【作者】 谭海波；

【导师】 林祥通； 管一晖； 尹端沚； 罗世能；

【作者基本信息】 复旦大学 ， 影像医学与核医学， 2008， 博士

【摘要】 一、选题的目的与设计思路:1、具有几千年用药历史的中药,对中华民族繁衍和发展起到了重要的作用,并日益引起世人的瞩目。中药药理的研究近50年来取得了长足的进步,并取得了一些可喜成果。但就整体而言,中药成分复杂,对其作用机制的诠释仍存在较大困难,因此,应在注意发扬我国以往优势基础研究的基础上,通过多学科交叉和应用先进的技术和方法平台,加强自主创新和前瞻性的研究,注意加强研究目标和技术选择,力争在某些领域取得突破,以推动中药新药的创新,这在国家对中药发展的中长科技规划中已经有战略构想。期望通过本世纪初叶的努力,使中医药的现代化战略目标能有所突破,使更多创新中药进入世界新药的高科技产品殿堂。更有意义的是,对13亿人口大国的国内市场真正建立起中西药平行的医疗保健市场体系,为13亿中国人民的健康保驾护航。开展中药的基础研究,不仅是阐明中药作用的的科学原理,以及研究开发现代中药新药物的需要,而且有助于寻求和发现现代医药学新的学科生长点。2、“工欲善其事,必先利其器”,现代药理学、药代动力学、毒理学、药物学、药剂学、药物分析学、生药学等等,均离不开相关的新方法和新技术进步,中医药的现代化进程也概莫能外。许多国家的政府主管机关和大企业,均能把新技术、新方法列入药学研究的前沿课题,并给予专项的资金投入。诸如基因工程动物模型、反义技术、基因芯片技术、单克隆抗体工程、计算机技术、受体分析技术、信息科学（包括遥感检测技术）、现代仪器分析技术（NMR、MS、HPLC—MS、LC—MS—MS等）,上述技术在验证创新药物疗效（药效）和安全性两个关键指标中起着关键作用。随着科学技术的不断进步,多学科交叉的现象越来越多。近年来,随着现代分子影像学（Molecular Imaging）的迅速发展,并与其他学科不断渗透、交叉和结合。如上世纪70年代CT问世以来,CT、MR、fMR、SPECT、PET以及PET/CT已在全球范围内广泛应用,使对人体疾病的诊断、分期、疗效和预后评价迈入“分子影像学”时代,医学影像学的进展是20世纪人类的重大科技成就之一。分子影像技术从临床人体应用的仪器设计到小动物专用仪器的设计思路,首见于1997年美国加州大学Cheng博士领导的研究小组的报导（Cheng SR,Shao Y,SilVelmanRW,et al.MicroPET:A high resolUtion PET scanner for imaging smallanimals.IEEE Trasaction on Nuclear Science,1997,44:1161-1166）,此是基于正电子断层成像（PET）临床成像技术发展起来的专门用于小动物PET成像的装置,称为microPET。它的特点是:①分辨率较高,达1mm;②体积小,造价低;③可用PET/CT、PET/MR进行解剖与功能（代谢、血流、受体、基因）图像融合;④在药物研究方面显示出优良前景,可缩短研制周期、节约开发成本,与相关技术形成的Translation Medicine（转化医学）已引起大企业集团的兴趣与投入研发。本课题旨在探索正电子核素标记中药的有效成分（先导物）,应用PET技术了解药物的代谢动力学的有关参数并与经典的药学方法对比,以观察PET技术应用的可行性及前景。3、神经精神疾病脑内受体和递质呈多靶点之关系,而中药药物作用往往也呈多靶点,而且一种药物即使作用于同一靶点其产生的效应在不同生理病理情况下亦呈现不同（比如上调-下调,激动-阻断作用等）,上述因素决定了中药药理、药效研究的复杂性和艰巨性,要探索研究PET技术在研究中药药理、药效、药代动力学参数方面的可行性,要突破如下技术问题:①正电子核素的选择及标记技术,诸如:要求易于标记、反应时间短、放化收率高、不影响构效关系（活性）、能满足实验观察正电子核素标记的药物在动物体内生物分布和代谢的时限要求、标记产物的质量控制和稳定性;②PET测量及图像ROI重建技术:包括仪器质控、测量的统计误差及重复性、ROI的勾画技术、图像重建技术和药代动力学参数数学模型和正确运算以及影响因素（麻醉等）;③中药先导物选择的依据:按已发现的新药国际上通常实行四级（A-D）分类,A为具有新化学结构,治疗有突破;B为已知化学结构,治疗有突破;C为新化学结构,治疗未有突破;D为已知化学结构,治疗未有突破。上述A及B无疑最具有创新性。在我国中药研究方面,知母皂甙元、石衫碱甲和左旋千金藤啶碱（1-SPD）已明确其有效化学结构及构效关系,临床应用有效,当属于A类的创新药物,尤其1-SPD,它是中药延胡索的有效成分,它具有D₁激动-D₂阻滞的双重药理作用,迄今为止国际上只发现1-SPD具有这种双重作用特征,1-SPD的双重药理作用机制为新型安定剂开辟了研究方向,并且在精神疾病、阿片类毒品成瘾的治疗方面有着发展前景。因此,1-SPD具有很大的二次开发价值。自然,从新结构和全新药理作用的创新（新药）是人们追求的目标。本研究的重点旨在探索验证PET技术从整体研究的角度应用于药物开发领域的可行性。4、¹¹C标记中药左旋千金藤啶碱尚未见文献报道,如能探索¹¹C标记中药有效成分的方法并进行生物学评价,无疑具有新颖性,同时利于探索中药有效成分在活体内的作用机理,此外也可为PET或micro PET药物开发进行先期准备工作,考虑本课题取材方面等因素,首先选择左旋千金藤啶碱进行探索性研究。旨在通过PET对左旋千金藤啶碱药代动力学进行研究作为第一阶段工作,通过该研究为今后中药的活体研究探索一条新的途径。本研究的主要目的:1)探索正电子核素标记中药有效成分的方法学可行性。2)探索¹¹C标记中药有效成分1-SPD在体内的生物学分布,为药物二次开发提供参考。3)探索PET显像用于中药有效成分药代动力学研究的可行性,为PET或micro PET药物开发进行先期准备工作,通过动态地观察动物体内生物分布,为中药在活体内的药代动力学研究探索一条新途径。