| The malignant tumors must establish a neovasculature to grow, to invade, and to metastasize, in which process the αvβ3 integrins play an important role. It is found that the αvβ3 integrins effect mainly by binding with triplet peptides Arg-Gly-Asp (RGD) sequence in the proteins of the extracellular matrix. Because the expression of αvβ3 integrins is reported to be strong on activated endothelial cells but restricted on normal cells , radiolabeled RGD antagonistic peptides could be used as a class of tumor specific markers in early stage cancer detection and therapy. Objective To establish a useful and stable method for direct radiolabeling of RGD-4CK with 99Tcm; To study the biodistribution of 99Tcm-RGD-4CK in vivo of nomal mice and bearing mice. Methods The method to label RGD-4CK with 99Tcm is based on the following initial experiment: In the reaction tube,10 μg of RGD-4C peptide,100 μl buffer (containing 45 mmol/L sodium potassium tartrate, 10 mmol/L potassium hydrogen phthalate, PH 5.0～5.2),750 μg ascorbic acid and 100μg stannous tartrate were added,the PH value of the reaction system was adjusted to 3.0,and the head space was filled with nitrogen after mixing.Then the reaction tube was sealed and had been incubated at 37℃for 6 hours.Finally 100 μl of 99Tcm pertechnetate solution was added , the tube was sealed again and had been incubated at 90℃for 30 min.When the reaction mixture was cooled, aliquots were taken for labeling yield analysis.To find the optimum labeling condition, various parameters, such as the mass of peptide, ascorbic acid and stannoustartrate, volum of 99TcmO4-, incubation period at 37℃,temperature of labeling reaction, were studied. The vitro stability of 99Tcm-RGD-4CK in room temperature was analyzed by determining the radiochemical purity from 2 to 12 hours after labeling was completed.Finally in vivo biodistribution of nomal mice and bearing mice was carried out in the optimum labeling condition with labeling yield at 95% or more. 99Tcm-RGD-4CK (200 μl, 370 KBq) was injected via caudal vein,and the mice were sacrificed and dissected in scheduled time after injection. The organs of interest and tumours were removed and weighed. The radioactivity in the tissue was measured using a gamma counter. Results are expressed as the percentage of the injected dose per gram of tissue (%ID/g) in normal and bearing mice.Tumor/tumorouse tissue ratios (T/NT) were calculated in the organs of interest in the bearing mice. For blocking study the bearing mice were injected with 150μg RGD-4CK 30 minutes before injection of 99Tcm-RGD-4CK,and restrained efficiency was calculated by comparing the percentage of the injected dose per gram of tumor of blocking and unblocking bearing mice at the same phase. Results 1.The optimum labeling condition of the pretinning direct Method to labele RGD-4Ck with 99Tcm in 100 μl buffer is as the following: ⑴The mass of ascorbic acid is 500 μg or more;⑵the mass of stannous tartrate is 150μg to 250 μg;⑶the PH value of the reaction system before pretinning is 3.0;⑷the reaction temperature is 90℃;⑸the reaction time is 30 minutes;⑹the volume of 99TcmO4-is 100 μl;⑺the mass of RGD-4CK is 10 μg;⑻the pretinning period is 6 hours.The labeling yield will be 92% to 95% in the optimum conditions. The radiochemical purity of 99Tcm-RGD-4CK is more than 90% when having been placed for 4 hours in room temperature, And it is more than 80% when having been placed for 6 hours in room temperature. 2.In vivo biodistribution of 99Tcm-RGD-4CK in normal Kunming strain mice indicated that radiolabeled peptide was cleared rapidly from blood, highest activity concentration was observed in kidneys, and activity concentration in liver was second; Biodistribution in C57BL/6 mice bearing murine Lewis pulmonary carcinoma is like that in normal Kunming strain mice in main organs. Activity accumulation in the tumour was 1.64±0.13,1.94±0.09 and 2.31±0.22%ID/g in unblocking bearing mice 1h, 2h and 4h postinjection, respectively, and restrained efficiency was 57%,66% and 69% respectively in blocking bearing mice comparing with unblocking bearing mice at same phase. The resulting T/NT (tumour/nontumorous tissue) ratios in blood, muscle,lung and brain increased gradually, and it was 5.97±1.07, 6.92±2.29, 8.27±1.93 and 19.62±5.07 respectively in unblocking bearing mice 4h postinjection,and was 2.49±0.31, 2.89±0.82, 2.08±0.61,and 2.31±1.08 respectively in blocking bearing mice at the same phase. Conclusions 1. The optimum condition of radiolabeling of RGD-4C with 99Tcm wasmade certain when all of the studying radiolabeling parameters were optimum,and the radiolabeling yield was 92% to 95%.The radiolabeling peptide was stable in vitro in room temperature. 2. In vivo biodistribution of 99Tcm-RGD-4CK in normal mice indicated that radiolabeled peptide was cleared rapidly from blood and excreted mainly via urinary system and partially via digestive system. Biodistribution in muscles,lungs,and small intestine were low-level,and that in brain was low-level at all times.This revealed that radiolabeled peptide could not enter into blood-brain barrier. Biodistribution in C57BL/6 mice bearing Lewis murine pulmonary carcinoma was like that in normal Kunming strain mice in main organs. Activity accumulation in the tumour was higher than the majority of the interest organs in unblocking bearing mice and decreased obviously in control by blocking with nature RGD-4CK. Thist indicates that the activity accumulation in tumour in different observing phases should be receptor-dependent, which confirmed that 99Tcm-RGD-4CK should have the same biologic activety as the nature RGD-4CK,and this study can help the farther imaging study with this labeling peptide.
|
PDF Full Text Request