环形精氨酸-甘氨酸-天冬氨酸多肽的核素标记及在健康动物体内的实验

Radiolabeling of cyclic RGD peptide and its experimental study in normal animals

目的:探讨99Tcm直接标记环形RGD-4CK九肽的可行性,观察99Tcm-RGD-4CK在健康动物体内的药代动力学、生物分布特点及显像表现。方法:实验于2004-10/2005-11在解放军第三军医大学西南医院核医学中心完成。①采用预锡化法99Tcm直接标记RGD-4CK,3MM色谱纸层析测定标记率,通过HPLC分析、SepPakC18柱层析、体外稳定性实验、半胱氨酸置换实验及血清蛋白结合实验,评价99Tcm-RGD-4CK的放射化学性质。②选取雄性日本大耳兔9只,每只静脉注射37MBq99Tcm-RGD-4CK,分别于1.5,3.0,5.0,10,30,60,90,120,180,240min采血、称重并测定血样品放射性计数,结果经参考源校正后以MBq/L表示,所得数据应用DAS软件处理,结合αvβ3受体在正常体内实际表达情况判断室模型。③选取小鼠40只,随机分为8组(n=5),每只静脉注射0.74MBq99Tcm-RGD-4CK,分别于1,5,20,60,90,120,180,240min处死小鼠,采集血液、心、肺、肝、肾、肠、肌肉、骨、脑,称重并测定放射性计数,结果经参考源校正后以每克组织百分注入剂量(%ID/g)表示。④选取雄性日本大耳兔3只,每只静脉注射37MBq99Tcm-RGD-4CK,应用SPECT进行显像,结合感兴趣时间-放射性曲线分析,观察家兔体内放射性的动态分布变化。结果:40只小鼠和12只兔全部进入结果分析。①99Tcm-RGD-4CK标记率为(97.8±0.4)%,比活度为(11.90±0.05)PBq/mol;HPLC保留时间与洗脱液放射峰值时间基本一致,室温放置6h的放射化学纯度仍>95%;SepPakC18柱层析游离99Tcm仅比纸层析高0.5%;与300mmol/L半胱氨酸37℃温育1h,游离99Tcm仅增加1.88%;99Tcm-RGD-4CK与血清蛋白无明显结合。②99Tcm-RGD-4CK在健康家兔体内的药代动力学符合权重数为1/C的二室模型,分布相半衰期为(4.18±2.17)min,消除相半衰期为(69.32±0.00)min。③小鼠血液放射性清除迅速,通过肾脏排泄且快,其余组织器官放射性均随时间逐渐降低,而脑始终呈最低放射水平。④家兔SPECT显像示:各组织器官T-A曲线均随时间逐渐下降,与肾、心、肝相比,肺、胃、肌肉曲线呈低水平;1min双肾即显影,5min心、肝影开始减弱,肺放射性分布均匀,强度低于肝脏,膀胱显影;5min后膀胱影持续增强,20min后软组织影逐渐减弱;胆囊未显影,腹部呈持续低放射分布,胃区始终呈放射性缺损,颈部未见明显核素浓聚。结论:99Tcm-RGD-4CK制备方法简便,标记率高(>95%),具有良好的放射化学性质,体内稳定性好,具有比较理想的体内动力学。

AIM： To investigate the feasibility of ^99Tc^m direct labeling of cyclic RGD- 4CK peptide, and observe the pharmacokinetics, biodistribution characteristic and imaging manifestation of ^99Tc^m-RGD-4CK in normal animals. METHODS： This experiment was conducted in Nuclear Medicine Center, Southwest Hospital, Third Military Medical University of Chinese PLA between October 2004 and November 2005.①RGD-4CK was labeled directly with ^99Tc^m by pretinning method, and the labeling rate was determined with 3 MM chromatography. The radiochemical property of ^99Tc^m-RGD-4CK were evaluated by HPLC analysis, Sep Pak C18 chromatography, the stability experiment in vitro, the cysteine challenge experiment and the serum incubation test.②Nine male Japanese flap-eared rabbits were selected and injected with 37 MBq ^99Tc^m-RGD-4CK via auris vein, then the blood was sampled at 10 phases during 1.5-240 minutes, then weighted and measured by γ scintillation counter. The result was corrected by referenced source and finally expressed as MBq/L. The obtained data were processed by computer with DAS software and the compartment model ,sas judged by combining the actual expression of etch3 receptor in normal body.③Forty mice were divided into 8 groups randomly with 5 in each group. After vein injection of 0.74 MBq ^99Tc^m-RGD-4CK, mice were executed at 1, 5, 20, 60, 90, 120, 180 and 240 minutes r espectively, then blood, heart, lungs, liver, kidneys, intestine, muscle, bone and brains were collected, weighted and determined by γ counter. The result was corrected by referenced source and finally expressed as %ID/g. ④The scintigraphy of ^99Tc^m-RGD-4CK was performed respectively in three male Japanese flap-eared rabbits, combining the ROI T-A curve analysis and SPECT imaging, the dynamic distribution manifestation of ^99Tc^m-RGD- 4CK was observed. RESULTS： Totally 40 mice and 12 rabbits were involved in the result analysis. ①The labeling rate of ^99Tc^m-RGD-4CK was （97.8±0.4）% and the specific activity was （11.90±0.05） PBq/mol. The retention time was essentially identical as the radiative peak of eluent from an analytical HPLC. The radiochemical purity was still greater than 95% after placing for 6 hours at room temperature. The amount of free ^99Tc^m measured by Sep Pak C18 chromatography was only more 0.5% than that by 3 MM paper. The free ^99Tc^m merely increased 1.88% after incubation with 300 mmol/L cysteine for 1 hour at 37 ℃ There was unobvious combination found between ^99Tc^m-RGD-4CK and serum protein.②The pharmacokinetics of ^99Tc^m-RGD-4CK matched the two compartment model in normal rabbits （Weighted=l/C）, and T1/2α was （4.18＋2.17） minutes in blood while T1/2β was （69.32：t0.00） minutes. ③The biodistribution showed that the radioactivity was removed rapidly from blood in mice and renal excretion was also fast, whereas other tissues and organs were gradually reduced with time, in which the brain was the lowest radiation of all phases. ④The SPECT imaging displayed that the ROI T-A curves of tissues and organs were all descended with time, in which the lungs, stomach and muscle were lower than the kidneys, heart and liver; Kidneys image appeared at 1 minute postinjection; At 5 minutes, both heart and liver radioactivity began to weaken, the radioactivity distribution was homogeneous in lungs but was lower than that of liver. In addition, there was a nuclide accumulation in bladder; The radioactivity was continuously increased in bladder after 5 minutes and gradually decreased in soft tissues after 20 minutes; Gallbladder had not radioactive uptake, there was always lower level of radioactivity distribution in abdomen, the stomach region presented radioactive defect all the time, and there was no obvious nuclide aggregation in neck. CONCLUSION： ^99Tc^m-RGD-4CK can be readily prepared, and the labeling yields are quite high （〉 95%）. Moreover, ^99Tc^m-RGD-4CK has excellent radiochemical characters in vitro and stability in vivo, thus it has an ideal dynamics in vivo.