The Investigation Of Integrin α_vβ₃-Selective Radiotracer ^99mTc-3PRGD₂ In Breast Tumor

Objective:This report describes the synthesis of a new radiotracer: ^99mTc-HYNIC-PEG₄-E [PEG₄-c (RGDfK)]₂(^99mTc-3PRGD₂) and the evaluation of its safety and feasibility for integrinα_vβ₃-positive imaging by vivo and in vitro experiments.Methods:3PRGD₂ was prepared with HYNIC as bifunctional coupling agent; its integrinα_vβ₃ binding affinity was detected by half inhibitory concentration (IC50) in competitive binding experiments. ^99mTc-3PRGD₂ was prepared with tricine and trisodium triphenylphosphine-3, 3′, 3″-trisulfonate (TPPTS) as ligand. The average radiochemical purity of ^99mTc-3PRGD₂ was determined by radio-HPLC and radio-ITLC (the Rf value of ^99mTc-3PRGD₂ and ^99mTcO₄^- are 0⁰.1 and 0.9¹.0 respectively in acetone system; the retention time of ^99mTc-3PRGD₂ and ^99mTcO₄^- is 21.89 min and 3⁵ min in radio-HPLC). ^99mTc-3PRGD₂ was placed in 0.9%saline and 1mg/mL cysteine solution respectively for observing its stability in vitro. The pharmacokinetics in normal mice,biological distribution and SPECT imaging of ^99mTc-3PRGD₂ in the MDA-MB-435 human breast tumor models and HT29 human colon tumor models were evaluated. The safety and accuracy of ^99mTc-3PRGD₂ were analyzed in 40 breast tumor patients by observing their reactions after administration and calculating the radioactivity ratio of tumor and non-tumor (T/NT) in regions of interest. The expression of integrinα_vβ₃ was assessed by integral optical density of the tumor in pathological and immunohistochemical analysis, and the Pearson correlation coefficient r² of T/NT and IOD value was calculated. Results:Competitive binding experiments showed that 3PRGD₂ has a higher integrinα_vβ₃ binding affinity than HYNIC-E[c(RGDfK)]₂ and c(RGDfK). The IC50 of 3PRGD₂,HYNIC-E[c(RGDfK)]₂,c(RGDfK) were 3.4nM,9.6nM and 58.1nM respectively. The radiochemical purity of ^99mTc-3PRGD₂ was over 95% and even over 99% after purifie by radio-ITLC and radio-HPLC. It's radiochemical purity remained above 95% after incubation 6 hours at 37℃in 0.9%saline and 1mg/mL cysteine solution respectively.^99mTc-3PRGD₂ cleared rapidly from blood. The percentage values were 19.32%, 7.21% and 1.27% at 10min, 20min and 60min after injection. The biodistribution showed: at 0.5h, 1h, 2h and 4h after administration, the uptake value of MDA-MB-435 human breast tumor models were significantly higher than that in higher than that in HT29 human colon tumor models (6.073,5.877,4.718 and 2.787%ID/g vs 1.506,1.208,0.913 and 0.899%ID/g, P<0.05). There was high uptake in kidney, its value were 11.697 and 11.512% ID/g at 4h after injection in MDA-MB-435 human breast tumor models and HT29 human colon tumor models. The tumor/muscle ratio in MDA-MB-435 human breast tumor models was higher than that in HT29 human colon model at 2h after injection(5.866 vs 1.008, P<0.05). The imaging appeared from 15min after administration in MDA-MB-435 human breast tumor models and was clearest at 2h till 4h; there was no tumor imaging in HT29 human colon model.There was no adverse reactions (such as nausea, bronchospasm, itching, flushing, hives, chills, coughing, bradycardia, muscle cramps, or dizziness) within 24 h after injection of ^99mTc-3PRGD₂ in 40 breast tumor patients.25 malignant lesions in 40 patients were found by pathology, 23 lesions (sensibility=92.0%) were detected by ^99mTc-3PRGD₂ imaging. 1 patient with multiple bone metastases confirmed by wholebody bone scan and biopsy was also found. 30 benign lesions in 40 patients were also found by pathology. There was no uptake in 20 lesions, slight uptake in 2 lesions and high uptake in 8 lesions (specificity=73.3 % ). The expression of integrinα_vβ₃ was found in neovascularization of all malignant lesions and 8 benign lesions. The expression of integrinα_vβ₃ was not found in left benign lesions.There was a significant positive correlation between T/NT ratio and IOD value (r²=0.927).Conclusion:3PRGD₂ has a character of simple preparation and high integrinα_vβ₃ binding affinity. The radiochemical of ^99mTc-3PRGD₂ was high and its properties in vivo and vitro,pathology consistent for breast tumor imaging were good. There was a positive correlation between T/NT and IOD. But its potential for breast tumor target imaging will be tested by increasing the sample number.