| PET/CT is currently one of the most widely used clinical molecular imagingdetection technique which can realize the advance of medical imaging function fromreflecting human body structure change to the metabolic changes. For its sensitivity,accuracy, specificity and precise positioning, this technique can be used to monitor thedistribution and variation of radioactive drugs labeled by positron nuclide in the bodyquantitatively, dynamically and non-invasively, reflect the physiological and bio-chemical changes in vivo at the molecular level, provide us with an ideal platform fordrug screening. The choice of positron radiopharmaceuticals is of great importance forPET/CT imaging scanning.Parkinson’s Disease, one of the three common kinds of nervous systemdegenerative diseases, is a kind of chronic, insidious disease among the elder people.Parkinson’s Disease has high heterogeneity. Patients will undergo different diseaseprogression. Without any specific diagnostic methods existing, the diagnosis mainlyrely on the medical history, clinical symptom and physical sign. It is more difficult todiagnose when patients only have some atypical clinical symptoms, especially whenpatients are asymptomatic (subclinical type). Most patients’ symptoms can be wellcontrolled through medical treatment at early stage. Patients’ condition will be out ofcontrol due to the patients’ poor response to drugs at middle and advance stage. Thesymptom of stiffness will appear and the patients would not be able to look afterthemselves, and their life quality will severely deteriorated. Therefore early diagnosisand treatment are particularly important.With the rapid development of molecular nuclear medicine and new positrondrugs, a lot of specific positron radioactive imaging agents have been developed,aimed directly at Parkinson’s Disease. Through PET/CT scanning, abnormal changescan be found in time from metabolism and function imaging before the anatomicalstructure change happens so that the goal of early diagnosis and timely treatment can be achieved.1. ObjectiveThe main objective of this work is to develop a new type of18F-FethypridePET/CT imaging agent for the early diagnosis of Parkinson’s Disease. Each product inthe process of the precursor synthesis will undergo nuclear magnetic resonance (NMR)and high resolution mass spectrometry analysis in order to determine whether theproducts are target products. Grope for the radioactive method by18Fˉlabeling, theoptimum reaction conditions and a series of quality control tests of the final products.Investigate the clinical application feasibility of radioactive imaging agent18F-Fethypride through pre-clinical acute toxicity test, stability test, biodistribution testand pharmacokinetic test.2. Methods2.1A new type of PET/CT imaging agent with vanillic acid methyl ester as rawmaterial was prepared, involving7-step conventional reactions and1step radioactive18Fˉlabeling. This new agent will have great potential application prospect was for thesimple synthesis method and the cheap and widely available raw material. What’smore, the substituent on nitrogen can be further optimized.2.2Positron Radiopharmaceutical18F-Fethypride Quality Control2.2.1Clarity DetectionDetect the clarity by turbidimetric method. It was clear and qualified when theturbidity is below0.5turbidity standard solution.2.2.2pH DetectionDetermine pH value of the solution by using pH test strips.2.2.3Radiochemical Purity DetectionDetect the radiochemical purity of18F-Fethypride solution by HPLC method andTLC method.2.2.4Bacterial Endotoxin DetectionMeasure the bacterial endotoxin of18F-Fethypride solution by TachypleusAmebocyte Lysate(TAL) reagent method. It was qualified when the content ofbacterial endotoxin was below10EU/ml. 2.2.5Sterility Test18F-Fethypride solution was vaccinated in two different medium respectively andplaced at different temperatures to cultivate for14days. It was qualified if no bacterialgrew in the medium tube.2.2.6Abnormal Toxicity TestLab animals, including mice and guinea pigs, were intraperitoneally injected withdifferent doses of radiopharmaceutical18F-Fethypride and were observed for sevendays. During the period of observation, the test was qualified if the animals were ingood health, without abnormal reactions observed and gaining in weight.2.2.7K2.2.2Content DetectionDetect