In Vitro And In Vivo Properties Of ¹⁷⁷Lu-labeled RM26 Modified By Oligo Phenylene Ethynylene

Objective:Gastrin Releasing Peptide Receptor（GRPR）is overexpressed in a variety of malignant tumors,such as prostate and lung cancers.GRPR antagonists have been found to be superior to agonists in biological properties.Hence,GRPR antagonists have become a research hotspot in GRPR positive tumors.In order to solve the problem of the low internalization characteristics of antagonists,this study intended to introduce penetrating oligo phenylene ethynylene（OPE）with the GRPR antagonist RM26 to achieve higher tumor cell penetration and cell targeting.A chelating agent NOTA was used for radionuclide ¹⁷⁷Lu or ⁶⁸Ga labeling.In vitro stability,GRPR positive cells uptake,in vivo distributions in tumor-bearing mice were explored using¹⁷⁷Lu-NOTA-OPEs-RM26.Thetherapeuticeffectof¹⁷⁷Lu-NOTA-OPE-2-RM26 was performed using tumor-bearing mice.Micro-PET/CT was used to investigate the in vivo distribution of⁶⁸Ga-NOTA-OPE-2-RM26 in tumor-bearing mice.Methods:（1）NOTA-OPEs-RM26 were synthesized in multiple steps,then were characterized by Ultra Performance Liquid Chromatography-Mass Spectrum（UPLC-MS）,and the final products were purified by HPLC.（2）NOTA-OPEs-RM26 were labeled with radionuclide ¹⁷⁷Lu and the optimal labeling conditions were explored.The in vitro stability of¹⁷⁷Lu-NOTA-OPEs-RM26 in newborn calf serum（NBCS）,DMEM-H（serum-free）and physiological saline were determined.（3）The uptake,internalization and cellular specific binding of ¹⁷⁷Lu-NOTA-OPEs-RM26 in human prostate cancer cells PC3 were studied.The cellular binding in human lung adenocarcinoma cells A549 and human lung cancer lymph node metastasis cells H292 of ¹⁷⁷Lu-NOTA-OPEs-RM26 was developed.（4）PC3 tumor-bearing mice were established by using SPF BALB/c nude mice.3.7 MBq of⁶⁸Ga-NOTA-OPE-2-RM26 was injected into the PC3 tumor-bearing mouse via the tail vein（n=1）,and Micro-PET/CT imaging was performed after 1 hour.（5）0.37 MBq of ¹⁷⁷Lu-NOTA-OPE-1-RM26 and ¹⁷⁷Lu-NOTA-OPE-2-RM26 were injected into PC3 tumor-bearing mice along the tail vein（n=2）,respectively,and the biodistributions were performed at 2 hours or 24 hours.0.37 MBq of¹⁷⁷Lu-NOTA-OPE-1-RM26 and ¹⁷⁷Lu-NOTA-OPE-2-RM26 were injected into A549 tumor-bearing mice through the tail vein（n=1）,respectively,and biodistributions were performed at 4 hours or 50 hours.（6）3.7 MBq of ¹⁷⁷Lu Cl₃and ¹⁷⁷Lu-NOTA-OPE-2-RM26 were injected in situ into the tumors of PC3tumor-bearing mice（n=5）,respectively,to study the therapeutic effects of these radiopharmaceuticals.Results:（1）The NOTA-OPEs-RM26 were successfully synthesized by multi-step synthesis method.The target products were separated and purified by HPLC,and identified as the correct target products by UPLC-MS.（2）The optimal labeling reaction conditions of ¹⁷⁷Lu-NOTA-OPEs-RM26 were determined as follows:sodium acetate（Na OAc）（0.5 M,p H 5.5）buffer,80°C,1h.The radioactive purity detected by thin layer chromatography（TLC）above99%.The radioactive purities of the two radionuclides were over 99%in NBCS,DMEM-H（serum-free）,and physiological saline after 72 hours at room temperature.（3）The cellular uptake experiment of ¹⁷⁷Lu-NOTA-OPEs-RM26on PC3 cells:（1）The cellular binding of membrane surface reached the maximum value first and then decreased over time.（2）The internalizing dose of the cells gradually increased with time.（3）The total cell uptake reached the maximum value at 6 h and basically maintained unchanged,which was mainly the membrane surface binding turned into cell internalization.The cellular internalization assay on PC3 cells:At 24 hours,the internalization of the control group ¹⁷⁷Lu-NOTA-RM26 without introducing OPE reached 28%,while the internalization of¹⁷⁷Lu-NOTA-OPE-1-RM26 and¹⁷⁷Lu-NOTA-OPE-2-RM26 reached 30%,39%,respectively,indicating that the introduction of OPE improved the internalization ability.Cellular specific binding experiment on PC3 cells:A 1000-fold of bombesin（BBN）,which was the standard GRPR specific expressing agonist peptide,as a blocking agent was used,and the experimental results showed that the introduction of OPE did not change the specific binding ability of the compounds.Cellular binding experiment on lung cancer cells:¹⁷⁷Lu-NOTA-OPES-RM26 had higher cellular binding in A549 cells,and lower cellular binding in H292 cells.（4）The Micro-PET/CT imaging of ⁶⁸Ga-NOTA-OPE-2-RM26 in the PC3tumor-bearing nude mouse showed that the radioactive dose was mainly concentrated in the liver,but not in the tumor.（5）The biodistributions of PC3and A549 tumor-bearing nude mice indicated that ¹⁷⁷Lu-NOTA-OPE-1-RM26was mainly enriched in liver,spleen and lung,while¹⁷⁷Lu-NOTA-OPE-2-RM26 was mainly distributed in lung,neither of which was accumulated in the tumor.（6）The therapy experiment of¹⁷⁷Lu-NOTA-OPE-2-RM26 on the PC3 tumor-bearing nude mice demonstrated that ¹⁷⁷Lu-NOTA-OPE-2-RM26 had a certain therapeutic effect.Conclusion:Two NOTA-OPEs-RM26 compounds had been successfully developed and labelled with ¹⁷⁷Lu or ⁶⁸Ga in this study.In vitro cell experiments showed that the introduction of OPE could increase the internalization of the compounds while maintaining the specific binding ability of the compounds.In vivo animal experiments showed that the introduction of OPE reduced the solubility of the compounds which affected the targeting abilities of tumors to a certain extent.The ideas of this research were helpful for the development of new targeted radiopharmaceuticals with high affinity and high internalization that integrated diagnosis and treatment.