重组人纤溶酶原Kringle 5的体内药代动力学及应用价值

In vivo pharmacokinetics and application of recombinant human plasminogen Kringle 5

目的研究纤溶酶原Kringle5体内药代动力学及其显像和治疗价值。方法应用基因工程技术重组人纤溶酶原Kringle5(rhK5),Iodogen法和直接法分别制备125I-rhK5、131I-rhK5和99mTc-rhK5。MTT比色法检测rhK5、125I-rhK5和99mTc-rhK5的活性。应用3p87程序分析125I-rhK5的药代动力学。制作肺癌移植瘤裸鼠模型,按3.7MBq经尾静脉注射99mTc-rhK5,于不同时间点观察显像情况。按7.4MBq经尾静脉注射131I-rhK,观察肿瘤生长及其微血管密度(MVD)和血管内皮生长因子(VEGF)表达,同时设立单纯rhK5蛋白治疗组和阴性对照组进行比较分析。结果125I-rhK5、131I-rhK5和99mTc-rhK5的标记率分别为86%、84%、90%,放化纯度均>90%;活性检测发现,标记与未标记rhK5之间无显著差异;125I-rhK5经尾静脉单剂量给药后,选择二室模型拟合药-时曲线,分布相半衰期(T1/2(α))为(0.31±0.03)h,清除相半衰期(T1/2(β))为(14.48±0.73)h,药-时曲线下面积(AUC)为(436.58±34.60)ng·mL-1·h-1。注射99mTc-rhK5后1h可见放射性浓聚,4h显像更趋清晰,肿瘤部位呈"热区"。与rhK5蛋白治疗组和阴性对照组比较,131I-rhK5治疗组肿瘤生长明显受到抑制(抑瘤率为34.14%),且MVD和VEGF表达显著减少。结论核素标记rhK5进行肿瘤显像和治疗是可行的。rhK5体内应用每天注射1次即可,为体内用药提供了实验依据。

Objective To investigate the in vivo pharmacokinetics of plasminogen Kringle 5 and the feasibility of radionuclide imaging and therapy. Methods Recombinant human plasminogen Kringle 5 （rhK5） was produced by gene engineering technique. 125^Ⅰ-rhK5 and 131^Ⅰ-rhK5 were prepared by Iodogen method, and 99m^Tc-rhK5 by direct method. The bioactivity of rhK5, 125^Ⅰ-rhK5 and 99m^Tc-rhK5 were detected by MTT assay. The pharmacokinetics of 125^Ⅰ-rhK5 was analyzed. The lung cancer-bearing nude mice model was established, and 99m^Tc-rhK5 was injected at 3.7 MBq. The imaging presentation was observed with time. 131^Ⅰ-rhK was injected at 7.4 MBq, and tumor growth, microvessel density（MVD） and expression of vascular endothelial growth factor（VEGF） were detected. The single rhK5 protein treatment group and negative control group were set for comparison analysis. Results The labeling yield of 125^Ⅰ-rhK5, 131^Ⅰ-rhK5 and 99m^Tc-rhK5 were 86% , 84% and 90% , respectively, and the radiochemical purity was 〉 90% for all groups. There was no significantdifference between labeled and unlabeled rhK5. The pharmacokinetics of 125^Ⅰ-rhK5 in rats was in correspondence with twocompartment model, the T1/2（α） was（0.31 ±0.03） h,T1/2（β） was（ 14.48 ±0.73） h and area under the curve（AUC） was （436.58 ±34.60） ng· mL ^- 1· h ^- 1. Serial scintigraphy after 99m^Tc-rhK5 injection confirmed that tumor uptake could be clearly visualized by 1 h, with gradual improvement in contrast achieved over 4 h. Hot spot imaging was observed in the tumor site. Compared with rhK5 protein treatment group and negative control group, tumor growth was significantly inhibited by 131^Ⅰ-rhK5 with the inhibition rate of 34.14% , and MVD and VEGF expression were significantly decreased. Conclusion Radiolabeled rhK5 can be used as a tumor imaging agent and therapy radionuclide. It may be sufficient that rhK5 be taken once daily, which provides the experimental evidence for in vivo clinical application.