the K2.2.2content of18F-Fethypride by Spectrophotometric method. Itwas qualified if the figure was not above25mg/ml.2.3Lipo-hydro Partition CoefficientDetermine the radiocounting of Radiopharmaceutical18F-Fethypride with γ-radioactive counter when it was in n-butyl alcohol phase and water phase. The lipo-hydro partition coefficient(Log P) was the logarithm of both radiocountings ratios.2.4The Stability TestTo detect, analyze and evaluate its stability,18F-Fethypride solution was placed atroom temperature and in static state for60min,90min,120min,180min,240min,300min respectively after radioactive labeling. It was proved to be stable if theradioactive chemical purity(RCP) was not less than90%.2.5Acute Toxic TestKunming mice were injected with different doses of radiopharmaceutical18F-Fethypride into the tail vein. After7-day’s observation, they were killed andpathological section was done to check whether the main organs were normal.2.618F-Fethypride Biodistribution Test in the Main Organs in RatRats were injected with radiopharmaceutical18F-Fethypride solution into the tailvein and were killed10min,30min,60min,90min and120min respectively afterinjection. Adequate amount of samples from heart, liver, spleen, lung and kidney wererinsed in saline, blotted up moisture with filter paper. Weigh the samples with thebalance and determine the radiocounting with γ-radioactive counter. Hereby thepercentage of injected dose per gram (ID%/g) was calculated.2.718F-Fethypride Biodistribution Test of Brain Tissue in Rat Rats were injected with radiopharmaceutical18F-Fethypride solution into the tailvein, were killed10min,30min,60min90min and120min respectively after injection.Adequate amount samples from frontal lobe, parietal lobe, temporal lobe, striatum,hippocampus, hypothalamus and cerebellum were taken and rinsed in saline, blotted upmoisture with filter paper. Weigh the samples with balance and determine theradiocounting with γ-radioactive counter. Hereby the percentage of injected dose pergram (ID%/g) was calculated.2.818F-Fethypride Pharmacokinetic TestRats were injected with radiopharmaceutical18F-Fethypride solution into the tailvein.50ml blood sample was taken at different time and was analyzed by using liquidchromatography-mass spectrometry(LC-MS).2.9PET/CT ScanNormal rats and PD rats were injected with radiopharmaceutical18F-Fethypridesolution into the tail vein and were fixed in center field of examination bed afteranesthesia with10%chloral hydrate, supine position for PET/CT scanning.3. Results3.1Positron Radiopharmaceutical18F-Fethypride Quality Control18F-Fethypride solution was colorless, clear, transparent, without impurities andwith pH value of7, comforming to the rules. RCP was100%by TLC method, the Rfvalue was0.48; RCP was99%by HPLC method, t(R) was3.09min. The content ofbacterial endotoxin was below5EU/ml by Tachypleus Amebocyte Lysate reagentmethod through the conversion, conforming to the rules. Traceability sterility testresult showed that18F-Fethypride solution was qualified and no bacterial grew in twodifferent medium at different culture temperature. Animal abnormal toxicity testshowed that mice and guinea pigs were alive after7-day’s experiment. The animalshad increase in weight and no abnormal reactions were observed. The averageKryptofix2.2.2(K2.2.2) content was13.7mg/ml, less than25mg/ml in Pharmacopoeia,conforming to the standard. Quality control tests indicated that positron radio-pharmaceutical18F-Fethypride was qualified and safe.3.2Lipo-hydro Partition CoefficientLogP of radiopharmaceutical18F-Fethypride were2.4and2.2, which indicatedthat18F-Fethypride had good fat-solubility which helped radiopharmaceutical 18F-Fethypride to pass through the blood-brain barrier into the brain tissue.3.3The Stability TestRadioactive chemical purity(RCP) still exceeded95%by TLC method5hoursafter keeping at room temperature. There were some defluorization phenomenon4hours later, but the percentage was very small, less than5%. So the stability ofradiopharmaceutical18F-Fethypride was good, and suitable for clinical application.3.4Acute Toxic TestCompared with negative control group, no abnormal phenomenon were observedin livers and kidneys through pathological section. The maximum injection doses formice was as many as500times of that for the human being, which indicated