5、正电子发射断层（PET）基础与临床研究是我院影像医学与核医学博士点学科及教育部重点学科主要研究方向之一,而正电子核素标记中药有效成分的探索研究又系此方向的一个分领域,并且是系列性博士课题研究,包括¹⁸F-知母皂甙元（2006年）、¹¹C-石衫碱甲（2007年）、¹¹C-左旋千金藤啶碱（2008年）以及天花粉蛋白等,曾得到影像医学与核医学国家重点学科（211工程1期）、复旦985神经病学重点项目及卫生部核医学国家重点实验室开放基金（WK006001及WK006002）的少额研究基金支持。本课题为上述总课题（2006年-2008年）的重要组成部分。二、研究结果:1.¹¹C标记中药左旋千金藤啶碱（1-SPD）的制备与质量控制目的:探索¹¹C标记中药延胡索的有效成分左旋千金藤啶碱（1-SPD）方法学,并对其进行质量控制。方法:由美国CTI公司RDS111加速器生产¹¹CO₂,用碘代甲烷模块将。¹¹CO₂转化成。¹¹C-碘代甲烷(¹¹C-CH₃I),再转化为¹¹C-Triflate—甲烷后传入到溶有1-SPD的二甲基亚砜溶液中,在常温下反应得到产物。与合成的标准品¹²C-1-SPD比较,鉴定通过液相色谱仪[流动相甲醇:乙腈:三乙胺=60:20:20（三乙胺0.05mol/1,用磷酸调节至3.0）,流速为1ml/min。],质谱及核磁氢谱鉴定,放化纯度、稳定性检测。并对新制剂¹¹C-1-SPD进行质量控制。结果:¹¹C-1-SPD放化合成时间约为10～20min,¹¹C-1-SPD注射液为无色溶液,半衰期在20±5分钟范围内。PH值为6.5±0.3,注射液在2h内不同时间的放射性化学纯度均大于95%,化学纯度大于90%。生物学质控亦符合正电子放射性药物质量要求。结论:正电子核素¹¹C标记中药延胡索有效成分左旋千金藤啶碱在方法学上是可行的,合成的¹¹C-1-SPD注射液符合进一步的动物或人体探索性研究要求。2.¹¹C-1-SPD在正常动物体内分布及药物代谢动力学实验目的:研究¹¹C-1-SPD在动物体内的生物学分布及其药物代谢动力学特性。方法:经大鼠尾静脉注射¹¹C-1-SPD,每组5只,分别于不同时相点5、15、30、60、90min测量血液及各主要组织器官的放射性计数（cpm）,计算每克组织百分注射剂量率（%ID/g湿组织）。通过大鼠尾静脉取血进行药物代谢动力学分析;大鼠尾静脉注射给药,于注射后不同时间分别将各组大鼠断头处死,迅速解剖取脑,分离额叶、顶叶、颞叶、枕叶、小脑、海马、纹状体、丘脑、脑干等脑区,称组织湿重后,用γ计数器测组织放射性,计算每克组织百分注射剂量率（%ID/g湿组织）。结果:¹¹C-1-SPD在体内吸收迅速,分布广泛,以肝、肾、心、脑、肺分布最多。静脉注入¹¹C-1-SPD 5min后放射性分布即可达到高峰,5min后各组织呈明显下降的趋势,给药后60min,各脏器内放射量均有显著下降。脑内分布在5min即可达到高峰,各脑区内的放射性分布差异无统计学意义（p＞0.05）。药物经肝脏代谢,肾组织是¹¹C-1-SPD的主要排泄器官,肝、肾组织在5min放射性分布分别为1.484±0.350%ID/g、1.323±0.153%ID/g,90min分别为0.478±0.039%ID/g、0.394±0.165%ID/g。在胃、脾、肠、肌肉等组织放射性分布较少。大鼠时间-放射性曲线在权重为1/CC时二室模型最佳,对¹¹C-1-SPD大鼠血时间.放射性曲线进行二室模型拟合,经分析计算,中央室的消除速率常数为0.19/min,表观分布容积为0.36ml/mg,分布半衰期为1.39min,清除半衰期为26.2min,清除率为0.07ml/min/mg。药时曲线下面积为1613.72（kbpm/ml）*min。结论:静脉注射¹¹C-1-SPD在体内呈现快速摄取快速清除的特性。1-SPD在脑内广泛分布,清除快速。1-SPD主要经肝胆系统代谢,肾脏是主要排泄器官。血药浓度-时间曲线符合二型房室模型特征。3.¹¹C-1-SPD在正常动物体内的PET/CT（临床用机型）显像研究目的:探索PET/CT显像（临床用机型）在研究¹¹C标记中药有效成分1-SPD在体内的生物学分布及药物代谢动力学特征的可行性。方法:SD大鼠戊巴比妥钠麻醉后固定于木板上,尾静脉注射37 MBq¹¹C-1-SPD。分别于注射后5min、15min、30min、45min、60min、90min行PET/CT显像。使用Multi Modality工作站获得脑、心脏、肺脏、肝脏、肾脏、肠腔、膀胱的容积分布比值（DistributionVolume Ratios,DVR）。结果:¹¹C-1-SPD大鼠PET/CT显像显示在5分钟时在肝脏、肾脏分布最多,心脏、肺、脑可见放射性分布。肝肾是¹¹C-1-SPD的主要代谢及排泄器官,肝、肾、肠腔、膀胱在5min放射性分布分别为1.37±0.42%、1.10±0.19%、0.89±0.18%、0.97±0.111%,90min分别为0.65±0.11%、0.54±0.05%、5.49±1.44%、9.86±1.88%。结论:PET/CT（临床用机型）显像可以初步的直观、动态的观察药物的分布及代谢特点,然而由于轴向视野及临床用机型PET分辨率远不如MicroPET（小动物专用型）理想,现有的结果尚不能得出PET（临床型）显像完全取代处死动物的体内分布实验的结论,有待小动物专用型MicroPET的进一步对照研究。三、本研究论文可以得出以下结论:1.通过亲核取代反应制备中药左旋千金藤啶碱¹¹C标记化合物:¹¹C-1-SPD,放化纯度＞95%,体外稳定性好,主要质量控制指标达到要求,可满足进一步探索性实验的要求。2.静脉注射¹¹C-1-SPD在体内呈现快速摄取快速清除的特性,¹¹C-1-SPD在脑内广泛分布,清除快速,各脑区的放射性分布差异性无统计学意义（p＞0.05）。1-SPD主要经肝脏代谢,肾脏是主要排泄器官。血药浓度-时间曲线符合二型房室模型特征。¹¹C-1-SPD在体内、脑内分布及药代学参数若获得不同单位方法学重复性的验证,以及有条件用小动物专用型MicroPET单位所获得数据吻合,从整体动物获得的参数,可有望为该药的二次开发提供参考。3.试用PET/CT（临床用机型）显像用于中药研究可行性,试图动态的观察¹¹C-1-SPD在动物体内的分布及代谢特点,并用DVR（Distribution Volume Ratios）进行定量分析,然而由于PET分辨率及轴向视野等方面的因素,目前资料未能得出¹¹C-1-SPD活体动物PET（临床用机型）显像能取代处死动物的体内分布实验的结论,这有待今后小动物专用MicroPET的进一步对比研究。同时,我们首先提出在中药研究开发中建立转化医学（Transition Medicine）及移动式MicroPET技术平台（toolbox）的建议。4.¹¹C-1-SPD的制备、药代动力学观察,以及PET/CT（临床用机型）小动物显像,目前未见文献报导,选题及研究工作具有新颖性。更多还原

