^(99)Tc^m直接标记α_yβ_3受体拮抗剂环形RGD多肽

A direct radiolabeling of ^(99)Tc^m cyclic RGD peptide,an antagonist of α_vβ_3 receptor

目的探讨^(99)Tc^m 直接标记α_vβ_3受体配体——含2个二硫键的精氨酸-甘氨酸-天冬氨酸(RGD)-九肽(RGD-4CK)的可行性和不同标记条件对标记率的影响。方法采用预锡化法^(99)Tc^m 直接标记 RGD-4CK,3 MM 色谱纸层析测定^(99)Tc^m-RGD-4CK 的 Rf 值,计算标记率与比活度。研究各种标记条件对^(99)Tc^m-RGD-4CK 标记率的影响。通过高效液相色谱仪(HPLC)分析,Sep-Pak C18柱层析,进行体外稳定性实验、半胱氨酸置换实验以及血清蛋白结合实验,评价^(99)Tc^m-RGD-4CK 的放射化学性质。结果 ^(99)Tc^m-RGD-4CK 在以丙酮和V(氨水):V(乙醇):V(水)=1:2:5为流动相中的 Rf 值分别为0与0.8～0.9,标记率为(97.8±0.4)%,基础标记条件下的比活度为(11.90±0.05)TBq/mmol。在以下条件下放化纯均>95%:(1)酒石酸亚锡为150～300 μg;(2)预锡化反应 pH 值为2.0～3.5,温度60℃,保温时间6 h 以上;(3)^(99)Tc^m O_4^-活度为37～185 MBq,体积在200μl内;(4)标记温度为95℃,标记时间30 min。^(99)Tc^m-RGD-4CK HPLC 的保留时间与其洗脱液的放射峰基本一致;^(99)Tc^m-RGD-4CK 室温放置6 h,放化纯仍>95%;Sep-Pak C18柱层析测定的^(99)Tc^m O_4^-峰仅比3 MM 纸层析高0.5%;标记物与300 mmol/L 半胱氨酸37%保温1 h,^(99)Tc^m O_4^-峰仅增加1.88%;^(99)Tc^m-RGD-4CK 与血清蛋白无明显结合。结论预锡化法^(99)Tc^m 直接标记 RGD-4CK 方法简便、标记率高,无需分离纯化即可直接应用,还可用于制备冻干品药盒。标记产物^(99)Tc^m-RGD-4CK 具有良好的放射化学性质。

Objective It has been repotted that Cys-Asp-Cys-Arg-Gly-Asp-Cys-Lys-Cys （RGD） peptides, as integfin αvβ3 receptor antagonists, might inhibit angiogenesis of tumor. The aim of the present study was to investigate the feasibility of direct labeling of ^99Tc^m to a cyclic nine peptide containing RGD sequence （ RGD-4CK）, to observe the effects of different conditions on labeling efficiency, and to evaluate the radiochemical property of ^99Tc^m-RGD-4CK. Methods The peptide RGD-4CK contained the sequence of Cys-Asp-Cys-Arg-Gly-Asp-Cys-Lys-Cys. It was labeled directly by 99Tern with ＂pretinning＂ method. The Rf value of ^99Tc^m-RGD-4CK and ^99Tc^mO4 were determined in 3 MM paper chromatography. The labeling efficiency and specific activity were calculated. The influences of various conditions to the labeling rate were studied. High performance liquid chromatography （HPLC） analysis and Sep-Pak C18 chromatography were used to assess the property of radiolabeled ^99Tc^m-RGD-4CK. The stability in vitro, the cysteine replacement and the serum protein combination were also tested. Results The Rf values of ^99Tc^m-RGD-4CK were 0 and 0.8 ～ 0.9 respectively in mobile phase of acetone and V（ NH4 OH ） ： V（ethanol） ： V（water） = 1 ： 2： 5. The radiochemical yield was （97.8 ± 0.4）% and the specific activity （11.90 ± 0.05 ） TBq/mmol in the basic condition of labeling. The labeling efficiency remained over 95% under the condition of 150± 300μg stannous tartrate, pH value 2.0 ± 3.5, temperature 60℃ and incubation time over 6 h in the process of pretinning reaction, the activity of ^99Tc^mO4- was 37 - 185 MBq within volume of 200 μl. With the labeling temperature of 95℃ in 30 min, the retention of ^99Tc^m-RGD-4CK was in accordance with the major peak of timeactivity curve from the analytical HPLC. The radiochemical purity of 99Tc^m -RGD-4CK remained above 95% after 6 h at room temperature. The amount of ^99Tc^m O4^- increased only by 0.5% as measured with Sep-Pak C18 chromatography. The free ^99Tc^mO4^- from the labeled product increased by 1.88% after incubation with 300 mmol/L cysteine for 1 h at 37℃. There was no obvious binding of ^99Tc^m-RGD-4CK to serum protein. Conclusions This study indicated that direct ＂ pretinning＂ techniques was practical in preparing ^99Tc^m- RGD-4CK with acceptable labeling efficiency and radiochemical characteristics, and might be potentially useful in future preparation of ^99Tc^m-RGD-4CK kit.