Synthesis Of Novel Bifunctional Chelating Agent 3,4-HOPO And Treatment For Triple Negative Breast Cancer With¹⁷⁷Lu-3,4-HOPO-Cetuximab

Part ? Synthesis and characterization of 3,4-HOPOObjective:To synthesize a novel bifunctional chelating agent-3,4-HOPO,to detect the complexation constant with each metal ion and to explore the chelation ability compared with DTPA.Methods:Based on maltol,3,4-HOPO was synthesized by a series of chemical methods.The correctness and purity of the structure were detected by HPLC,mass spectrometry,nuclear magnetic and infrared;and the complexation constant with each metal ion was detected by potentiometric titration.And compared with DTPA,the species distribution and stability performance under different pH conditions were analyzed.Results:The purity of 3,4-HOPO was 98.6%by HPLC and the molecular weight was 183 by mass spectrometry.The structure of nuclear magnetic and infrared analysis was correct.The dissociation constants of pKa1 and pKa2 of 3,4-HOPO were analyzed by Hyperquad 2008 software are 10.1±0.1 and 3.7±0.1.The dissociation constants pKa1,pKa2,pKa3,pKa4 and pKa5 of DTPA are 2.5±0.2,2.5±0.2s 4.6±0.2,8.7±0.2 and 10.1±0.2,respectively.In physiological environment pH 7.4,Lu3+ exists in the form of Lu2LH and Lu2L,respectively.The combined constants are 26.6±0.1 and 21.0±0.2,respectively.Conclusion:Self-synthesis of bidentate 3,4-HOPO,compared with DTPA,it shows stronger chelation ability for metal Lu3+.If it can synthesize four teeth or even more teeth 3,4-HOPO based on double-toothed 3,4-HOPO,the chelating ability for lanthanide metal ions and uranyl may be stronger.Part ? Study of 177Lu-3,4-HOPO-Cetuximab killing triple negative breast cancer cellsObjective:To establish a method for the identification of 177Lu-labeled 3,4-HOPO-cetuximab,and to compare its physicochemical properties with DOTA,and to verify the targeted effect of cetuximab and the lethal ability of 177Lu by CCK-8 assay and cell-binding assay of 4T1 cells.Methods:3,4-HOPO and DOTA were used as bifunctional chelating agents,respectively coupled with Cetuximab and labeled with 177Lu to determine the labeling rate,radiochemical purity and stability of 177Lu-3,4-HoPo and 177Lu-DOTA.Cell viability and cell binding rate were measured by CCK-8 assay and cell binding assay after 177Lu-3,4-HOPO-Cetuximaband 177Lu-DOTA-Cetuximabwere applied at different times or different doses.Results:The labeling rates of 177Lu-3,4-HOPO-C etuximab and 177Lu-DOTA-Cetuximab were(89.7±0.9)%and(92.6±1.0)%,respectively,and the radiochemical purity after ultrafiltration separation was greater than 95%;The radiochemical purity of 177Lu-3,4-HoPo and 177Lu-DOTA is(96.85±0.9)%and(98.69±1.0)%,respectively.The storage is most stable at 4oC,and the radiochemical purity can reach 92%after 72h,the difference was statistically significant(P<0.01);177Lu-3,4-HoPo-Cetuximab was administered with the dose of 500 Bq/mL,5 kBq/mL,50 kBq/mL,500 kBq/mL and 5 MBq/mL,respectively.The cell viability after 48 h were(93.91±1.24)%,(91.01 ±0.92)%,(84.84±0.74)%,(60.15±1.84)%and(17.82±F 1.03)%at the dose of 5 MBq/mL.The cell viability after 8 h,24 h,48 h,72 h and 96 h were(50.47± 1.83)%,(24.18±0.74)%,(17.2±1.03)%,(12.75±0.91)%and(9.24±0.68)%respectively.The binding rate of 4T1 cells after 177Lu-3,4-HoPo treatment for 1 h,2 h,4 h,8 h and 24 h(1.67±0.02)%,(2.06±0.08)%,(3.72±0.19)%,(6.44±0.51)%and(10.17±0.37)%respectively.Conclusion:177Lu-3,4-HOPO-Cetuximab has good stability in vitro,and is most suitable for storage at 4oC.Cetuximab is targeted to 4T1 cells positive for EGFR receptor,177Lu-3,4-HOPO-Cetuximab can effectively kill 4T1 tumor cells,and as the dose increases,the killing ability of the cells rises in a nearly exponential form.Part ? 177Lu-3,4-HOPO-Cetuximab in the treatment for triple-negative breast cancerObjective:To investigate the effect of 177Lu-3,4-HOPO-Cetuximab on the radionuclide internal irradiation of triple-negative breast cancer by tail vein injection of tumor-bearing nude mice,and to verify the effective treatment of tumor sites by immunohistochemistry.Methods:A subcutaneous metastasis model of triple negative breast cancer was established.The 177Lu-3,4-HOPO-Cetuximab was injected into the tail vein 100 ?L/well(about 500 ?Ci/body),177Lu-DOTA-Cetuximab and normal saline were set as the control group.One-time injection was administered,and tumor volume growth and body weight changes were regularly monitored.After radiotherapy,the radioactivity distribution in nude mice was analyzed and HE staining were used to monitor the expression of EGFR in untreated tumor tissues.Results:After 20 days of treatment,the body weight of nude mice in the 177Lu-3,4-HOPO-Cetuximab group and the 177Lu-DOTA-Cetuximab group did not change,and the weight of the control group was significantly reduced;177Lu-3,4-HOPO-Cetuximab group and 177Lu-DoTA The tumor inhibition rates of the Cetuximab group were(37.03±11.16)%and(38.73±5.08)%,respectively.The bone radioactivity of the 177Lu-3,4-HoPo-Cetuximab group was significantly lower than that of the 177Lu-DOTA-Cetuximab group with statistical significance(P<0.05).HE staining showed that tumor cells were affected by 177Lu and caused necrosis.Conclusion:177Lu-3,4-HOPO-Cetuximab has obvious inhibitory effect on the growth of 4T1 tumor.3,4-HOPO as a bifunctional group complexes Lu3+has less deposition in bone than DOTA.HE staining proves that 177Lu causes tumor cells deadly killing.