【Abstract】 Purpose and design of the study1.Traditional Chinese pharmacy,a splendid pearl in the treasures of world traditional medicine,has a history of thousands of years,and it has played an important role in the development of Chinese nation.For a long time,traditional Chinese pharmacological research has been developed from scratch,and great achievements have been made.The elucidation of efficacy of traditional Chinese drugs and their underlying mechanisms,however,is still a great challenge.Therefore we should take full advantage of the current technology and use the multi- discipline crossing methods to help the innovation of traditional Chinese pharmacy. Basic researches for traditional Chinese drugs are of great importance for the elucidation of scientific principles of effects of traditional Chinese drugs,and for the development of modern new traditional Chinese drugs,and are helpful for investigating the new growth point for modern medicine and pharmacy.2."A better tool is needed for good work,a workman must sharpen his tools if to do his work well".Modern pharmacology,pharmacokinetics, toxicology,materia medica,and so on,all depend on the development of new methods and new technology,as well as traditional Chinese pharmacy.With the development of science and technology,multiple-discipline crossings are more and more common.With the repaid development of modern medical imaging,and its intercrossing and integration with other disciplines,the knowledge,techniques and methods in the field of medical imaging are more and more introduced into modern pharmacology to address issues in traditional Chinese pharmacology,such as CT,MR,fMR,SPECT,PET, PET/CT,which is necessary for the modernization of traditional Chinese medicine and pharmacology,and is essential for the development of traditional Chinese drug research,microPET is a device of imaging for small animal,its characteristic is:①high resolution.②small volume, and low price.③it can fuse the anatomical and the functional images.④it can reduce time and cost of the reserch in new drug.3.Neuropathy and psychosis show the relationship of multi-target between receptor and transmitter,as well as the traditional Chinese medicine,lf we want to use PET to explore the pharmacology,curative effect,pharmacokinetics of the traditional Chinese medicine,we must resolve the three problems:①the selection and radiolabeling of positron-emitting radionuclide.