfK)]2 (99mTc-3PRGD2) was made of joint hydrazine nicotinamide (HYNIC) as bifunctional coupling agent, trimethylol glycine (tricine) and three sodium triphenylphosphine (TPPTS) as synergistic ligands, and was labeled with radionuclide technetium. A large number of in vitro and animal experiments were done with 99mTc-3PRGD2, and on this basis to further study the pharmacokinetics, biodistribution and assessment of radiation dose in healthy people, meanwhile to observe the biochemical and physical examinations and vital signs of the healthy volunteers. The purpose was to prove the safety and reliability of 99mTc-3PRGD2 application in healthy people. Then do diagnosis studies with 99mTc-3PRGD2 in patients with solitary pulmonary nodule. The aim of this study was to evaluate the in vitro and in vivo characteristics of 99mTc-3PRGD2 as imaging agent of integrinαvβ3 positive tumors.Methods:1. A radiotracer of 99mTc-3PRGD2 was prepared.2. Quality control:to calculate the labeling yield of 99mTc-3PRGD2 by radioactive thin layer chromatography (ITLC) and radioactive high performance liquid chromatography (HPLC). While the lipid-water partition coefficient of determination,99mTc-3PRGD2 with membrane receptor binding, dissociation kinetics, cell internalization and efflux were investigated. And to image with SPECT in nude mice bearing human lung cancer xenografts. 3. Approved by our hospital independent ethics committee, 99mTc-3PRGD2 free of pathogenic microorganisms was injected into 5 healthy volunteers after informed consent, and acquired the healthy volunteers'data about the safety testing, the pharmacokinetics, biodistribution and assessment of radiation dose.4. The 99mTc-3PRGD2 free of pathogenic microorganisms was injected into 10 cases of solitary pulmonary nodules patients, by SPECT imaging analysis, immunohistochemical analysis of patients with pathological results conducted a preliminary study, to evaluate the diagnosis significance in solitary pulmonary nodules.Results:1. In vitro study, the labeling yield of 99mTc-3PRGD2 was over 95% by ITLC analysis, and was over 95% by HPLC method, too.2. The lipid-water partition coefficient LogPo/w of 99mTc-3PRGD2 was-4.16±0.05. It had a good water-solubility. It was also observed the binding and dissociation rate of 99mTc-3PRGD2 with integrinαvβ3 cell surface receptor. After ten minutes of the reaction, the combination speed of 99mTc-3PRGD2 with integrinαvβ3 receptor obtained the maximum, and as time prolonging, remained at the maximum binding. Adding an excess of unlabeled HYNIC-3PRGD2, the speed of the dissociation was fast firstly, then become slow. In the initial twenty minutes,50% of the maximum binding capacity had been dissociated, after one hour nearly 60% of the maximum binding capacity had been dissociated.3. Human lung adenocarcinoma cells A549 internalization and efflux by 99mTc-3PRGD2 were investigated. It is the fastest reaction to markers internalization at 37℃, and the fastest reaction of the maximum amount of 45.3% for the total binding within twenty minutes. After reaction two hours, the internalized percentage of the 99mTc-3PRGD2 basically unchanged. The outflow speed of internalized markers through the cell membrane is quickly, more than 20% of internalized markers gone out into the extracellular in the first quarter. Two hours later,57.1% of the internalized 99mTc-3PRGD2 was retained within the cells, and with the increase of holding time, the internalized labeled 99mTc-3PRGD2 were flowed out gradually.4. SPECT imaging was carried out on nude mice bearing A549 lung cancer after intravenous injection of 99mTc-3PRGD2,0.5,1,2 and 4 hours respectively. After thirty minutes, the tumor could be clearly imaging. After two hours, the clearest part of imaging was tumor, kidney and bladder. And in the heart, liver, lungs and other parts, there was no obvious radioactive concentration. With the increase of holding time, the stomach and intestine had slightly intake. After injection four hours, tumor was still clearly visible, while the general background radioactivity gradually decreased.5. After injection, five healthy volunteers' vital signs were stable within 24 hours; blood and urine of index was normal; the heart, the lung, the abdomen and other specific neurological examination had no abnormalities, had no abnormal electrocardiogram; and we had not found the clinical adverse reactions, such as