Radioiodinated Small-Molecule Tyrosine Kinase Inhibitors For HER2-Selective SPECT Imaging Of Breast Cancer

Breast cancer is one of the most common malignant tumors in women all over the world.Breast cancer in our country has been the highest incidence of cancer in women.Breast cancer occurs in epithelial tissues and is a highly heterogeneous tumor with distinct individual differences in pathological morphology,molecular immunophenotype and tumor biological behavior,even in patients with the same tumor stage.Both the effect and the prognosis may be completely different.Among them,human epidermal growth factor 2(erbB2/HER2/neu)is a member of the human epidermal growth factor(EGF)receptor(EGFR)family,which is overexpressed in 20-30%of breast cancers.A tyrosine kinase inhibitor(TKI)is a small molecule that binds to the cell membrane and binds to the HER2 kinase domain,reflect the level of tyrosine kinase inside the cell.The purpose of this paper is to develop a radioactive iodine-labeled TKI,and to study the physical and chemical properties and a series of in vitro and in vivo bioassays to investigate the prospects of this probe for breast cancer imaging applications targeting HER2.Firstly,the analog IBA-CP of the small molecule inhibitor CP724,714 was successfully synthesized,and 125/131 I-IBA-CP was prepared by the iodine-labeled iodine method.The radiochemical yield of 65%,radiochemical purity of>98%.The molar activity can reach 42 GBq/?mol.At the same time,125/131 I-ICP was prepared by direct labeling of CP724,714 by Iodogen method with radiochemical yield of>90%and radiochemical purity of>99%.Comparing the in vivo stability of the two radioactive probes,the in vivo stability of 131I-IBA-CP was significantly better than that of 131 I-ICP within 1h.Secondly,four common human breast cancer cells were selected.The positive expression of HER2 in BT-474 and MDA-MB-453 cells and the negative expression of HER2 in MCF-7 and MDA-MB-468 cells were confirmed by flow cytometry and Western Blot analysis.Among them,MDA-MB-468 cells have strong positive expression of EGFR.Subsequently,by WB assay,the expression of phosphorylation after compound and cell co-incubation,we found that IBA-CP and CP724,714 can inhibit the expression of HER2 phosphorylation,but has no effect on EGFR phosphorylation,inhibition experiments show that IBA-CP has Good specificityFinally,specific experiments and imaging experiments were performed on the radiolabeled two probes.In SPECT/CT imaging,HER2-positive tumor sites had significant probe uptake after injection of 125I-IBA-CP,which reached the highest at 3 h,and the tumor/meat ratio reached 3.9,while no significant uptake was observed in HER2-negative tumors..In inhibition experiments,uptake of HER2-positive tumors was inhibited by non-radioactive IBA-CP,but not by erlotimib(EGFR inhibitor),demonstrating that 125I-IBA-CP has good HER2 selectivity.The directly labeled 125I-ICP also had a certain uptake in HER2-positive tumors,reaching the highest at 1 h of dosing,but the tumor/meat ratio was only 2.3.In the biodistribution experiment of tumor-bearing mice,MDA-MB-453 had the highest tumor uptake 3 h after injection of 131I-IBA-CP,which was 2-3 times that of MCF-7 and MDA-MB-468 tumors(1.05±0.25).%ID/g vs.0.32±0.13%ID/g and 0.38±0.05%ID/g,P<0.001),co-injection with non-radioactive IBA-CP significantly reduced tumor uptake(from 1.05±0.25%ID/g to 0,19±0.04%ID/g,P<0.001),co-injection with erlotinib did not reduce tumor uptake,and biodistribution results were consistent with small animal SPECT imaging The results of biodistribution experiments of 131I-ICP showed that after 3 h of injection,MDA-MB-453 tumor uptake was 0.7±0.5%ID/g,which was lower than 131I-IBA-CP On the contrary,thyroid uptake was significantly higher.131I-ICP was observed.A large degree of deiodination occurred in the body to enrich the thyroid gland,demonstrating that the direct labeling method obtained radioactive iodine-labeled probes with poor stability in vivoIn summary,two radiolabeled TKI probes were successfully synthesized and prepared in this study:125/131I-IBA-CP and 125/131I-ICP.Through a series of experiments,it was confirmed that the 125/131I-IBA-CP probe has good specificity,HER2 selectivity,and has certain stability during in vivo circulation.It can be selected to targeting of HER2-positive tumors in SPECT/CT imaging of tumor-bearing mice.This probe deserves further study and is expected to be used for the diagnosis and treatment monitoring of HER2-positive breast cancer.