②PET imaging and image reconstruction.③Selection Principle of the traditional Chinese medicine primer.In the shudy of the traditional Chinese medicine,L-Stepholidine（1-SPD） is one of the active ingredients of corydalis,which is a traditional Chinese herb.L-SPD has a dual pharmacological effect of D1 agonism and D2 blockade. So far,1-SPD is the only substance found to have this kind of dual effect. The dual pharmacological effect of 1-SPD will be a new research field for the development of new neuroleptic agents,and will be promising in the treatment of mental disorders and opioid addiction.Therefore,the second development of 1-SPD is of great value.4、On the whole,it has not been reported in literature to label an active ingredient of 1-SPD with ¹¹C.If the method to label an active ingredient of traditional Chinese herb with ¹¹C is investigated,and biological evaluation is conducted,these will be of great novelty,and will be beneficial to the investigation of in vivo mechanism of active ingredients of Chinese herbs.In addition,these studies of labeling methods may be used as preparatory studies for future possible PET or microPET assisted drug development.Based on factors relating to material selection in this project,L- Stepholidine is chosen to conduct an exploratory study.The pharmacokinetic study of 1- Stepholidine using PET will be conducted as first stage work,by which a new method may be provided for future in vivo investigations for active ingredients of Chinese herbs.The objectives of this study are as follow:1) To investigate method feasibility of labeled active ingredients of Chinese herb imaging with PET.2) To investigate in vivo distribution of ¹¹C labeled 1-SPD to provide reference for future possible second development. 3) To investigate the feasibility of PET imaging in the study of pharmacokinetics of active ingredients of Chinese herbs to conduct preparatory work for future possible PET or micro PET assisted drug development;To explore a new investigation method for the study of in vivo pharmacokinetics of traditional Chinese drugs by dynamically monitoring biological distribution in animals.5、Preliminary And Clinical Studies On Positron Emission Tomography was one important research of Batch of doctoral program of Imaging medicine and Nuclear medicine and Key Discipline of MH.To label an active ingredient of traditional Chinese herb with 11C was one branch of serial studies.The serial