nausea, bronchospasm, pruritus, flushing, urticaria, chills, cough, bradycardia. muscle cramps or dizziness, etc. An intravenous injection 99mTc-3PRGD2 in healthy volunteers within 60 minutes, the blood clearance results:we toke the blood radioactive activity of post injection 2 min for initial radioactivity 100%, the data showed that 99mTc-3PRGD2 cleared rapidly in the blood, in 10 minutes,30 minutes and 60 minutes of blood intake value were 45%.18% and 12% respectively. Plasma clearance rate had a similar result with blood clearance kinetics, P> 0.005. After intravenous injection of 99mTc-3PRGD2 the percentage of urine excreted over time showed that urine radioactivity half time was (16±2.6) hours. The accumulated radionuclide excretion by urine in 24 hours was (52.9±6)% of total injection dose. From 10 minutes to 24 hours after injection, we got all the body anterior and posterior high-quality SPECT imagings of healthy volunteers. With the continuous planar imaging method deducting the background, we estimated the intake percentage of general and major organs at different time points compared with the total radionuclide count of the anterior and posterior imaging at ten minutes. Kidney intake was the highest point through checking all the time, meanwhile the radioactivity accumulation in the bladder was also very evident. In 10 minutes,30 minutes,1 hour,2 hours,4 hours,24 hours respectively, the general intake percentage was 100.00±0.00,99.28±0.53,97.350±2.00,66.98±2.98,57.470±4.81,51.090±7.23; the intake percentage of kidney was 10.22±1.89,7.84±1.26,6.89±1.24,6.06±1.31,6.93±2.46,5.07±0.90; the intake percentage of bladder was 13.95±3.91,23.72±6.46,26.15±5.84,3.15±1.40,1.86±0.83. 0.08±0.03; the intake percentage of cardiac was 1.92±0.52,1.40±0.33,1.07±0.20,0.40±0.11,0.36±0.09,0.24±0.11:the intake percentage of lung was 2.12±0.30,1.65±0.10,1.42±0.10.1.20= 0.18,1.05±0.21,1.12±0.16; the intake percentage of liver was 7.85±1.07,5.90±0.82,5.97±1.81,5.27±1.80,5.58±2.53.3.92±0.91. Then can we suggest that 99mTc-3PRGD2 excrete mainly through the renal pathway, and small amount of metabolism in liver, in the heart and lung and other parts, there were no significant radioactivity intake. Salivary glands and thyroid intake were low and only were observed slow perfusion during the first 4 hours. With the increase of holding time, the general background radioactivity decreased. The radioactive count of the RGD at different times in the source organs was input into the OLINDA/EXM 1.0 software, to estimate the radiation absorbed dose of the source organs, the effective data of the source organs were used to estimate the effective dose. Because the tracer excreted mainly through urinary pathway, so the maximum of the absorbed dose was in the kidney (3.50E-02mSv/MBq). In addition to the kidney, other organs absorbed dose is in a relatively low level. The systemic average effective dose is 3.92E-03mSv/MBq. If your weight was 70 kilograms, injected 11.1MBq/kg of 99mTc-3PRGD2 intravenously, the radiation absorbed dose of an inspection was 3.05mSv.6.10 cases of solitary pulmonary nodules patients underwent 99mTc-3PRGD2 SPECT imaging.6 patients were positive with pathologically diagnosed cancer,1 case of sclerosing hemangioma was positive too, then the other 3 patients had benign pulmonary nodules were negative.99mTc-3PRGD2 SPECT positive cases had positive Integrinαvβ3 expression. It is showed there was the correlation between intake of 99mTc-3PRGD2 andαvβ3 expression.Conclusion:This topic through a series of basic and clinical studies confirmed that 99mTc-3PRGD2 as a new radioactive molecular probe targeting to integrinαvβ3 receptor was feasible. All the data of 99mTc-3PRGD2 in healthy human, such as pharmacokinetics, in vivo stability, metabolite analysis, tissue distribution and radioactivity distribution in vivo were laid the foundation for clinical application. Through its clinical application in solitary pulmonary nodules, lung cancer patients had a higher intake of 99mTc-3PRGD2. The intake level of 99mTc-3PRGD2 correlated with the expression of integrinαvβ3 receptor. There were no serious systemic and local side effects. However, the diagnostic value of 99mTc-3PRGD2 still remains a large sample, long observation and research. I believe that with the deepening of this study,99mTc-3PRGD2 is expected to be the new way to diagnose and treat integrinαvβ3 positive tumors.
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