thatradiopharmaceutical18F-Fethypride had less toxic effects and was safe in use.3.518F-Fethypride Biodistribution Test of Main Organs in RatsRadiopharmaceutical18F-Fethypride was mainly distributed in the liver andkidney and radioactivity gradually decreased with the passage of time. The intake ofheart was in a lower level. Clearance rates in kidney, spleen and lung were faster, butslower in hepatic. It indicated that18F-Fethypride was metabolized through the liver,and excreted through the kidney, which was coincided with benzamide compounds.3.618F-Fethypride Biodistribution Test of Brain Tissue in RatsRadiopharmaceutical18F-Fethypride was mainly distributed in the thalamus, thefrontal lobe and parietal lobe, temporal lobe and the least in cerebellum. Clearance ratein the cerebellum was the fastest, with the slowest clearance rate in the striatum.Striatum/Cerebellum gradually increased with the extension of time, which indicatedthat18F-Fethypride had specific and high affiliative bind with the dopamine D2receptor in the striatum.3.718F-Fethypride Pharmacokinetic TestThe half-life of18F-Fethypride in vivo was47.62min, less than the half-life of109.8min in vitro, it illustrated18F-Fethypride eliminated quickly in vivo afterintravenous drug, reduced the side effects, which was consistent with the results ofacute toxicity experiment.Hepatic extraction yield (ER=CL/Q) of18F-Fethypride inrats was3.36, it showed that the liver intake of radiopharmaceutical18F-Fethypridewas larger, which was consistent with the results of biodistribution test in vivo in rats.3.8PET/CT ScanPET/CT scan showed that radiopharmaceutical18F-Fethypride rapidly entered into all the major organs of rats and quickly cleared. The liver was the major metabolicorgan, and the kidney was the major excretory organ. Radiopharmaceutical18F-Fethypride could quickly pass through the blood brain barrier into the brain tissueand specific uptake was observed in the striatum. Clearance rate was low. It iscoincided with the results of biodistribution test in the brain tissue and major organs invitro. The right side of the striatum (damaged) of Parkinson’s rats obviously took inmore18F-Fethypride than normal rats, but no obvious differences were observed in theleft side (healthy side). That may be because the clinical type PET/CT was mainly usedfor human being, not for small animals. It had lower spatial resolution and poorvolume effect for those relatively small animals.4. Conclusions4.1A new precursor compound of18F-Fethypride with vanillic acid methyl ester as araw material was successfully synthetized after7-step conventional reaction. Productsat different stages all undergo nuclear magnetic resonance (NMR) and high resolutionmass spectrometry analysis. The precursor compound was (s)-3-[5-{(1-ethylpyrrolidin-2-yl) methylcarbamoyly}-2,3-dimethoxy phenyl] propyl-4-methyl benzemide.4.2Positron radiopharmaceutical18F-Fethypride was successfully labelled throughnucleophilic substitution reaction. Quality control tests indicated it was qualified andsafe.4.3Lipo-hydro partition coefficient of radiopharmaceutical18F-Fethypride wasappropriate.It could quickly pass through the blood brain barrier and specifically bindwith the corresponding receptor. It was stable, and had low toxic effect. So it issuitable for clinical intravenous injection.4.4Radiopharmaceutical18F-Fethypride was metabolized through the liver, andexcreted through the kidney. It had specific and high affiliative bind with the dopamineD2receptor in the striatum.4.5The kinetic courses of18F-Fethypride in rats were described by an open twoartment model with pharmacokinetic software DAS3.0, according to the principle ofthe minimum AIC and the maximum R2.4.6The right side of the striatum (damaged) of Parkinson’s rats obviously took inmore18F-Fethypride than the normal rats. The reason was that dopamine receptorlevels increased in the damaged side. This research involved a series of pre-clinical experimental tests forradiopharmaceutical18F-Fethypride, including precursor design, chemical synthesis,radioactive labeling, quality control, biodistribution test, pharmacokinetic test andPET/CT scan, etc. It laid an experimental foundation for the application ofradiopharmaceutical18F-Fethypride in clinical diagnosis of Parkinson’s Disease. |
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