【作者】 李英华；

【导师】 陆景彬； 林英杰；

【作者基本信息】 吉林大学 ， 粒子物理与原子核物理， 2014， 博士

【摘要】 正电子发射计算机断层显像-计算机断层成像(positron emission tomography-computed tomography，PET/CT)，是目前临床应用最广泛的分子影像学检测设备之一。它实现了医学影像学从解剖结构变化向功能代谢变化的根本转变，PET/CT具有灵敏、特异、定位精确等特点，可以无创、定量、动态监测正电子核素标记的放射性药物在体内的分布，从分子水平反映活体内生理、生化改变，是理想的药物筛选平台。正电子放射性药物的选择是PET/CT成像的基础和关键。帕金森氏病是三大常见的神经系统变性疾病之一，是一种慢性、隐袭性疾病，多见于老年人。帕金森氏病具有高度异质性，没有特异的诊断方法，主要依靠病史、临床症状和体征进行诊断，部分临床症状不典型或无症状的患者诊断更加困难。早期患者通过药物治疗大多可很好地控制症状，至疾病中、晚期由于对药物反应差，症状得不到控制，可出现全身僵硬，生活不能自理，严重影响生活质量，因此早诊断、早治疗显得尤为重要。随着分子核医学迅速发展和新的正电子药物不断开发，针对帕金森氏病已经研发出多种特异的正电子放射性显像剂，通过PET/CT扫描，可以在解剖结构发生改变之前，从代谢和功能显像方面及时发现异常变化，从而达到早期诊断、及时治疗的目的。1.目的自行设计、研发一种新型的PET/CT显像剂18F-Fethypride，用于诊断帕金森氏病。对前体化合物合成中的每种产物进行核磁共振和高分辨质谱表征分析，以确定合成的是目标产物；探索放射性18Fˉ标记方法及最适反应条件，并对终产物进行一系列质量控制；对正电子放射性显像剂18F-Fethypride进行临床前急性毒性实验、稳定性实验、生物学分布实验和药代动力学实验，探讨18F-Fethypride临床应用的可行性。2.方法2.1以香草酸甲酯为原料，经7步常规反应和1步放射性18Fˉ标记合成一种新型的具有潜在应用前景的PET/CT显像剂18F-Fethypride，该化合物具有合成方法简单，原料价廉、易得，而且氮上的取代基还可以做进一步优化等优点。2.2正电子放射性药物18F-Fethypride质量控制2.2.1澄清度检测：通过比浊法检测18F-Fethypride溶液的澄清度，不超过0.5号浊度标准液的浊度为澄清，即合格。2.2.2pH值检测：pH试纸检测18F-Fethypride溶液的pH值。2.2.3放射化学纯度检测：用高效液相色谱法(HPLC)和薄层色谱法(TLC)检测18F-Fethypride溶液的放射化学纯度。2.2.4细菌内毒素检测：用鲎试剂法检测18F-Fethypride溶液的细菌内毒素，细菌内毒素含量<10EU/ml，判定合格。2.2.5无菌实验：18F-Fethypride溶液分别接种于2种不同培养基，放置不同温度培养14天后，各管培养基均无杂菌生长，判定合格。2.2.6异常毒性实验：包括小鼠实验和豚鼠实验，将不同剂量的放射性药物18F-Fethypride腹腔注射受试动物，观察7天，观察期间动物应全部健存，无异常反应，到期后每只体重应增加，则供试品判定合格。2.2.7氨基聚醚(K2.2.2)含量测定：分光光度法测定18F-Fethypride溶液中K2.2.2的含量，K2.2.2含量不得超过25μg/ml，判定合格。2.3脂水分配系数测定用γ-计数器分别测定放射性药物18F-Fethypride在正丁醇相和水相的放射性计数，两者计数比值的对数为脂水分配系数(Log P)。2.4稳定性实验将18F-Fethypride溶液室温静置，分别于放射性标记后60min、90min、120min、180min、240min、300min测定放射性化学纯度并进行分析，评价18F-Fethypride溶液的稳定性，放化纯度应不低于90%。2.5急性毒性实验小鼠尾静脉注射不同浓度的18F-Fethypride溶液，观察7天，处死，病理切片，检查主要脏器是否正常。2.618F-Fethypride大鼠主要脏器生物学分布实验大鼠尾静脉注射18F-Fethypride溶液，分别于注射后10min、30min、60min、90min和120min处死，取适量心、肝、脾、肺、肾等样本，生理盐水冲洗干净，滤纸吸干水分，天平测量样本质量，γ-计数仪测量放射性计数，计算每克组织百分注射剂量率(ID%/g)。2.718F-Fethypride大鼠脑组织生物学分布实验大鼠尾静脉注射18F-Fethypride溶液，分别于注射后10min、30min、60min、90min和120min处死，取大脑额叶、顶叶、颞叶、纹状体、海马、丘脑和小脑等样本，生理盐水冲洗干净，滤纸吸干水分，天平测量样本质量，γ-计数仪测量放射性计数，计算每克组织百分注射剂量率(ID%/g)。2.818F-Fethypride药代动力学实验大鼠尾静脉注射放射性药物18F-Fethypride溶液，分别于注射后不同时间尾尖部取血50μl，液相色谱-质谱联用仪进行分析测量。