studies included ¹⁸F-ZMS（2006）,¹¹C-HupA（2007）, ¹¹C-1-SPD（2008）,Trichosanthin,and so on.The serial studies had been Sponsored by 985 Key Project of neurology and State Key Laboratory of Nuclear Medicine（WK006001,WK006002）.This subject was the important part of the serial studies.Results of the studyPartⅠ.Synthesis and quality control of ¹¹C-radiolabeled 1-SPDObjective:To expore the method and quality control of ¹¹C -radiolabeled 1-SPD.Methods:¹¹CO₂ was produced by CTI RDS 111 cyclotron, converted ¹¹CO₂ to ¹¹C- CH₃I by CH₃I die-block,and then converted it to ¹¹C-Triflate—CH₄,imported ¹¹C-Triflate—CH₄ to 1-SPD which was dissolved in Dimethyl sulfoxide[（CH3）₂SO],reacted at common temperature and then obtained the product.Compared with ¹²C-1-SPD which was produced at the same condition.The labeled product was analyzed by HPLC on a reverse-phase C18 column.Contents and analytical methods of quality control for ¹¹C-1-SPD were investigated and the main quality criteria were achieved through strict control of the determining parameters by standard procedures.Results:The Synthesis time of ¹¹C-1-SPD was 15 to 20 min with chemical purity＞90%,and PH values 6.5±0.3,radiochemieal purity＞95% in 2 hours.All quality criteria of ¹¹C-1-SPD met the requirements of the positron radio-pharmaceuticals.Conclusion:¹¹C-1-SPD injections can be used to further study in the animal or human study. PartⅡ.Pharmacokinetics and biodistribution of ¹¹C-1-SPD in the normal animalObjective:To exolpre the pharmacokinetics and biodistribution of the new agent,¹¹C-1-SPD in vivo.Methods:Take the sample of blood and organs in different time,5 min,15 min,30 min,60 min,90 min after injecting ¹¹C-1-SPD by vail in mice and measure the cpm,then the%ID/g was calculated.Analysed the pharmacokinetics by taking the blood sample from tail of rats and analysed the biodistribution in the different regions of brain,such as frontal lobe,apical lobe,temporal lobe,occipital lobe,cerebellum,hippocampus,striatum,thalamencephalon and brain stem in the rats.Results:¹¹C-1-SPD was of the characteristic that quickly discharge from the blood,,metabolism through the liver,kidney was the main eccrisis organ,which was 1.323±0.153%ID/g at 5 min and decreasing graduately,it was 1.484±0.350ID/g in liver at 5 min. Pharmacokinetics of ¹¹C-1-SPD in the rats corresponded to two-compartment model.The half-time was 26.2min,apparent volume of distribution was 0.36ml/mg,clearance rate was 0.07ml/min/mg.Conclsions:¹¹C-1-SPD could quickly discharge and decrease to the low level from background of issues in vivo.There was not significant