Experimental Study On Radionuclide Imaging And Targeting Therapy In Nude Mice Bearing Human Lung Cancer With Radionuclide Labeled Peptides K237

【Objective】Studies have demonstrated neovascularization as a requirement for primary tumor growth,invasion,and metastasis.Because cells in solid tumors must receive oxygen and other nutrients to survive and grow.In the absence of a mechanism for inducing angiogenesis,solid tumors are incapable of growing beyond 1-2 mm³.Dozens of natural pro-angiogenic and antiangiogenic factors have been identified.Inhibitors of angiogenesis are among the most promising new chemotherapeutic agents under investigation.Inhibitors of angiogenesis induced significant delay in tumor growth,which can disturb tumor growth and inhibit tumor progression.A lot of clinical trials have been used to determine the efficacy of angiogenesis inhibitors.Peptides K237 is a novel inhibitor of angiogenesis, which was isolated from a phage-displayed peptide library,can inhibit tumor growth and metastasis by blocking the binding of vascular endothelial growth factor(VEGF) to it's KDR receptor.1.The aim of this study was to explore the method of the radiolabeling of peptides K237 with ⁹⁹Tc^m and to investigate the imaging in vivo and biodistfibution characters of ⁹⁹Tc^m-K237 as tumor receptor imaging agent in mice;2.To investigate the targeted therapeutic efficiency of ¹³¹I radiolabeled peptide K237 (¹³¹I-K237) on nude mice bearing human lung cancer.【Methods】1.Peptide K23 7 was labeled directly with ⁹⁹Tc^m.The labeling rate,the radiochemic purity and the in vitro stability of ⁹⁹Tc^m-K237 were studied under various condit- ions.The bioactivity of ⁹⁹Tc^m-K237 was tested by human umbilical vein endothelial cell proliferation inhibitory assay.The affinity of ⁹⁹Tc^m-K237 was examined by competition binding studies.⁹⁹Tc^m-K237 was injected through tail vein of normal mice with 2.96 MBq in a volume of 0.1 ml.Those mice were killed at different stages after injection and their blood and other major organs were taken out,then the radioactivity count of these organs were measured to investigate the biodistribution.⁹⁹Tc^m-K237 of 0.1 ml 5.55 MBq was injected into the tail vein of tumor bearing nude mice,SPECT imaging and using regoin of interest technique for semiquantitativeanalysis of tumors and opposite side muscle organs' uptake at 1,2,3,5 and 8 h after injection.2.Iodogen method was used for ¹³¹I labeling K237.The bioactivity of ¹³¹I-K237 was tested by HUVEC proliferation inhibitory assay and the affinity of ¹³¹I-K237 was examined by competition binding studies.¹³¹I-K237 was injected through tail vein of nude mice with 2.96 MBq in a volume of 0.1 ml to investigate the biodistribution.Human lung cancer xenografts with positive KDR expression were established in nude mice.All 25 mice models were divided into five groups randomly, including physiologic saline group,¹³¹I-K237 group intravenously,¹³¹I-K237 group intratumorally,K237 group and ¹³¹I group.Thirty-one days after infusion,the tumor growth inhibition rate was calculated.The inhibited effect and the radiation breakdown of ¹³¹I-K237 on nude mice were determined by biochemical,immunohistochemical and pathohistology methods.【Results】1.Under the conditions of SnCl₂·2H₂O 100μg,K237 100μg,pH value 6.0,reaction time 10～30 min and reaction temperature 4～37℃,the labeling rate and the radiochemic purity of ⁹⁹Tc^m-K237 were more than 95%.Tthe inhibition rate of HUVEC proliferation had no significant difference between radiolabeled K237 and unlabeled K237(t = 2.83,P＞0.05).After ⁹⁹Tc^m-K237 was placed in physiologic saline and in blood serum,the radiochemic purity was respectively(89.1±1.4)%and (88.3±1.1)%at 24 h,while there was no significant difference between the two groups(t=1.56,P＞0.05).In the first 24 h postinjection,the biodistribution in mice showed rapid blood and renal clearance with gradually excreted in other organs or tissues including heart,liver,lungs,stomach and muscle,most of the radioactivity was observed in renal.In imaging,the uptake ratios of tumor to background(T/B) at 1,2,3,5 and 8 hwere 1.25±0.11,3.13±0.26,3.97±0.31,1.34±0.29 and 1.18±0.25, respectively;2.The labeling rate of ¹³¹I-K237 was 60.16%,radiochemical purity was above 95%.Tthe inhibition rate of HUVEC proliferation had no significant difference between radiolabeled K237 and unlabeled K237(t = 1.67,P＞0.05).The ratios of tumor to muscle were 2.12,2.32,2.51,2.70,3.03,4.31and 4.19 at 30min,1h,2h,4h,8h,12 h and 24 h,respectively.The growth of transplanted lung cancer was inhibited by 75.01%,78.99%,31.15%and 12.61%at groups treated with ¹³¹I-K237 intravenously,¹³¹I-K237 intratumorally,K237 and ¹³¹I,respectively.The average tumor volume of the ¹³¹I-K237 trial groups less than that of the control,K237 and ¹³¹I group (P＜0.01).Irreversible destruction of tumor cells in ¹³¹I-K237 trial groups and K237 group was found under light and electron microscope.There was no effect of radiation breakdown on liver,kidney,spleen and blood-cell in nude mice.【Conclusion】1.⁹⁹Tc^m-K237,which is an easily prepared and labeled compound with high labeling rate and stability,will be a potential tumor imaging agent;2.¹³¹I-K237 can effectively inhibit the growth of tumor in nude mice beating human lung cancer,with little obvious side effects.