2.9PET/CT扫描正常大鼠与帕金森氏病大鼠分别尾静脉注射放射性药物18F-Fethypride溶液，10%水合氯醛麻醉、固定，置检查床中心视野进行PET/CT扫描。3.结果3.1正电子放射性药物18F-Fethypride质量控制18F-Fethypride溶液无色、澄清、透明、无杂质，pH值为7，符合规定。TLC法测得的放化纯度为100%，Rf值为0.48；HPLC法测得的放化纯度为99%，保留时间t(R)为3.09min。通过换算，鲎试剂法测得的细菌内毒素含量<5EU/ml，符合规定。追溯性无菌试验结果显示，两种不同培养基在不同的培养温度无菌试验均合格，未有杂菌生长。动物异常毒性实验表明，7天后小鼠和豚鼠均健康存活，体重增加，无异常反应。氨基聚醚(K2.2.2)含量平均值为13.7μg/ml，小于药典规定的25μg/ml，符合标准。质量控制实验说明放射性药物18F-Fethypride是合格且安全的。3.2脂水分配系数18F-Fethypride脂水分配系数(Log P)分别为2.4和2.2，说明18F-Fethypride脂溶性大，有利于通过血脑屏障进入脑组织。3.3稳定性实验室温放置5h后，TLC法测得放化纯度仍大于95%，有部分脱氟现象，多在4h以后出现，所占比例很小，低于5%。可见，放射性药物18F-Fethypride稳定性很好，适合临床应用。3.4急性毒性实验病理切片结果显示：与阴性对照相比，肝脏、肾脏未见异常。小鼠的最大注射剂量相当于人用注射剂量的500倍，表明18F-Fethypride毒性作用较小，应用安全。3.518F-Fethypride大鼠主要脏器生物学分布实验18F-Fethypride主要分布在肝脏和肾脏，随时间延长而逐渐减少，心脏的摄取一直很低。肾脏、脾脏、肺部的药物清除速率较快，肝脏的清除速率较慢，说明18F-Fethypride通过肝脏代谢，肾脏排泄，与苯甲酰胺类化合物的代谢和排泄途径相符。3.618F-Fethypride大鼠脑组织生物学分布实验在脑组织中18F-Fethypride主要分布在纹状体，丘脑、大脑的额叶、顶叶、颞叶和小脑分布很低。药物的清除速率小脑最快，纹状体最慢。随着时间的延长，纹状体/小脑(靶/非靶)摄取比值逐渐增加，说明18F-Fethypride能与纹状体中的多巴胺受体特异性结合，且亲和力高。3.718F-Fethypride药代动力学实验用DAS3.0药代动力学软件对血药浓度进行房室模型拟合，根据AIC最小，R2最大原则，放射性药物18F-Fethypride在大鼠体内的动力学过程符合二室模型。18F-Fethypride在大鼠体内表观分布容积大，说明其脂溶性大，有利于通过血脑屏障进入脑组织。18F-Fethypride在大鼠体内的半衰期小于体外半衰期，提示其在体内消除较快，降低了毒副作用，这与急性毒性实验结果相一致。3.8PET/CT扫描PET/CT扫描显示：18F-Fethypride快速进入大鼠各个主要脏器，并快速清除；肝脏为主要代谢器官，肾脏为主要排泄器官；可通过血脑屏障迅速进入脑组织，在纹状体有特异性摄取，清除速率较慢，与脑组织和主要脏器的生物学分布实验结果基本一致。帕金森氏病大鼠纹状体对18F-Fethypride的摄取明显高于正常大鼠，但右侧纹状体(毁损侧)与左侧(健侧)相比，没有显著变化，可能由于本实验使用的是临床型PET/CT扫描仪，主要用于人体扫描，相对于体积较小的大鼠，空间分辨率低、容积效应差。4.结论4.1以香草酸甲酯为原料，经7步常规反应，对每步产物进行核磁共振和高分辨质谱表征分析，成功合成18F-Fethypride前体化合物(s)-3-[5-{(1-乙基-2-吡咯烷基)甲基胺甲酰基}-2,3-二甲氧基苯基]丙基-4-甲基苯磺酸酯。4.2通过亲核取代反应，放射性18Fˉ成功标记，合成正电子放射性药物18F-Fethypride，质量控制实验表明其安全、合格。4.318F-Fethypride脂水分配系数适宜，能快速通过血脑屏障，该放射性药物稳定、对动物毒性作用小，适合临床静脉注射。4.418F-Fethypride主要通过肝脏代谢，肾脏排泄，能与纹状体中多巴胺受体特异性结合，亲和力高。4.5用DAS3.0药代动力学软件对血药浓度进行房室模型拟合，根据AIC最小，R2最大原则，正电子放射性药物18F-Fethypride在大鼠体内的动力学过程符合二室模型。4.6帕金森氏病大鼠纹状体对放射性药物18F-Fethypride的摄取明显高于正常大鼠，可能是多巴胺受体水平上调所致。本课题完成了正电子放射性药物18F-Fethypride的前体设计、化学合成、放射性标记、质量控制实验、生物学分布实验、药代动力学实验以及PET/CT扫描等一系列临床前实验工作，为其应用于临床帕金森氏病的诊断奠定了实验基础。更多还原

【Abstract】 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.更多还原