difference of ¹¹C-1-SPD that distribute in the different brain regions,there were more distribution in brain.Pharmacokinetics of ¹¹C-1-SPD in the rats corresponded to two-compartment model.PartⅢ.Biodistribution of ¹¹C-1-SPD in rats assessed by PET/CT（dedicated PET/CT） ImagingObjective:To exolpre the feasibility to assess the biodistribution of ¹¹C-1-SPD in vivo by PET/CT（dedicated PET/CT） Imaging.Methods:Perform PET/CT scan in different time,5 min,15 min,30 min,45min,60 min,90 min after injecting ¹¹C-1-SPD by vail in rats and transfer the information of brain,heart,lung,liver,kidney,intestine,bladder to the Multi Modality Workststion.Obtain the Distribution Volume Ratios（DVR）of the above tissues.Results:¹¹C-1-SPD was keeping in a relative higher level in liver and kidney at 5min.metabolism through the liver,kidney was the main eccrisis organ.The distribution of ¹¹C-1-SPD in liver, kidney,intestine,bladder was 1.37±0.42%,1.10±0.19%,0.89±0.18 %,0.97±0.111%respectively at 5min and was 0.65±0.11%,0.54±0.05 %,5.49±1.44%,9.86±1.88%respectively at 90min.Conclsions: PET/CT（clinical type） imaging could observe the distribution and metabolism of ¹¹C-1-SPD dynamic and directly.On account of resolution and FOV of the PET/CT（dedicated PET/CT）,we could not draw the conclusion that PET/CT imaging can take the place of biodistribution experiment ex vivo.Conclusions（PartⅠ-PartⅢ）:1.Synthesis of ¹¹C-1-SPD was of high radiochemical purity and good stability in vitro.All quality criteria of ¹¹C-1-SPD met the requirements of the positron radionuclide-radiolabeled pharmaceutical, and can be used to further study.2.¹¹C-1-SPD could quickly discharge and decrease to the low level in vivo.Pharmacokinetics of ¹¹C-1-SPD in the rats corresponded to two-compartment model.There was not significant difference of ¹¹C-1-SPD that distributed in the different brain regions of normal rats.If the data could be repeated by other departments,the distribution and pharmacokinetics of ¹¹C-1-SPD in brain and body could offer references to second exploitation of drugs.3.Try to assesse the biodistribution of ¹¹C-1-SPD in rats by PET/Cr（dedicated PET/CT） Imaging,but on account of resolution and FOV of the PET/CT（dedicated PET/CT）,we could not draw the conclusion that PET/Ctimaging can take the place of biodistribution experiment ex vivo.We firstly bring forward a proposal to establish "toolbox" of Translation Medicine in the research of traditional chinese herb.4.Synthesis of ¹¹C-1-SPD and pharmacokinetics study by PET/CT （dedicated PET/CT）in small animals had not been reported in literature.更多还原